An assessment of nucleic acid amplification testing for active mycobacterial infection
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- Nucleic acid amplification test (NAAT) for active mycobacterial infection
- Commercial NAAT
Numbers in bold highlight the three most common pulmonary and extrapulmonary NTM species that were responsible for significant disease in 2005. Source: Thomson (2010) The incidence of pulmonary disease due to NTM has been increasing worldwide. Some of the reasons for this increase include greater awareness of NTM as pulmonary pathogens, the introduction of new technologies and improvements in existing methods, enabling better detection and more-accurate identification of NTM isolates. In addition, NTM is more prevalent in an ageing population. NTM organisms originate from environmental sources such as food, other animals, soil or water. Pulmonary NTM infections are the most common and are usually caused by the MAC group. M. kansasii, M. xenopi and M. malmoense are the next most common causes, with their prevalence varying among American and European countries (Borchardt & Rolston 2013; Martin-Casabona et al. 2004). Skin and soft-tissue NTM infections, often originating from a cut or graze, manifest clinically as rashes, ulcers, nodules, granulomas, cellulitis or abscesses. NTM skeletal infections of bones, joints and tendons primarily occur following accidental trauma, surgery, puncture wounds or injections. These infections can be localised or multifocal, and can progress to septic arthritis, osteomyelitis and even bacteraemia. Disseminated NTM infections are almost exclusively limited to severely immunocompromised persons (Borchardt & Rolston 2013).
In-house NAATMost in-house NAAT methods are polymerase chain reaction (PCR)-based. The PCR process amplifies DNA via a temperature-mediated DNA polymerase, using specific primers that are complementary to the ends of the targeted sequence. PCR is carried out with a series of alternating temperature steps or cycles: (1) 92–95 °C to denature the DNA so that the two strands separate, (2) a lower temperature, usually between 45 °C and 60 °C, to allow annealing of the primer sequences to the single-stranded DNA and (3) an amplification step at the optimal temperature for the DNA polymerase, usually 65 °C. PCR can be used to amplify targeted gene sequences that vary in length from 100 bases to over 20,000 bases. For the detection of DNA sequences specifically associated with MTB or NTM, the targeted sequence is usually small, around 100 bases, but may be as large as 500 bases. A commonly occurring problem with PCR is that primers can bind to incorrect regions of the DNA, for example to a related gene from another bacterial species, resulting in unexpected non-specific products. Several modified PCR methods are used to overcome this problem. Nested PCR involves two sets of primers used in two successive runs of PCR; the second set amplifies a secondary smaller target region within the first PCR product. Thus, the second region is only amplified if the first product was amplified from the intended target sequence and not from a non-specific sequence. Real-time PCR is a quantitative method where the amplified product is detected as the reaction progresses. This method often uses fluorescent dyes to detect the PCR product. The number of cycles required and the quantity obtained of the product can be used to determine if the amplified product is due to the specific target. Products that require additional cycles and are slow to amplify are often non-specific. Reverse transcription is used to detect and amplify RNA sequences using an enzyme called reverse transcriptase, which transcribes the RNA of interest into its DNA complement. Subsequently, the newly synthesised complementary DNA is amplified using traditional PCR. Reverse-transcription PCR can be combined with quantitative real-time PCR for quantification of RNA. Multiplex PCR consists of multiple primer sets within a single PCR mixture to produce products of varying sizes that are specific to different DNA sequences. By targeting multiple genes at once, additional information may be gained from a single test run. Thus, one PCR run could be used to both identify MTB using an MTB-specific target and detect the presence of specific mutations that confer antibiotic resistance, such as the well-documented mutation in the rpoB gene that confers rifampicin resistance. Loop-mediated isothermal amplification (LAMP) is an isothermal non-PCR-based amplification method in which isothermal amplification is carried out at a constant temperature. This method employs a DNA polymerase and four to six specially designed primers that recognise a total of six to eight distinct sequences on the target DNA (Figure a).
Figure Principles of the LAMP method (a) Primer design of the LAMP reaction (b) Starting structure producing step (c) Cycling amplification step Source: Tomita et al. (2008) LAMP is then initiated by the binding of an inner primer containing sequences of the sense and antisense strands of the target DNA. Strand displacement DNA synthesis is primed by an outer primer causing the release of a single-stranded DNA, which serves as a template for DNA synthesis that is primed by a second primer pair that hybridise to the end of the target to produce a stem–loop DNA structure (Figure b). In subsequent LAMP cycling, one inner primer hybridises to the loop on the product and initiates the displacement DNA synthesis (Figure c). This results in the accumulation of 109 copies of the target in less than an hour. LAMP is relatively new and less versatile than PCR, and the primer design is much more difficult than for PCR, requiring computer programs as it is subject to numerous constraints (Torres et al. 2011). LAMP may also be combined with a reverse transcription step to allow the detection of RNA.
The Xpert assay is a semi-quantitative, nested real-time PCR test that uses a cartridge containing all elements necessary for the reaction (Association of Public Health Laboratories 2013; Lawn et al. 2013). The Xpert assay detects MTB and rifampicin resistance (considered to be a reliable proxy for MDR-TB) in sputum samples or concentrated sediments prepared from induced or expectorated sputa that are either AFB microscopy positive or negative. The Xpert assay system simplifies molecular testing by fully integrating and automating sample preparation, real-time PCR amplification and detection using a six-colour laser (Association of Public Health Laboratories 2013). The assay simultaneously detects MTB-complex and the genetic mutations associated with rifampicin resistance by amplifying an MTB-complex-specific 81-bp sequence from the core region of the rpoB gene. The assay is based on this region as it accounts for 95% of all known rifampicin-resistant mutations in MTB, and all known mutations in this region confer rifampicin resistance (El-Hajj et al. 2001; Lawn et al. 2013). It then uses five differently coloured fluorogenic nucleic acid probes, which fluoresce only when bound to their target sequence. Each probe is highly specific and binds to a different segment of the amplified core region, as shown in Figure . If the amplified sequence differs from the target rifampicin-susceptible sequence by as little as a single nucleotide substitution, the probe will not bind (El-Hajj et al. 2001). The assay also includes a sample-processing control probe, which will fluoresce even if the assay cannot detect any MTB in the sample, to distinguish between a true negative result and test failure (El-Hajj et al. 2001).
Figure The 81-bp MTB-specific rifampicin-resistance determining region of the rpoB gene The hybridisation sites of the five Xpert fluorogenic probes are shown. The single letter codes for the amino acids encoded by this region and the common single amino acid substitutions that confer rifampicin resistance are also shown. Changes in codon Ser531 and His526 account for more than 70% of the mutations in this region. Sources: Adapted from Cepheid Xpert MTB/RIF brochure (Cepheid), Casali et al. (2014) and Rattan et al. (1998) Thus, the interpretation of the Xpert NAAT results (assuming the sample-processing control probe is positive, indicating that the test has not failed) is as follows:
There are no commercially available kits for the detection of NTM listed on the Australian Register of Therapeutic Goods (ARTG). However, there are nine listed by the US Food and Drug Administration; three of these kits detect M. avium, one kit each detect M. kansasii, M. gordonae, M. intracellulare, and three kits are rapid diagnostic systems for mycobacteria4. Recently, NAAT has also been used for diagnosis of TB from extrapulmonary specimens (Lawn et al. 2013). Yüklə 3,88 Mb. Dostları ilə paylaş:
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