Genotypic Methods Nucleic Acid Amplification Tests Biology Essay

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Rapid and accurate diagnosis is a corner stone in effective clinical management and infection control of tuberculosis [26,27]. Despite recent advances in conventional TB laboratory tests such as smear microscopy and culture methods, they still lack either accuracy or speed needed to fight the emerging TB infections. Although Acid-fast bacilli (AFB) smear microscopy is rapid, specific and inexpensive, it has limited sensitivity (45% - 80% with culture-confirmed pulmonary TB cases) and poor predictive value (50% - 80%) especially in settings where NTM are commonly isolated [28,29]. On the other hand culture methods, the gold standard in the diagnosis of tuberculosis, are capable of detecting as few as 10 - 100 bacilli per mL [30], but are slow, requiring 4-8 weeks to yield results. Although liquid culture techniques reduced identification time to 10 - 14 days [31], all culture methods still fall short from fulfilling the need of rapid tests. Therefore direct detection of Mycobacteria in clinical samples suffers a major imbalance between accuracy of the used detection methods and turnaround time. Current methods are either fast but unreliable such as smear microscopy or reliable but time consuming such as culture methods. An overview of the general diagnostic algorithm for clinical pulmonary specimens is presented in Fig. 1.

Nucleic acid probe technology offered solution for direct, specific and rapid detection of viral and microbial pathogen specific targets in clinical samples. However, the performance of nucleic acid probes is dependent on the target concentration, low target concentration leads to unacceptably low sensitivity [32]. The advents of target amplification technologies, primarily polymerase chain reaction, offered a substantial breakthrough in detection of pathogens and were rapidly adopted by researcher in quest for developing rapid and sensitive direct detection method for Mycobacteria.

Over the past few decades nucleic acids amplification tests (NAATs) had been widely accepted as gold standard for the detection of different pathogenic bacteria and viruses, especially in setting when it is difficult to culture the pathogen or too dangerous and require advanced biosafety facility such as HIV [33,34]. In NAATs approach DNA or RNA strain specific target sequence is amplified by polymerase chain reaction or other techniques followed by amplicon detection step [33]. Table 1 shows selected studies using nucleic acid amplification tests for detection of Mycobacteria

Polymerase Chain Reaction

Different varieties of PCR had been developed for target amplification such as conventional PCR, nested PCR, multiplex PCR and real time PCR. Amplicon detection was also performed in different formats such as agarose or polyacrylamide gel electrophoresis, probe hybridization or fluorescent detection of the amplicon in case of real time PCR [31].

Conventional PCR

In the early 1990's single step polymerase chain reaction was extensively investigated as potential approach that encompass both speed and accuracy for the direct detection of Mycobacteria as it was already established as gold standard of many viral and bacterial pathogens [33,34]. In these studies different in-house protocols had been described for sample pre-treatment, Mycobacteria DNA extraction, amplification conditions and amplicon detection [27].

The results of these studies showed reduced sensitivity especially in smear-negative specimens in addition to variable specificity. In 1993 Clarridge et al conducted a large-scale investigation (5000 samples) to evaluate the use of conventional PCR; targeting 317 bp of IS6110 insertion sequence; for the detection of Mycobacteria in clinical samples, they compared the overall sensitivity and specificity to culture on Lowenstein Jensen and MiddleBrook agar. The results variation was correlated to reaction inhibitors, PCR contamination and non-specific amplification [27,35]. The performance of PCR was also correlated to the number of Mycobacteria bacilli in the sample, low load sample with <50 CFU/ml showed 52% positive PCR, while samples with >100 cfu/ mL showed 98% positive PCR in comparison to culture [32,35]. In general the studies showed result variations of the conventional PCR for the direct detection of Mycobacteria specially in paucibacillary settings in either smear negative or extrapulmonary TB, thus the conventional PCR was left behind because of its low sensitivity [33]. Are there any studies more recent?

Nested PCR

In quest for increased sensitivity nested PCR was adopted as it can enhance the sensitivity up to 1000 fold compared to single-step PCR [36]. In nested PCR the first reaction amplify target region using pair of specific primers "outer primers" followed by a second amplification reaction that amplifies a smaller region within the amplicon of the first reaction. This is done using a second pair of primers placed internal to the outer primers referred to as "nested" or "internal" primers. The second reaction uses the product of the first reaction as template. In some cases only a single internal primer is used in conjugation with one of the outer primers from the first reaction, in this case it is called "Hemi-nested" or "semi-nested" PCR. But this increase in sensitivity came on a price of increased risk of cross contamination and false positives due to amplicon handling, as the tube of the first reaction is opened to transfer part of the reaction mix to be used as template for the second reaction. The cross contamination problem was addressed by the single-tube nested PCR approach, in this approach the inner primers are designed to have a lower melting teempreture than the outer primers and are incorporated in the same reaction tube with the outer primers. During the first phase of the single-tube nested PCR only the outer primers anneal and amplify their specific region as the annealing temperature is higher than the inner primers melting temperature. During the second phase the annealing temperature is lowered to allow the annealing of the inner primers. Chan et al [37], evaluated the usefulness of single-tube nested PCR in the diagnosis of tuberculosis in clinical samples, they tested 1497 pulmonary and 536 extrapulmonary specimen utilizing two sets of primers with different melting temperatures incorporated in the same reaction(melting temperatures were 88°C for outer primers and 70°C for inner primers). During the first 15 cycles of the reaction, the annealing temperature was 72°C to allow only the annealing and extension of the outer primers, the annealing temperature was then lowered to 55°C to allow the annealing of the for extrapulmonary tuberculosis specimens [37].

Multiplex PCR

Multiplex PCR assays were applied for rapid simultaneous detection and identification of different mycobacterial strains. In a recent study Perez-osorio et al developed multiplex PCR assay for identification and drug resistance screening, which allows identification of Mycobacterium tuberculosis complex, Mycobacterium avium complex with simultaneous detection of rifampin, isoniazid and pyrazinamide genes. the reaction also amplifies a region of heat shock protein 65 (hsp65) for further confirmation or identification of other strains by DNA sequencing [38].

Anilkumar et al developed a tetraplex PCR assay that can differentiate between Mycobacterium tuberculosis complex (MTBC) and NTM and can easily categorize clinical isolates into MTBC or NTM [39]. The assay simultaneously amplifies four DNA regions (IS6110, recA intetin, plcA and 16rRNA gene) for differentiation between MTBC and NTM strains. The insertion sequence IS6110 is a common target for identification and typing of MTB strains, however since strains lacking IS6110 has been reported, Anilkumar et al targeted a MTBC specific region of the recA gene in addition to IS6110. plcA gene is deleted in M. bovis and M. bovis BCG, while present in the three other MTC members, thus it allow differentiation between M. bovis and BCG from other MTBC strains. To comply to the guidelines of the American Thoracic Society, and Infectious Disease Society of America, that recommend the identification of clinically important NTMs to species level, authors targeted genus specific region in the 16s rRNA, thus NTM strains were identified by the presence of the 16s rRNA genus specific amplicon, while the other bands are absent. MTBC strains except M. bovis and M. bovis BCG gives typical four band pattern [39].

Real-time assays

The early 2000,s have witnessed major development in the application of real-time PCR techniques for the detection of different infectious agent including Mycobacteria [33]. Real-time PCR technology provides a major advantage over conventional PCR by offering a closed system for real-time monitoring of PCR amplicon formation during the reaction in comparison to end point detection and post PCR handling in conventional PCR. Different chemistries are available for real-time detection of PCR amplicons. These can be classified into three basic signaling systems, DNA binding dyes, dye-primer based systems and , probe-based systems [40]. All three systems employ fluorescent dyes. The reaction starts by initial low fluorescent signal that increase exponentially during succeeding PCR cycles in response to increase in amplicon formation [40].

DNA binding dyes

DNA binding dyes are fluorescent dyes that emit undetectable fluorescent when in free form or in the presence of single stranded DNA. During the PCR these dyes are incorporated in the newly formed ds DNA and emit high fluorescent signal [41].

SYBR® Green I (Molecular Probes - Invitrogen) is the most commonly DNA binding dye in real-time PCR reaction. It bound to the minor Groove of the newly formed double stranded DNA, DNA-dye complex results in dramatic increase (about 2000-fold) in the fluorescent signal compared to the free dye [40,41]. Other types of DNA binding Dyes were recently introduced to market such as EvaGreenâ„¢ (Biotium, Hayward, CA) which was reported to have some advantages over SYBR Green such as lower PCR inhibition, greater stability in high temperature and stronger fluorescent signal [40]. Another type of DNA Binding dye is BOXTO (TATAA Biocenter, Gothenburg, Sweden), that is optimally excited in wave length higher than SYBR Green or EVAGREEN and thus can be used in the same reaction with FAM conjugated probe to detect primer-dimers or melting curve analysis [40]. A detailed of these dyes are beyond the focus of this document for further details reader can check "Real-Time PCR", edited by M. Tevik Dorak, 2006 [40].

The SYBR green assays advantage of are low cost of assay, simple assay development in comparison to other real-time assays and successful assay only need a carefully designed pair of primers. On the other hand SYBR green assay has a major limitation as the fluorescent signal is non-specific, and dsDNA will generate signal such as primers-dimer and non-specific amplification [40].

Dye Primer based system

In these systems the one of the primers is labeled by fluorescent dye which is quenched in one of different format according to the primer type. When the primer is specifically annealed to its target the quencher is released and the fluorescent signal develops in response to amplicon formation. Specificity of dye-primer based assay depends on the annealing of the primers with their complementary sequence in the target region. These systems also provide multiplexing capability by using primers labeled with different dyes for different target, this multiplexing capability cannot be achieved with DNA binding dyes [40]. LUXâ„¢ (light upon extension, Invitrogen) primers and scorpions are example of Dye-primer based system that will be briefly discussed.

LUXâ„¢ (light upon extension, Invitrogen) system consists of two primers one of which is labeled with fluorescein dye. The labeled primers is designed to contain short 4-6 base extension on 5' end that is complementary to internal region near the 3' end that overlap with the position of the fluorescein dye. These self-complementary stretch leads to the formation of stem loop secondary structure that quenches the fluorescein signal. During the PCR reaction the primers are linearized and incorporated in the newly formed strands, this structural change leads to increase in the fluorescent signal.

Scorpion primers are physically coupled to a probe that is held in hairpin structure by complementary short strand at 5' and 3' sides. The probe is complementary to target region downstream the primer and is labeled by fluorescent dye at the 5' end that is quenched by quencher joined to the 3' end of the probe. The probe is linked to the 5' end of the primer by PCR stopper. During the PCR the probe bind to its specific target in amplicon opening the hairpin structure and separating the fluorescent dye from the quencher, which release fluorescent signal. Scorpions are very sensitive for the detection of single base mutation [41,42].

Probe based assays

Probe-based assays involve fluorescently labeled oligonucleotide located between the forward and reverse primers. TaqMan® probes, Molecular Beacons®, and hybridization probes are the most commonly used probes under this category.

TaqMan Probe

specifically target internal region between the two primers, it contains both fluorescent dye at 5' end and quencher at 3' end, the fluorescent dye and the quencher are in close proximity and the signal is quenched. TaqMan probes are designed to bind to their targets prior to primers, during PCR reaction the TaqMan probes are cleaved by the Taq DNA polymerase 5' exonuclease activity during the new strand synthesis. The cleavage of the probe releases the fluorescent dye and distances it from the quencher and fluorescence signal is generated [40,41].

Molecular Beacons

Molecular beacons are similar to TaqMan probes in having one end labeled with fluorescent probe and the other with quencher, but their signal doesn not depend on cleavage with DNA polymerase. Molecular beacon are designed as a stem-loop structure with short self-complementary sequences on each side of the probe, thus molecular beacon form perfect stem structure that brings both dye and fluorescent into close proximity and thus the signal is quenched. In PCR reaction the probes are unfolded and specifically bind to their target, this separate the dye from quencher and the signal is generated [40].

Hybridization probes

Hybridization probes consist of two probes specifically target the internal region between the two primers. Signal generation depend on FRET rather than quenching, one probe with donor fluorophore (3' end) and other probe with acceptor fluorophore (5' end). When the probes anneal to target both the donor and acceptor come in close proximity, in this case the donor fluorophore (e.g a fluorescein dye) absorb the energy from light source and transfer it to the acceptor fluorophore (e.g. a rhodamine dye) by FRET. The acceptor dye emit fluorescent signal that is detected.

Hybridization probe can be used for fluorescence melting curve analysis which is a useful method for typing microbial strains based on genomic sequence variations, mutations or polymorphisms. Melting curve analysis is based on the fact that perfectly matched probes melt from their target at temperature (Tm) higher than mismatched or partially annealed probes. For this type of analysis a pair of hybridization probe is designed in the same concept of hybridization probes. First probe covers the region of polymorphism and called the sensor probe and a second probes covers more conserved region and called anchor probe. Melting curve analysis starts by a denaturation step at 95°C for complete dissociation of all probes, primers and target double strands, this is followed by low annealing temperature (around 40°C) to ensure binding of both perfect and mismatched probes finally incremental increase in temperature to melt the hyberdization probes. When the sensor probe melts off its target fluorescence signal change and Tm calculated.

Application of Real-Time PCR in detection of Mycobacteria

In the recent years real-time PCR has been widely used as powerful tool in Mycobacteriology laboratories for several applications that includes, identification of Mycobacteria in clinical specimens, identification of species and subspecies, differentiation of MTBC from non-MTBC strains, detection of antimicrobial resistance and quantitation of Mycobacteria [27,41].

Pathogenic strains of Mycobacteria can be identified and differentiated according to SNPs and mutations in specific genes or genomic regions such as 16S rDNA gene, ITS region, rpoB and hsp65 [27]. In the method reported by Kim et al, hybridization probes were used to detect and differentiate seven Mycobacterial pathogenic strains according to mutation in hsp65 gene through melting curve analysis approach. This method was successfully applied to standard strains and clinical isolates with 100% sensitivity and specificity. it was also applied to sputum samples with AFB score from trace to 3+ showing sensitivity 94.3% with AFB score 2+ and above while sensitivity with AFB score trace was 76% compared to rpoB PCR-restriction analysis [43].

The Beijing family of Mycobacteria tuberculosis is a large genetically related group of Mycobacteria highly prevalent in Asia and former Soviet Union countries [44]. This Mycobacteria genotypic family lineage is considered highly virulent and transmissible strains associated with multi-drug resistant in several areas and thus are closely monitored and studied [45]. Identification of Beijing family strain is based on genetic variation and several polymorphic or hypervariable genetic markers [44], IS6110-restriction fragment length polymorphism (IS6110-RFLP) and spacer oligonucleotide typing (Spoligotyping) are commonly used genotyping method for the Beijing strain detection based on their banding or hybridization pattern [44,45]. Absence of RD region , intact open reading frame in the pks15/1 gene and recently single nucleotide polymorphism (SNP) in the Rv2629 gene were also proposed as useful markers [45,46]. Alonso et al reported a novel real-time PCR based method for the detection of Beijing strain targeting Rv2629 SNP by real-time PCR followed by high resolution melting analysis (HMR) approach. First real-time PCR was performed on DNA extracted from the samples using primers spanning the SNP site (A191C for Beijing genotype and A191A for non-Beijing genotype) and two molecular beacons labeled with two different fluorophores, one specific for 191C and one specific for 191A as described by Chakravorty et al [47]. Melting curve profiles were generated at temperature range from 65°C to 95°C at an increment of 0.05 °C/s. Melting curve analysis showed higher Tm for Beijing strains (92.4 ± 0.02°C) than non-Beijing strains(91.9 ± 0.03°C). the method sensitivity was evaluated by testing 44 respiratory clinical samples with different AFB load with overall sensitivity 84.1%. sensitivity obtained with high and intermediate/low specimens were 100% and 26.9 respectfully [45].

In another approach to identify and preliminary genotype Mycobacteria clinical isolates according to RD750 polymorphism, cheah et al combined TaqMan/SYBR green assay [48], they differentiate M. tuberculosis complex (MTBC) from non-tuberculous Mycobacteria (NTM) by real-time PCR targets 16s rDNA gene using MTBC-specific 16s TaqMan probe. The assay also identified the global lineage-defining RD750 polymorphism by use of hemi-nested PCR and RD750+ -specific TaqMan probe to differentiate MTBC strais with intact (RD750+) or deleted (RD750-) RD750 regions, SYBR green was used in both assay for total amplicon detection [48].

Isothermal Nucleic acid amplification methods

Strand displacement amplification

Strand displacement amplification (SDA) is an isothermal amplification method first reported by Walker et al 1992 [49], this method utilizes two enzymes HincII restriction endonuclease and exonuclease deficient polymerase (exo-Klenow) for isothermal amplification of DNA targets. This assay is based on the ability of HincII to nick unmodified recognition sequence bound to its complementary hemiphosphorothioate modified strand, which is followed by the polymerase activity of (exo-Klenow) that extend 3' end at the nick site with consequent displacement of the downstream strand which act as template for the reverse strand [49,50], the detailed principle of SDA is illustrated in Fig.2. In their report Walter et al applied SDA for the detection of genomic DNA samples of Mtb and M. bovis using a portion of IS6110 as target and showed 106 fold ? amplification [49].

SDA had a major development few years later by the introduction of BDProbeTec ET system (Becton Dickinson, Sparks, Md.) that combines the isothermal DNA amplification based on SDA and real-time fluorescence detection for target DNA detection [50,51]. The main modification was using BsoBI restriction enzyme instead of HincII and Bst DNA polymerase instead of exo-Klenow. This system was employed for direct detection of Mycobacterium tuberculosis in clinical samples by co-amplification of a 95-bp region in IS6110, highly specific MTBC, and 16S rRNA gene, common to most Mycobacteria. Real-time fluorescence detection is achieved by Förster resonance energy transfer FRET based stem-loop beacon type probes containing fluorescein and rhodamine labels. The probe loop region compromise BsoBI restriction enzyme recognition site. The probe also contain a target specific sequence attached 3' to the rhodamine label. During the SDA in the presence of the target the probe is linearized and incorporated in the double strand amplicon, thus exposing the restriction enzyme recognition site, which is cleaved by restriction enzyme releasing the fluorescein, which leads to generation of fluorescent signal Fig 2 [50,51].

BDProbeTec ET MTBC direct detection assay provides a rapid semi-automated platform for the direct detection of M.tuberculosis in clinical samples [52], with overall sensitivity of 90-100 % & specificity of 92% [31], in another study sensitivity and specificity shown to be 92.7% and 96.0% respectively in respiratory specimens [27]. With smear positive samples BDProbeTec ET showed 100% f sensitivity and specificity and 87.1% sensitivity and 96.5%.specificity for smear negative samples [52].

The performances of the BD ProbeTec ET was compared to COBAS AMPLICOR MTB (Roche) for detecting Mycobacterium tuberculosis complex in various respiratory specimens, in this study 824 specimens of various respiratory clinical specimen were tested, these included 109 culture positive and 715 culture negative BACTEC and MGIT culture system were used as reference. The results showed sensitivity of 86.2% and 78% for BD ProbeTec ET and Amplicor respectively while both assays showed similar specificity of 99.9 % [27,53].

Compared to AMTD Test (Gen-Probe, CA, USA) Table 2 for detection of M. tuberculosis in respiratory and extrapulmonary specimens, BD ProbeTec ET showed sensitivity and specificity of 94.5% and 92.3% respectively vs 88.0% and 74.3% for AMTD. The presence of internal amplification control in BD ProbeTec was an advantage that allowed detection of false negatives due to reaction inhibitors [27].

Nucleic acid sequence based amplification (NASBA)

Nucleic acid sequence based amplification (NASBA) is isothermal nucleic acid amplification method currently owned by bioMérieux Inc., Boxtel, The Netherlands (Table 2). This method that produces high level of amplification in order of 109 copies, at constant temperature that can be achieved by heat block or water bath eliminating the need of expensive thermal cyclers [54].

NASBA uses three enzymes AMV-reverse transcriptase, RNase H and T7 RNA polymerase and two primers to amplify RNA target. The reaction start by annealing the first primer complementary to the 3' end of the target this primer contains promoter site of T7 bacteriophage polymerase at its 5' end. AMV-RT enzyme produce cDNA copy of the target by elongating the annealed primer, this forms DNA /RNA heteroduplex which is substrate for the RNAse H, at this point RNAse H hydrolyze the RNA strand leaving the ssDNA. Second primer anneals to the ssDNA and extended by the action of reverse transcriptase enzyme synthetizing dsDNA and rendering the T7 promoter double stranded and transcriptionally active. T7 RNA polymerase start recognize the active promoter site in the dsDNA and produce numerous copies of RNA [55]. NASBA is reported to as sensitive as PCR or more [54], it was used to amplify of RNA target for the detection of Mycobacteria in conjugation with ELISA [56]. Figure 3 illustrate the principle of commercial assays using NASBA

Transcription mediated amplification (TMA)

Transcription mediated amplification (TMA) is another isothermal amplification method very similar to NASBA that works in the same way except for the degradation of the template initial RNA strand following reverse transcription, in NASBA template RNA strand in the RNA/DNA hetero duplex is degraded by RNAse H while in TMA it is degraded by the RNAse activity of the reverse transcriptase [55]. TMA is utilized for the amplification of 16S RNA target in AMPLIFIED MTD test (Gen-Probe Inc., San Diego, CA) (Table 2). The commercial assays using TMA is illustrated inFig 3.

DNA Probe Technology

Culture identification DNA probes technology (Direct Hybridization probes)

Culture identification probe technology is one of the successful and most widely used technologies for identification of Mycobacterium culture. The AccuProbe (Gen-Probe,San Diego, CA, USA) Table 2, Fig 3, was the first commercially available molecular system for the Mycobacterium culture identification [31]. This assay can rapidly identify and differentiate several clinical important Mycobacteria such as M.tuberculosis complex, M. avium complex, M. avium, M. Kansasii, M. gordonae from culture [27,31]. The assay utilizes single stranded DNA oligonucleotide labeled with acridinum ester that targets species specific ribosomal RNA (rRNA), as a chemiluminescent probe. rRNA is released from harvested cells by sonication, followed by hybridization phase in which the probes are added to cell lysates. The probes bind to target organism rRNA forming stable DNA/RNA hybrid. Hybridization protection assay; a selective chemical degradation process of acridinium label associated with single stranded DNA; is used to differentiate hybridized and unhybridized probes. In the final step the DNA/RNA hybrid concentration is measured using GEN-PROBE luminometer to detect the chemiluminescence after addition of appropriate substrate [27,57,58]. Accuprobe for Mycobacteria culture identification was approved by the US Food and Drugs Association (FDA) in early 1990's, it was widely evaluated with both liquid and solid cultures and recognized as rapid, highly specific and sensitive procedure with turnaround time less than 2hrs [27,31].

Line Probe assays

Line probe assays utilize solid phase reverse hybridization technology in which specific probes are immobilized in parallel lines on solid support; usually nitrocellulose or nylon membranes strips; to detect labeled target DNA in solution. In this assay approach the target DNA region is amplified with labeled primers, the labeled amplification product is then incubated with the solid support bearing the specific probes. Following incubation and proper washing steps to remove unbound DNA, the hybridized probes are revealed by color development through enzymatic reaction utilizing the amplicon label [59,60].

Inno LiPA Mycobacteria v2, (Innogenetics, Ghent, Belgium) Table 2, Fig 3, assay is based on on the above mentioned approach, it detects and identifies genus Mycobacteria and 16 Mycobacterial species based on variation in 16S-23S internal transcribed spacer (ITS) region [27,61]. In this assay the 16S-23S ITS region is amplified by biotinylated primers, the biotinylated amplicon is then hybridized with the specific probes immobilized on the membrane strips. Following hybridization, streptavidin-labeled alkaline phosphatase is added which binds specifically to any biotinylated DNA-probe hybrid. The color is then developed by incubation with 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium (BCIP/NBT) chromogen which develop purple/ brown precipitated at positive probe bands, this result in specific banding pattern for each strain.

Inno LiPA Mycobacteria v2, (Innogenetics, Ghent, Belgium) assay is applied on solid and liquid culture media and has been evaluated in various studies in comparison to biochemical and molecular tests. In their study evaluating the performance of DNA probe assays for the identification of Mycobacterial species isolated from clinical specimen, padilla et al [62] reported 100% assay sensitivity with the Mycobacterium genus probe and accuracy of 99.1% for correctly identifying genotypes [62]. In another study by Tortoli et al [63], assay senstivity and specificity were 100% and 94.4% respectively. The study also showed that the probes specific for Mycobacterium fortuitum complex, for the Mycobacterium avium-intracellulare-scrofulaceum group, and for Mycobacterium intracellulare type 2 cross-reacted with several Mycobacteria rarely isolated from clinical specimens [27,63]. InnoLipa showed heterogeneity in the identification of Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum (MAIS) complex isolates and sequencing methods are suggested for the identification of theses strains [64-66].

GenoType® Mycobacteria product series (Hain Lifescience, Nehren, Germany) Table 2, Fig 3, are another commercial assays for the identification of Mycobacteria based on reverse hybridization technology, these includes different assays for the identification of Mycobacteria such as GenoType® Mycobacterium CM, GenoType® Mycobacterium AS, GenoType® MTBC for identification of Mycobacteria from positive solid and liquid culture and GenoType® Mycobacteria Direct for direct identification of Mycobacteria from decontaminated clinical samples. The CM assay for more frequently isolated mycobacterial strains and AS assay for less common species [67]. Both assays target the 23S rRNA gene, the line probes are fragment of 23s rRNA shared by different strains thus results are based on band pattern generated by different positive probes specific for each strain [60]. MTBC assay differentiate the members of M.tuberculosis complex based on gyrB DNA sequence polymorphisms and the RD1 deletion from positive liquid and solid cultures [27,60,68]. GenoType® Mycobacteria Direct is specific for direct detection of RNA in clinical sample compromising NASBA amplification and DNA strip technology, it allow simultaneous detection and differentiation of MTBC and M. avium, M. intracellulare, M. kansasii, M. malmoense as four of the most clinically important NMT strains.

Genotype® assays are rapid and easy to perform and allow identification of several mycobacterial strains eliminating the need of further sophisticated tests [27]. GenoType® Mycobacterium CM assay and AS assay showed 92.6% and 89.9%, respectively, concordant results with 16S rRNA sequencing when evaluated by Richter et al [69]. In another study sensitivity and specificity of GenoType® Mycobacterium was 97.9% and 92.4% for CM assay and 99.3% and 99.4% for AS assay compared to 16S rRNA sequencing [67]. In their evaluation study Russo et al reported system limitation represented in the inability to differentiate some strains due to similar band pattern [67]. In a recent study evaluating the ability of commercial probe assay to identify less frequent isolated strains Tortoli et al reported that, Genotype® misidentified 28 taxa out of 317 strains, belonging to 136 species, 61 of which had never been assayed before [66]. Neonakis et al evaluated the ability of Genotype® MTBC to differentiate 120 clinical Mycobacterium tuberculosis complex clinical isolates identified with biochemical tests and culture, MTBC showed 100% agreement with the previous identification of the strains [66].

Nanoparticles and novel approaches

Gold nanoparticles (AuNPs) exhibit unique optical and physical characteristics that qualified them as potential candidates for novel biomarker detection platform. The typical structure of AuNPs is nanosized spherical gold particles or thin gold shell surrounding dielectric core such as silica range in size from 0.8 nm to 250 nm. AuNPs are characterized by easily tuned physical properties, high surface area, high absorption coefficient and unique optical properties. AuNPs can be easily synthesized and surface functionalized with different biomolecules such as oligonucleotides, peptides, and antibodies [70,71]. AuNPs' unique optical properties arise from phenomena known as Surface Plasmon Resonance (SPR), when the AuNPs are hit with electromagnetic radiation with a wave length larger than the diameter of the particle coherent, resonant oscillation of metal electron across the particle is induced and result in the characteristic exceptionally high absorption coefficient of AuNPs, colloidal solution of 20 nm AuNPs exhibit a SPR band with absorption maxima at approximately 520 nm and appear red in color [71]. When AuNPs aggregate SPR layer of locally adjacent particles interact (Plasmon-Plasmon interaction) resulting in shifting the absorbance maxima of the SPR layer to higher wave length and changing the particle color to blue [71]. The color change induced by the aggregation of AuNPs is the base for colorimetric assays utilizing AuNPs for detection of specific analyets. There are numerous methods for the detection of AuNPs such Scanometric, Fluorescence and surface-enhanced Raman scattering that had been used in different assays, discussing the basic concept of these method will be beyond the focus of this review.

Over the past two decades gold nanoparticles have been subject of intense research for the development new biomedical assays for the detection of clinically significant bioanalytes [72]. The main approaches for utilizing AuNPs in detection are modified non cross-linking, modified cross-linking and unmodified AuNPs Fig 4. In the modified non cross-linking approach AuNPs are functionalized with oligonucleotides, in the presence of complementary target modified AuNPs bind to their targets. When salt is added to induce AuNPs aggregation solution remains red in case of positive samples as complementary target prevent aggregation of AuNPs, while in absence of target AuNPs aggregate and solution turns blue. In modified cross-linking approach a pair of AuNPs linked oligonucleotides for each target is designed, in the presence of target AuNPs probes bind to their targets which brings the AuNPs in close proximity and thus the solution color turn to blue, in absence of target AuNPs remain dispersed and solution remain red. In unmodified AuNPs approach, ssDNA-probes stabilize the dispersed AuNPs and prevent salt induced aggregation, in presence of target ssDNA-probes anneal to their target leaving the AuNPs liable for salt-induced aggregation. In positive samples solution turn blue upon addition of salt while in negative samples solution remain red upon addition of salt. In the quest for fast, rapid and simple detection of Mycobacteria, AuNPs were resorted to for the development of new Mycobacteria detection and identification assays.

Costa et al reported the use of AuNP in a colorimetric approach for fast, specific and sensitive identification of Mycobacterium tuberculosis complex and differentiation of M. bovis and M. tuberculosis using gyrB gene PCR amplicon. In their work Costa et al utilized gold nanoparticles derivatized with thiol modified oligonucleotide in colorimetric non cross-linking approach to detect specific DNA target in gyrB PCR amplicon. Functionalized AuNPs are added to the PCR product and allowed to stand for 30 min at room temperature for hybridization followed by the addition of MgCl2 to induce AuNPs aggregation. The presence of complementary target stabilize the AuNPs and prevent the aggregation and solution remains red in color, while in absence of complementary target the AuNPs aggregate under the effect of salt and solution color change to blue. AuNPs aggregation can be monitored either visually by change of color or by spectrophotometric comparison of solution before and after addition of salt, as non-aggregated AuNPs absorbance peak at 526 nm, while aggregated particle shows absorbance maxima at 600 nm (red shift). The assay was evaluated using PCR amplicon of eight standard strain and fifteen clinical isolates and showed 100% concordance with gyrB-PCR-RFLP [73]. Sensitivity and specificity?

In another study Lindaris et al, utilized the same non-cross linking approach using thiol-linked ssDNA-modified gold nanoparticles for direct detection of Mycobacteria DNA without PCR amplification. The 16S-23S ITS region was targeted to allow collective detection of the main pathogenic strains of Mycobacteria M.tuberculosis complex (MTC), M.avium complex (MAV) and M.avium subspecies paratuberculosis (MAP) without the need of DNA amplification [74]. AuNPs aggregation was induced using 0.01 N HCl. First the assay was optimized on previously identified isolates DNA and showed positive results (red color) with all mycobacterial strains and negative (blue color) for non-mycobacterial strains. For performance evaluation the optimized assay was evaluated for the detection of MAP DNA in 12 goats faecal samples and results were compared to Real-time PCR, the assay showed 87.5% & 100% concordance with real-time PCR results for positive and negative samples respectively [74].

Soo et al [75] utilized the modified AuNPs cross-linking approach for the detection of MTB and MTBC targeting IS6110 and Rv 3618 in nested PCR product. In this approach a pair of thiol modified probes linked to AuNPs were designed for each target, in the presence of complementary target the two probes anneal bringing the AuNPs in close proximity which change the solution color from red to blue. In absence of target AuNPs remain dispersed and the solution color remains red The assay had 96.6% and 98.9% sensitivity and specificity respectively for MTBC samples, while MTB samples showed 94.7% and 99.6% respectively [75].

Expert commentary:

Tuberculosis is an ancient affliction that still claiming lives to date as a leading cause of death attributed to infectious diseases. In 2010 TB claimed more than 1.2 million lives and infected 9 million new and recurrent cases. The impact of global TB burden was intensified by HIV, poverty and limited laboratory capacity in high burden area. HIV pandemic worsen the situation as it increased the risk of death due to TB; in 2010 about 350,000 people died from HIV-associated TB which represent about 25% of deaths among HIV infected people most of them in sub-Saharan Africa. Poverty and undernourishment reduce both immunity and general hygienic living standard which consequently increase TB incidence and prevalence. according to FAO report 2010 65% of the world's hungry population lives in seven countries India, China, the Democratic Republic of Congo, Bangladesh, Indonesia, Pakistan and Ethiopia; all these seven countries are among the 22 TB HBC's (High burden countries) and contribute to about 68% of total TB notified cases in 2010. TB controlled efforts are tackled by limited inadequate capacity of both conventional and drug susceptibility testing (DST) laboratories; according to WHO only 14 out of 22 high burden country could meet the targets and provide 1 microscopy center per 100,000 population. While only 16 out of 36; representing high TB and MDR-TB burden countries; had provided one culture and sensitivity lab for every 5 million. These limited diagnostic capacities due to limited resources have a direct impact on TB control as only minority of the 9 million new case of TB each year receive laboratory confirmed diagnosis.

Reliable early diagnosis of TB is decisive in disease control and patient management. Furthermore detection of latent TB infection is pivotal in controlling disease spreading among vulnerable population especially in HIV pandemic era. Throughout the past decades TB diagnostics continues to rely on traditional approaches that still hold major limitations despite some developments that have been introduced. For example smear microscopy; still the cornerstone in active pulmonary TB diagnosis especially in low resource settings; is specific but highly insensitive especially in paucibacillary settings. Introduction of fluorescent microscopy improved the sensitivity but still unreliable. While culture methods are still considered the gold standard for their ability to detect very low bacillary load and extrapulmonary TB, are slow technically demanding and doesn't fit in low resource setting where the major TB problem reside. Automated culture systems reduced the turnaround time but still technically demanding. NAAT tests and DNA probe technology tests are highly specific while their sensitivity in paucibacillary settings such as smear-negative pulmonary infections, extrapulmonary infections and high HIV settings is variable and inconsistence. In HIV pandemic era there isn't really what can be called non-pathogenic mycobacteria; specificity of nucleic acid based tests to NTM's is challenged as more and more NTM's are isolated and correlated to tuberculosis in immunocompromised patients.

The few past years witnessed the promotion of researches and innovation in TB diagnostics driven by market potential, public and private funds and WHO initiative. To support TB control efforts, new diagnostics need to cover three types of tests; Active TB detection, latent TB detection, motoring treatment response and resistance detection. Major issues needed to be addressed in new diagnostics include improving molecular assays sensitivity and consistency in paucibacillary settings for reliable results in extra-pulmonary TB, smear-negative and HIV settings. Enhanced specificity of primers and probes of molecular assays against new strains of environmental mycobacteria associated with disease in HIV patients. There is urgent need for new point-of-care tests, capable of delivering rapid and accurate results in peripheral clinics and urban areas. The sophistication level, technical demand and intensity of labor needed by new diagnostics should be brought down to fit low resources setting countries were 90% of TB patients lies. Price affordability is also critical issue in new diagnostics.

Five-year view:

All the world's six regions are on track to achieve Millennium Development Goal target that TB incidence rates should be falling by 2015. All the world's region except Africa are on track of halving 1990's mortality rates. On the other hand halving the 1990's prevalence rate is not expected to be met.

During the next five years the TB diagnostics market will continue to expand, the economic potential in this growing market will attract more investment decisions which promote research and innovations in quest for new breakthrough product. The advent of Nanoparticles with their unique optical and physical characters holds great potentials for new TB diagnostics. Nanoparticle based diagnostics can combine sensitivity, specificity, low cost and simple technical demands in one test. More researches addressing utilization of nanoparticles in TB diagnostics are needed to unleash their potentials in the next years.

Further researches are required on NTM's isolated and correlated to infections in HIV patient to develop more specific tests. For latent TB detection the only available tests are Tuberculin sensitivity testing (TST) and the interferon gamma release assay. TST is unreliable while IGRA's is complicated and technically demanding. A less demanding accurate test of latent TB infection is required. Future researches should also address developing of new test for treatment monitoring as None of the current molecular tests are optimized for treatment monitoring.

Key issues:

Tuberculosis (TB) is a world leading cause of death , it is only second to HIV as cause of death attributed to infectious diseases

TB was declared as global emergency by WHO in 1993.

About 25% (1.4 billion) of world population maintain latent infection; 9.2 million cases in HIV negative patient and 1.2 million cases are reactivated each year due low socioeconomic conditions including malnutrition, poverty and co-endemicity with HIV.

TB causes about 1.2 million in 2010; that leads to about 10 million orphan children as result of parents' death by TB.

In 2010 there were about 1.2 million HIV positive new TB cases; more than 80% of them were in sub-Saharan Africa; with 350,000 deaths of HIV-associated TB.

The world's regions suffering the most of Tuberculosis are the world poorest undernourishment ones; 65% of the world's hungry population lives in seven countries; all these seven countries are among the 22 TB HBC's (High burden countries) and contribute to about 68% of total TB notified cases in 2010.

Rapid and accurate diagnosis plays a pivotal role in controlling the spreading of TB.

None of the current diagnostic method can provide satisfactory swiftness and accuracy.

Smear microscopy is a traditional diagnostic method that is easy to perform and highly specific but has a very limited sensitivity and poor predictive value especially in high prevalence HIV settings. It has a minimum detection limit of 10,000 bacilli/ mL.

Culture methods are gold standard for diagnosis and sensitivity testing of tuberculosis; capable of detecting as low as 10 bacilli per mL. However it time consuming and requires up to 21 days for results delivery. Automated culture system reduced the testing time to 9-14 days but it came with high cost and intensive labor settings.

Biochemical tests are useful in identifying and differentiating TB complex strains from Non-tuberculosis mycobacteria (NTM) which is crucial for selection of treatment policy; unfortunately they are still time consuming, labor intensive and require high cost central laboratories.

Nucleic acid amplification methods did not exactly meets the high exceptions in TB diagnosis from clinical samples; time is reduced to hours but as for sensitivity, it still not reliable especially in smear-negative samples and HIV settings. Negative samples still has to be confirmed by culture.

Nucleic acid amplification method require highly equipped laboratories with trained personnel; the thing which is not sufficiently available in high burden areas

Isothermal amplification methods eliminated the cost of PCR facility with accepted performance parameters except in smear-negative samples.

Due to the high prevalence of HIV the term "non-pathogenic mycobacteria" is not really applicable anymore. More and more case are reported caused by NTM's which is considered challenging for molecular based tests due the specificity of the used probes.

There is an urgent need to reconsider and evaluate the commercially available primers and probes used for testing clinical samples for their ability to detect and identify NTM's.

Nanoparticle has provided a new edge for the developing of novel nanoparticle based tuberculosis diagnostic tests that combine both speed and accuracy.

Nanoparticles interact on a one-to-one base with target molecules; which allows low detection limit that allow direct detection in clinical samples without prior isolation or target amplification.

The low detection limit can allow the detection of TB in smear-negative samples and high HIV settings.

Nanoparticles based method doesn't need sophisticated laboratories and can easily applied in low resources settings.