comparing the position of the DNA marker, having a size equivalent to the size expected for the PCR product of interest. The identification on the gel of the amplified product with the expected molecular weight indicates the presence of an MTBC member in the sample studied [55, 56].

therapy [66]. PCR multiplex is a rapid, sensitive and specific diagnostic test for acute bacterial meningitis. PCR is particularly useful for analyzing cerebrospinal fluid of patients who have been treated with antibiotics before lumbar puncture [64]. Furthermore, in 2002, Shah et al. [67] reported a multiplex PCR-based assay performing a rapid and specific differentiation of M. bovis and MTB. This procedure can be beneficial for medical and veterinary microbiological laboratories. The designed multiplex PCR to identify MTB or M. bovis is based on the differential amplification of pncA gene. This test is highly specific and sensitive. Only 20 pg of pure mycobacteria DNA are needed, and none PCR amplification product has been obtained from

Conventional and Molecular Diagnosis of Drug-Sensitive and Drug-Resistant Pulmonary Tuberculosis

http://dx.doi.org/10.5772/intechopen.75004

There is a series of requirements to be met to perform a good multiplex PCR analysis. These are as follows: (1) Accomplish the standard requirements for primer design. (2) Design one pair of primers for each expected amplicon. (3) Apply suitable temperatures, allowing DNA polymerase works accurately in every multiplex PCR cycle. (4) The primer design must be optimized; so that, all primer pairs work properly, at the same time and in a single reaction. (5) Apply similar alignment temperatures for all primers. (6) Choose the size of those DNA regions to be amplified so that be possible obtaining amplicons having clearly different sizes among them. Allowing, in this way, to distinguish the bands between them in the electrophoresis gel, (7) in case of needing to differentiate amplicons having the same size, the primers must be labeled with fluorescent dyes having distinctive colors. For certain multiplex analyses, commercial multiplexing kits are available. It has been informed that multiplex PCR has a sensitivity and

RFLP-PCR is a fast and reliable method for identifying non-TB mycobacteria [69]. This method is based on amplifying fragments of diverse genes; for instance, hsp65, the gene for histonelike protein hupB and pncA. Gene hsp65 are present in all species of mycobacteria. The selected target of hsp65 gene is a 439 bp fragment. Using hsp65 can be distinguished between MTB, M. avium and M. intracellulare, but not between MTB and M. bovis [70]. Gene hupB encodes for an histone-like protein. RFLP-PCR of hupB allows differentiation of MTB and M. bovis. The amplicons generated have 645 bp and 618 bp, respectively. pncA PCR assays were found specific in detecting MTB and M. bovis, as well as the M. avium complex in human

Real-time PCR, also known as quantitative PCR (qPCR), offers several advantages over endpoint PCR, which are as follows: (1) as its name indicates, it is quantitative; (2) reduces the risk of cross-contamination because it minimizes the need to manipulate samples after performing PCR; (3) the manual processing time is reduced; (4) the run preparation times are also reduced and (5) offers high sensitivity and specificity [72]. Since the last decade, qPCR for the detection of MTB target genes in clinical specimens has contributed to improved diagnosis and epidemiological studies of TB. This is because qPCR offers the advantage of combining amplification and detection of molecular targets in a single step; and there is no need to use nested PCR [73].

any atypical mycobacterial isolate.

specificity of 95% [68].

sputum [71].

6.3.4. Real-time PCR

6.3.3. Random fragment length polymorphism (RFLP)-PCR

## 6.3. Variations of PCR-final point of DNA

The main variations of the basic point final PCR are nested PCR, multiplex PCR, spoligotyping [57, 58] and RFLP-PCR. These variants were designed to improve the sensitivity and specificity of basic RFLP and PCR.

## 6.3.1. Nested PCR

Nested PCR is widely used, because this method significantly improves the sensitivity and specificity of basic PCR. The nested PCR consists of using two sets of primers, both directed against the same target DNA sequence, which may be a gene or some other region characteristic of a mycobacterial species [59]. The first set of primers is designed to hybridize to the most distal regions of the target sequence, the first amplification run is made and the resulting amplicons are used, in turn, to amplify a slightly inner region of the used original target sequence. Once performed the first series of PCR cycles, the PCR is used to carry out the second amplification procedure. Prasad et al. designed a nested PCR-based assay using the hupB gene of MTB (Rv2986c) and M. bovis (Mb3010c) as a method to differentiate these two closely related species [60]. Our research group developed a method for the diagnosis of TB based on nested PCR that targets the gene coding for the β-subunit of MTB RNA polymerase. This method is noticeably more specific and sensitive than all that have been published to date or have been approved by the FDA. Most current methods require cultivating mycobacteria for a few days and do not work with certain types of samples. In contrast, our method can work well with the genome of a single bacterium and samples of sputum [61] or cerebrospinal fluid [62], without the need to culture the biological samples. Currently, the patent is in a process.

### 6.3.2. Multiplex PCR

Multiplex PCR refers to the use of PCR to amplify, simultaneously, several different DNA sequences. That is to say, multiplex PCR allows performing many separate PCR reactions, all together, carrying out only one procedure. Otherwise, amplification by PCR of several DNA sequences would require several runs, as well as more reagents and longer time to perform them. On the other hand, if additional information is required from a particular DNA sequence, this may be gained by accomplishing a single, conventional PCR test.

PCR multiplex has enormous value in infectious disease that can be caused by a variety of microorganisms, for instance, meningitis. Meningitis can be caused by MTB [63], several other bacterial species (Streptococcus pneumoniae, Haemophilus influenzae type b, N. meningitidis, group b Streptococcus and Listeria monocytogenes) [64] and fungi [65]. Meningitis is a medical emergency, and immediate steps must be taken to establish the specific cause and initiate effective therapy [66]. PCR multiplex is a rapid, sensitive and specific diagnostic test for acute bacterial meningitis. PCR is particularly useful for analyzing cerebrospinal fluid of patients who have been treated with antibiotics before lumbar puncture [64]. Furthermore, in 2002, Shah et al. [67] reported a multiplex PCR-based assay performing a rapid and specific differentiation of M. bovis and MTB. This procedure can be beneficial for medical and veterinary microbiological laboratories. The designed multiplex PCR to identify MTB or M. bovis is based on the differential amplification of pncA gene. This test is highly specific and sensitive. Only 20 pg of pure mycobacteria DNA are needed, and none PCR amplification product has been obtained from any atypical mycobacterial isolate.

There is a series of requirements to be met to perform a good multiplex PCR analysis. These are as follows: (1) Accomplish the standard requirements for primer design. (2) Design one pair of primers for each expected amplicon. (3) Apply suitable temperatures, allowing DNA polymerase works accurately in every multiplex PCR cycle. (4) The primer design must be optimized; so that, all primer pairs work properly, at the same time and in a single reaction. (5) Apply similar alignment temperatures for all primers. (6) Choose the size of those DNA regions to be amplified so that be possible obtaining amplicons having clearly different sizes among them. Allowing, in this way, to distinguish the bands between them in the electrophoresis gel, (7) in case of needing to differentiate amplicons having the same size, the primers must be labeled with fluorescent dyes having distinctive colors. For certain multiplex analyses, commercial multiplexing kits are available. It has been informed that multiplex PCR has a sensitivity and specificity of 95% [68].

## 6.3.3. Random fragment length polymorphism (RFLP)-PCR

RFLP-PCR is a fast and reliable method for identifying non-TB mycobacteria [69]. This method is based on amplifying fragments of diverse genes; for instance, hsp65, the gene for histonelike protein hupB and pncA. Gene hsp65 are present in all species of mycobacteria. The selected target of hsp65 gene is a 439 bp fragment. Using hsp65 can be distinguished between MTB, M. avium and M. intracellulare, but not between MTB and M. bovis [70]. Gene hupB encodes for an histone-like protein. RFLP-PCR of hupB allows differentiation of MTB and M. bovis. The amplicons generated have 645 bp and 618 bp, respectively. pncA PCR assays were found specific in detecting MTB and M. bovis, as well as the M. avium complex in human sputum [71].

## 6.3.4. Real-time PCR

comparing the position of the DNA marker, having a size equivalent to the size expected for the PCR product of interest. The identification on the gel of the amplified product with the expected molecular weight indicates the presence of an MTBC member in the sample studied

The main variations of the basic point final PCR are nested PCR, multiplex PCR, spoligotyping [57, 58] and RFLP-PCR. These variants were designed to improve the sensitivity and specificity

Nested PCR is widely used, because this method significantly improves the sensitivity and specificity of basic PCR. The nested PCR consists of using two sets of primers, both directed against the same target DNA sequence, which may be a gene or some other region characteristic of a mycobacterial species [59]. The first set of primers is designed to hybridize to the most distal regions of the target sequence, the first amplification run is made and the resulting amplicons are used, in turn, to amplify a slightly inner region of the used original target sequence. Once performed the first series of PCR cycles, the PCR is used to carry out the second amplification procedure. Prasad et al. designed a nested PCR-based assay using the hupB gene of MTB (Rv2986c) and M. bovis (Mb3010c) as a method to differentiate these two closely related species [60]. Our research group developed a method for the diagnosis of TB based on nested PCR that targets the gene coding for the β-subunit of MTB RNA polymerase. This method is noticeably more specific and sensitive than all that have been published to date or have been approved by the FDA. Most current methods require cultivating mycobacteria for a few days and do not work with certain types of samples. In contrast, our method can work well with the genome of a single bacterium and samples of sputum [61] or cerebrospinal fluid [62], without the need to culture the biological samples. Currently, the patent is in a

Multiplex PCR refers to the use of PCR to amplify, simultaneously, several different DNA sequences. That is to say, multiplex PCR allows performing many separate PCR reactions, all together, carrying out only one procedure. Otherwise, amplification by PCR of several DNA sequences would require several runs, as well as more reagents and longer time to perform them. On the other hand, if additional information is required from a particular DNA

PCR multiplex has enormous value in infectious disease that can be caused by a variety of microorganisms, for instance, meningitis. Meningitis can be caused by MTB [63], several other bacterial species (Streptococcus pneumoniae, Haemophilus influenzae type b, N. meningitidis, group b Streptococcus and Listeria monocytogenes) [64] and fungi [65]. Meningitis is a medical emergency, and immediate steps must be taken to establish the specific cause and initiate effective

sequence, this may be gained by accomplishing a single, conventional PCR test.

[55, 56].

of basic RFLP and PCR.

6.3.1. Nested PCR

process.

6.3.2. Multiplex PCR

6.3. Variations of PCR-final point of DNA

82 Mycobacterium - Research and Development

Real-time PCR, also known as quantitative PCR (qPCR), offers several advantages over endpoint PCR, which are as follows: (1) as its name indicates, it is quantitative; (2) reduces the risk of cross-contamination because it minimizes the need to manipulate samples after performing PCR; (3) the manual processing time is reduced; (4) the run preparation times are also reduced and (5) offers high sensitivity and specificity [72]. Since the last decade, qPCR for the detection of MTB target genes in clinical specimens has contributed to improved diagnosis and epidemiological studies of TB. This is because qPCR offers the advantage of combining amplification and detection of molecular targets in a single step; and there is no need to use nested PCR [73]. Therefore, qPCR is rendering obsolete those techniques based on end-point PCR, which are much less sensitive. The qPCR technique involves the use of fluorescent chromophores (fluorophores) covalently attached to the primers. The fluorophore is placed at the 50 end of the primer (probe) and at the 30 end or internally, a fluorescence quencher is inserted covalently. During the PCR extension phase, the exonuclease activity of the Taq polymerase cuts the initiator in the 50 -30 direction, and the fluorophore and quencher are separated, which results in a detectable fluorescence that is proportional with the number of accumulated amplicons. A spectrofluorometer integrated with the thermocycler reads the emerging fluorescence. The PCR occurs in a dynamic way, and the results are read continuously. A computer integrated with the equipment, with the help of specialized software that accumulates and analyzes the data, generates a DNA amplification curve under study. The computer program allows monitoring the amplification curve on a screen. Usually, an analysis of qPCR is carried out in 1.5 h. The analysis described above can be performed with different platforms. SYBR green (cyanine colorant) is widely used for qPCR because it is very sensitive and high cost/benefit in uniplex formats. Besides of SYBR green, a number of options are available, such as TaqMan probes, molecular beacons, scorpion primers, fluorescence resonance energy transfer probes (FRETS) and primer-probe energy transfer. There are also different fluorescent chromophores, e.g. 6-carboxyfluorescein-aminohexyl amidite (6-FAM), carboxy-X-rhodamine (ROX) and cyanine (Cy5) as reporters at the 50 end and a quencher at the 30 end. Considering the advantages and disadvantages of IS6110 and of hsp65 discussed above, some authors have proposed using them in a multiplex qPCR format, which would presumably offer a more specific and inclusive diagnosis [2].

specific patterns. The DR locus is a member of the CRISPR (clustered regularly interspaced

Conventional and Molecular Diagnosis of Drug-Sensitive and Drug-Resistant Pulmonary Tuberculosis

http://dx.doi.org/10.5772/intechopen.75004

Spoligotyping is used to analyze genetic diversity of DR locus. It is useful for clinical, molecular epidemiology, evolution and population genetics laboratories [74]. This method is relatively easy to use, robust and allows numerical analysis. Spoligotyping was designed to detect biotinylated, amplified products from MTB and M. bovis from the DR locus. This method is based on reverse hybridization to the spacer sequences (43 synthetic oligonucleotides), which are covalently attached as lines to a nylon membrane. The hybridization is performed by turning 90 the amplicon template concerning the immobilized synthetic oligonucleotides. To immobilize the perpendicular samples, an acrylic device is used. So that, each homology will give rise to a tiny square of hybridization. Non-hybridizing products are eliminated with successive washes and will leave a blank space where the correspondent synthetic oligonucleotide is placed. On the other hand, those hybridized products will be detected using the streptavidin-peroxidase system, which binds to the biotin present in the amplified products. Results are visualized by incubating the hybridization template with luminol, in such a way that peroxidase catalyzes the oxidation of the reagent, resulting in light emission, which is developed by exposing the membrane to an X-ray film. The patterns obtained to reveal absence or presence of the spacers is read as a binary code, which can be easily interpreted and computerized. A commercial kit for MTB spoligotyping is available. In addition, a spoligotyping databases from MTB and M. bovis isolates from worldwide are available online.

Most PCR-based tests described above were designed exclusively to detect the presence of MTB in biological samples. Fortunately, there are other PCR-based tests designed to identify MTB in biological samples and to know whether these microorganisms are resistant to rifam-

The methods for detecting resistance to anti-TB drugs are based on the detection of mutations in the genes that are associated with that resistance condition and this is achieved by the application of sequencing or hybridization techniques. Recent advances in the rapid and direct detection of mycobacteria, with an emphasis on MTB, are based on the analysis of 16S rRNA gene sequence or oligonucleotide hybridization (oligohybridization), typing of strains and detection of patterns of drug susceptibility. Semi-automated systems for culture have greatly increased sensitivity and reduced the time required to perform the detection and identification of mycobacteria in clinical specimens. However, further research is still needed to assess the impact and cost/benefit of new diagnosis methods. In addition, well-designed clinical trials are still required to evaluate new diagnosis methods and thus enable medical staff to have methods to help them respond quickly [75]. PCR-based tests to identify mutations in the katG and rpoB genes that are associated with resistance to rifampicin and isoniazid, respectively, may assist in the early identification of resistance to these drugs in mycobacteria [40]. The WHO Stop TB Partnership's New Diagnosis Working Group and the Foundation for

picin or rifampicin and isoniazid. The most important tests are described below.

short palindromic repeats) family of the MTBC.

6.4. Methods to determine MTB-drug resistance

6.4.1. End-point PCR to determine MTB-MDR

#### 6.3.5. Real-time (RT) PCR

RT-PCR allows to amplify specific regions of the MTB genome or to obtain DNA starting from RNA by using a reverse transcriptase. This form of PCR is called RT-PCR (reverse transcriptase-PCR). The nested RT-PCR is a variation of the nested-PCR. Nested RT-PCR offers the same advantages than nested PCR, discussed above; with the difference that nested RT-PCR allows obtaining a cDNA strand, using the original mRNA sequence as template. A reverse transcriptase is used instead of a DNA polymerase to synthesize the cDNA. Then, like in nested PCR, the first set of primers is designed to hybridize to the most distal regions of the target sequence, the first amplification run is made and the resulting amplicons are used, in turn, to amplify a slightly inner region of the used original target sequence [59].

#### 6.3.6. Spoligotyping

Besides of PCR multiplex, spoligotyping offers the possibility of distinguishing between M. bovis and MTB. Forty-three types of mycobacterial spacers are known. Of these, 37 are typical of MTB and other 6 of M. bovis BCG. An edition of the international spoligotyping database namely SpolDB4/SITVIT was introduced containing 1939 different spoligotypes (ST) identified. Furthermore, spoligotyping allows grouping MTB isolates according to the presence or absence of intermediate regions of the DR locus (direct repeats) in each MTB isolate, forming specific patterns. The DR locus is a member of the CRISPR (clustered regularly interspaced short palindromic repeats) family of the MTBC.

Spoligotyping is used to analyze genetic diversity of DR locus. It is useful for clinical, molecular epidemiology, evolution and population genetics laboratories [74]. This method is relatively easy to use, robust and allows numerical analysis. Spoligotyping was designed to detect biotinylated, amplified products from MTB and M. bovis from the DR locus. This method is based on reverse hybridization to the spacer sequences (43 synthetic oligonucleotides), which are covalently attached as lines to a nylon membrane. The hybridization is performed by turning 90 the amplicon template concerning the immobilized synthetic oligonucleotides. To immobilize the perpendicular samples, an acrylic device is used. So that, each homology will give rise to a tiny square of hybridization. Non-hybridizing products are eliminated with successive washes and will leave a blank space where the correspondent synthetic oligonucleotide is placed. On the other hand, those hybridized products will be detected using the streptavidin-peroxidase system, which binds to the biotin present in the amplified products. Results are visualized by incubating the hybridization template with luminol, in such a way that peroxidase catalyzes the oxidation of the reagent, resulting in light emission, which is developed by exposing the membrane to an X-ray film. The patterns obtained to reveal absence or presence of the spacers is read as a binary code, which can be easily interpreted and computerized. A commercial kit for MTB spoligotyping is available. In addition, a spoligotyping databases from MTB and M. bovis isolates from worldwide are available online.

## 6.4. Methods to determine MTB-drug resistance

## 6.4.1. End-point PCR to determine MTB-MDR

Therefore, qPCR is rendering obsolete those techniques based on end-point PCR, which are much less sensitive. The qPCR technique involves the use of fluorescent chromophores (fluorophores) covalently attached to the primers. The fluorophore is placed at the 50

the primer (probe) and at the 30 end or internally, a fluorescence quencher is inserted covalently. During the PCR extension phase, the exonuclease activity of the Taq polymerase cuts the

in a detectable fluorescence that is proportional with the number of accumulated amplicons. A spectrofluorometer integrated with the thermocycler reads the emerging fluorescence. The PCR occurs in a dynamic way, and the results are read continuously. A computer integrated with the equipment, with the help of specialized software that accumulates and analyzes the data, generates a DNA amplification curve under study. The computer program allows monitoring the amplification curve on a screen. Usually, an analysis of qPCR is carried out in 1.5 h. The analysis described above can be performed with different platforms. SYBR green (cyanine colorant) is widely used for qPCR because it is very sensitive and high cost/benefit in uniplex formats. Besides of SYBR green, a number of options are available, such as TaqMan probes, molecular beacons, scorpion primers, fluorescence resonance energy transfer probes (FRETS) and primer-probe energy transfer. There are also different fluorescent chromophores, e.g. 6-carboxyfluorescein-aminohexyl amidite (6-FAM), carboxy-X-rhodamine (ROX) and cyanine

disadvantages of IS6110 and of hsp65 discussed above, some authors have proposed using them in a multiplex qPCR format, which would presumably offer a more specific and inclusive

RT-PCR allows to amplify specific regions of the MTB genome or to obtain DNA starting from RNA by using a reverse transcriptase. This form of PCR is called RT-PCR (reverse transcriptase-PCR). The nested RT-PCR is a variation of the nested-PCR. Nested RT-PCR offers the same advantages than nested PCR, discussed above; with the difference that nested RT-PCR allows obtaining a cDNA strand, using the original mRNA sequence as template. A reverse transcriptase is used instead of a DNA polymerase to synthesize the cDNA. Then, like in nested PCR, the first set of primers is designed to hybridize to the most distal regions of the target sequence, the first amplification run is made and the resulting amplicons are used, in turn, to amplify a slightly inner region of the used original target

Besides of PCR multiplex, spoligotyping offers the possibility of distinguishing between M. bovis and MTB. Forty-three types of mycobacterial spacers are known. Of these, 37 are typical of MTB and other 6 of M. bovis BCG. An edition of the international spoligotyping database namely SpolDB4/SITVIT was introduced containing 1939 different spoligotypes (ST) identified. Furthermore, spoligotyping allows grouping MTB isolates according to the presence or absence of intermediate regions of the DR locus (direct repeats) in each MTB isolate, forming

direction, and the fluorophore and quencher are separated, which results

end and a quencher at the 30 end. Considering the advantages and

initiator in the 50

84 Mycobacterium - Research and Development

(Cy5) as reporters at the 50

6.3.5. Real-time (RT) PCR

diagnosis [2].

sequence [59].

6.3.6. Spoligotyping

end of

Most PCR-based tests described above were designed exclusively to detect the presence of MTB in biological samples. Fortunately, there are other PCR-based tests designed to identify MTB in biological samples and to know whether these microorganisms are resistant to rifampicin or rifampicin and isoniazid. The most important tests are described below.

The methods for detecting resistance to anti-TB drugs are based on the detection of mutations in the genes that are associated with that resistance condition and this is achieved by the application of sequencing or hybridization techniques. Recent advances in the rapid and direct detection of mycobacteria, with an emphasis on MTB, are based on the analysis of 16S rRNA gene sequence or oligonucleotide hybridization (oligohybridization), typing of strains and detection of patterns of drug susceptibility. Semi-automated systems for culture have greatly increased sensitivity and reduced the time required to perform the detection and identification of mycobacteria in clinical specimens. However, further research is still needed to assess the impact and cost/benefit of new diagnosis methods. In addition, well-designed clinical trials are still required to evaluate new diagnosis methods and thus enable medical staff to have methods to help them respond quickly [75]. PCR-based tests to identify mutations in the katG and rpoB genes that are associated with resistance to rifampicin and isoniazid, respectively, may assist in the early identification of resistance to these drugs in mycobacteria [40]. The WHO Stop TB Partnership's New Diagnosis Working Group and the Foundation for Innovative New Diagnosis (FIND) ranked tools for diagnosing active and drug-resistant TB in three categories: (1) tools approved by WHO; (2) tools that are in the last phase of development or evaluation and (3) tools that are in the early stages of development [76]. Tools approved by WHO include LPAs for the diagnosis of MDR-TB, by GenoType® MTBDR plus, Hian Lifescience, Nehren, Germany; INNO-LiPA Rif. TB, produced by Innogenetics, Ghent, Belgium, an assay for rapid detection and speciation, whose manufacturer is Capilia TB-Neo, TAUNUS, Numazu, Japan and the GeneXpert MTB/RIF system, produced by Cepheid, CA, USA, a nucleic acid amplification test (NAAT) to screen for MTB-MDR. In the third group, tools that are in the early stages of development include an assay based on the detection of lipoarabinomannan, a breath analyzer, a loop-mediated isothermal amplification technology, called TB-LAMP, produced by Eiken Chemical Co Ltd, Tokyo, Japan, and a phage-based bioassay for the rapid diagnosis of MDR-TB [77]. Another very promising development is the one belonging to the company Hain Life-science, Nehren, Germany. This technique is used to screen resistance of MTB strains to second-line injectable drugs: amikacin, kanamycin and capreomycin.

needed to perform nucleic acid amplification. As the cartridges are individual and watertight, the possibility of cross-contamination and false results is eliminated. Concerning to usefulness and availability of GeneXpert MTB/RIF, van Rie et al. [77] stated that is the first system that has a true point of care [POC] tool in regions with limited resources. The automation and simplicity of the GeneXpert MTB/RIF system allow non-mycobacteriology personnel to obtain reliable results. According to WHO, GeneXpert MTB/RIF should be used as an initial test for the diagnosis of TB and in symptomatic patients having a high risk of be sick of MDR-TB. On the other hand, one of the main limitations that GeneXpertMTB/RIF has is the fragility of the equipment, especially in places where the power supply fails continuously. On the other hand, it is doubtful whether the detection of resistance only to rifampicin is sufficient to guide a suitable treatment

Conventional and Molecular Diagnosis of Drug-Sensitive and Drug-Resistant Pulmonary Tuberculosis

http://dx.doi.org/10.5772/intechopen.75004

Proteomics is one of the most recent technological advances, which will surely bring enormous benefits in the diagnosis of fast and simple PTB. Significant progress has already been made in this regard. The sources of MTB proteins are easier to obtain and analyze the serum or plasma of patients with TB and to control the serum of healthy controls. Several focused papers on the above-mentioned sense have already been published: (1) Deng et al. in 2011 [82]; (2) Liu et al. in 2010 [83]; (3) Zhang et al. in 2012 [84] and (4) Liu et al. in 2011 [85]. However, this approach is neither accessible nor suitable for microbiological laboratories. The reasons are as follows: currently, the instruments required to perform a TB diagnosis are costly, very sophisticated systems and software and a high degree of specialization are required, not only for the correct handling of the equipment [mass spectrometers, generally associated with high-performance liquid chromatography (HPLC) equipment]. In addition, once the main proteins are known, other specialized databases must be identified and consulted to determine their function.

Among molecular diagnosis methods, there are tools based on the detection of antibodies, for instance, the Enzyme Linkage Immuno Assay (ELISA). That is, these techniques are based on the use of serological tests [86]. The vast majority of patients with TB (90%) live in low- or middle-income countries. In these countries, the diagnosis of TB is based on the identification of AFB in unprocessed sputum samples and using conventional microscopes. Mycobacterial culture methods partially alleviate the low sensitivity of the ZN microscopy method. Nevertheless, we have already discussed the limitations of this method and those of techniques

Methods based on immunochromatography are suitable for use in low-income areas, because these tests can be done without specialized equipment and with minimal training. The most commonly used MTB detection tests are based on the detection of anti-MTB antibodies. One of

in areas where there is a high incidence of polyresistance [81].

6.4.4. Proteomic molecular tools

7. Immune molecular tools

based on nucleic acids amplification.

7.1. Methods based on immunochromatography

#### 6.4.2. RT-PCR and line probe assay (LPA) for detecting MDR

The LPA is based on the principle of reverse hybridization, in which the 16S-23S spacer region of the rRNA (ribosomal RNA) is amplified by PCR. The amplicons are hybridized with oligonucleotide probes which are placed on nitrocellulose strips and are detected by a colorimetric system (usually by the biotin/avidin system). The probe is biotinylated and the avidin is covalently attached to a chromophore. There is currently a system for an LPA called Inno-LiPA Mycobacteria, produced by Innogenetics, Belgium. Inno-LiPA Mycobacteria allows identification of CMTB species, M. kansasii, M. xenopi, M. gordonae, M. avium complex, M. intracellulare, M. scrofulaceum and M. chelonae-M abscessus complex species [78]. This system has demonstrated an accuracy of 99.2%. INNO-LiPA Rif has also proven its usefulness in examining the resistance/susceptibility of rifabutin in mycobacteria [79]. The LPAs allow obtaining results in 24 h, with a very high sensitivity (99%), as already mentioned above. This system is the only one under development that will allow detection of MTB resistant to second-line drugs. The limitation of LPAs is that in most resource-poor countries, the facilities required to avoid contamination of amplicons, laboratory supplies and the equipment necessary to perform amplification of genetic material by PCR are not available [80].

#### 6.4.3. GeneXpert MTB/RIF

The method called GeneXpert MTB/RIF is based on real-time PCR and RT-PCR. The target is the rpoB gene (encoding the beta subunit of RNA polymerase). This gene has in its sequence a hot zone, which is very susceptible to mutations associated with resistance to rifampicin. GeneXpert MTB/RIF is the fastest and safest system known until now, being able to produce reliable results in less than 2 h. Therefore, GeneXpert MTB/RIF allows physicians making accurate diagnoses and prescribing appropriate treatments, practically during the patient's visit to his office. GeneXpert MTB/RIF is fully automated. The system consists of an instrument, a personal computer, a bar code reader and specialized preloaded software to interpret the results. The system requires disposable GeneXpert cartridges that contain everything needed to perform nucleic acid amplification. As the cartridges are individual and watertight, the possibility of cross-contamination and false results is eliminated. Concerning to usefulness and availability of GeneXpert MTB/RIF, van Rie et al. [77] stated that is the first system that has a true point of care [POC] tool in regions with limited resources. The automation and simplicity of the GeneXpert MTB/RIF system allow non-mycobacteriology personnel to obtain reliable results. According to WHO, GeneXpert MTB/RIF should be used as an initial test for the diagnosis of TB and in symptomatic patients having a high risk of be sick of MDR-TB. On the other hand, one of the main limitations that GeneXpertMTB/RIF has is the fragility of the equipment, especially in places where the power supply fails continuously. On the other hand, it is doubtful whether the detection of resistance only to rifampicin is sufficient to guide a suitable treatment in areas where there is a high incidence of polyresistance [81].

## 6.4.4. Proteomic molecular tools

Innovative New Diagnosis (FIND) ranked tools for diagnosing active and drug-resistant TB in three categories: (1) tools approved by WHO; (2) tools that are in the last phase of development or evaluation and (3) tools that are in the early stages of development [76]. Tools approved by WHO include LPAs for the diagnosis of MDR-TB, by GenoType® MTBDR plus, Hian Lifescience, Nehren, Germany; INNO-LiPA Rif. TB, produced by Innogenetics, Ghent, Belgium, an assay for rapid detection and speciation, whose manufacturer is Capilia TB-Neo, TAUNUS, Numazu, Japan and the GeneXpert MTB/RIF system, produced by Cepheid, CA, USA, a nucleic acid amplification test (NAAT) to screen for MTB-MDR. In the third group, tools that are in the early stages of development include an assay based on the detection of lipoarabinomannan, a breath analyzer, a loop-mediated isothermal amplification technology, called TB-LAMP, produced by Eiken Chemical Co Ltd, Tokyo, Japan, and a phage-based bioassay for the rapid diagnosis of MDR-TB [77]. Another very promising development is the one belonging to the company Hain Life-science, Nehren, Germany. This technique is used to screen resistance of MTB strains to second-line injectable drugs: amikacin, kanamycin and

The LPA is based on the principle of reverse hybridization, in which the 16S-23S spacer region of the rRNA (ribosomal RNA) is amplified by PCR. The amplicons are hybridized with oligonucleotide probes which are placed on nitrocellulose strips and are detected by a colorimetric system (usually by the biotin/avidin system). The probe is biotinylated and the avidin is covalently attached to a chromophore. There is currently a system for an LPA called Inno-LiPA Mycobacteria, produced by Innogenetics, Belgium. Inno-LiPA Mycobacteria allows identification of CMTB species, M. kansasii, M. xenopi, M. gordonae, M. avium complex, M. intracellulare, M. scrofulaceum and M. chelonae-M abscessus complex species [78]. This system has demonstrated an accuracy of 99.2%. INNO-LiPA Rif has also proven its usefulness in examining the resistance/susceptibility of rifabutin in mycobacteria [79]. The LPAs allow obtaining results in 24 h, with a very high sensitivity (99%), as already mentioned above. This system is the only one under development that will allow detection of MTB resistant to second-line drugs. The limitation of LPAs is that in most resource-poor countries, the facilities required to avoid contamination of amplicons, laboratory supplies and the equipment necessary to perform

The method called GeneXpert MTB/RIF is based on real-time PCR and RT-PCR. The target is the rpoB gene (encoding the beta subunit of RNA polymerase). This gene has in its sequence a hot zone, which is very susceptible to mutations associated with resistance to rifampicin. GeneXpert MTB/RIF is the fastest and safest system known until now, being able to produce reliable results in less than 2 h. Therefore, GeneXpert MTB/RIF allows physicians making accurate diagnoses and prescribing appropriate treatments, practically during the patient's visit to his office. GeneXpert MTB/RIF is fully automated. The system consists of an instrument, a personal computer, a bar code reader and specialized preloaded software to interpret the results. The system requires disposable GeneXpert cartridges that contain everything

capreomycin.

86 Mycobacterium - Research and Development

6.4.3. GeneXpert MTB/RIF

6.4.2. RT-PCR and line probe assay (LPA) for detecting MDR

amplification of genetic material by PCR are not available [80].

Proteomics is one of the most recent technological advances, which will surely bring enormous benefits in the diagnosis of fast and simple PTB. Significant progress has already been made in this regard. The sources of MTB proteins are easier to obtain and analyze the serum or plasma of patients with TB and to control the serum of healthy controls. Several focused papers on the above-mentioned sense have already been published: (1) Deng et al. in 2011 [82]; (2) Liu et al. in 2010 [83]; (3) Zhang et al. in 2012 [84] and (4) Liu et al. in 2011 [85]. However, this approach is neither accessible nor suitable for microbiological laboratories. The reasons are as follows: currently, the instruments required to perform a TB diagnosis are costly, very sophisticated systems and software and a high degree of specialization are required, not only for the correct handling of the equipment [mass spectrometers, generally associated with high-performance liquid chromatography (HPLC) equipment]. In addition, once the main proteins are known, other specialized databases must be identified and consulted to determine their function.

## 7. Immune molecular tools

Among molecular diagnosis methods, there are tools based on the detection of antibodies, for instance, the Enzyme Linkage Immuno Assay (ELISA). That is, these techniques are based on the use of serological tests [86]. The vast majority of patients with TB (90%) live in low- or middle-income countries. In these countries, the diagnosis of TB is based on the identification of AFB in unprocessed sputum samples and using conventional microscopes. Mycobacterial culture methods partially alleviate the low sensitivity of the ZN microscopy method. Nevertheless, we have already discussed the limitations of this method and those of techniques based on nucleic acids amplification.

#### 7.1. Methods based on immunochromatography

Methods based on immunochromatography are suitable for use in low-income areas, because these tests can be done without specialized equipment and with minimal training. The most commonly used MTB detection tests are based on the detection of anti-MTB antibodies. One of the aspects to be considered in the evaluation of commercial methods or the design of new methods based on the detection of anti-TB antibodies is that the profile of antigenic MTB proteins that are recognized by the antibodies generated in the immune response of the host. A possible limitation of these methods consists of that the set of antibodies used as biomarkers varies according to the progression of TB [87]. Therefore, a reliable diagnostic tool based on an adequate combination of antigens is still required. Currently, there are about 40 commercial serological tests for rapid TB diagnosis. These systems use various antigen compositions to detect patients with anti-MTB antibodies. However, there are insufficient data on its reliability in patients with HIV/AIDS and uninfected persons.

7.4. Interferon (INF)-γ

nosis alternative.

7.5. Interferon-gamma release assays (IGRAs)

of patients with active TB have negative IGRA results.

7.6. Enzyme-linked immunospot (ELISPOT) assay

7.7. Quanti-FERON Gold In-tubeTM (QFT-GIT)

The basic utility of IFN-γ is the diagnosis of LTBI, although IFN-γ detection is also used intensively for the diagnosis of active TB. Tests based on the measurement of IFN-γ release for the diagnosis of TB are generically called IGRAs. The infection usually ends by infiltration into the lung tissue of CD4+ T lymphocytes, which release IFN-γ. In turn, IFN-γ activates macrophages [89]. Identification of LTBI is a difficult issue. Therefore, the current diagnostic methods are based on markers of infection. Because of this, the intradermal reaction described above has long been used. Currently, kits are available to measure the release of IFN-γ as an immunodiag-

Conventional and Molecular Diagnosis of Drug-Sensitive and Drug-Resistant Pulmonary Tuberculosis

http://dx.doi.org/10.5772/intechopen.75004

IGRAs are designed to diagnose LTBI. These detect a cellular immune response to MTB. A remarkable characteristic of IGRAs consists in that results produced by these methods are not affected by the status of vaccination with BCG. Thus, in contrast with Mantoux method, IGRAs are useful for evaluation of LTBI in BCG-vaccinated individuals, particularly in settings where BCG vaccination was administered after infancy or multiple times. On the other hand, the IGRA's limitations are as follows: (1) These are unable to distinguish between an LTBI and an active-TB disease. (2) A positive IGRA result may not necessarily indicate an MTB infection, because it can also be caused by a non-tuberculous mycobacteria infection. (3) A negative IGRA does not rule out an active TB disease; many researchers have shown that up to a quarter

The IGRAs mainly used in the world are base ELISPOT and Quanti-FERON Gold In-tube.

One of the best-known IGRA is ELISPOT, produced by Abcam. This system is used to determine the number of IFN-γ-producing T cells. ELISPOT employs monoclonal or polyclonal antibodies preferably monoclonal antibodies because of their greater specificity than polyclonal antibodies. The antibodies are immobilized under aseptic conditions in the bottom of polyvinylidene fluoride (PVDF) microplates. The microplates are blocked, usually with serum albumin, which does not react with any antibody in the assay. Patient's cells are deposited on the microtiter plates at different densities in the presence of an antigen (in this case, the ESAT-6 MTB protein or the CPF10 protein filtrate). Both targets are strongly specific for TH1 cells in an MTB infection and are absent in BCG [90], whereas M. kansasii, M. szulgai, M.marinum, M. flavensens and M. gastric react with MTB-ESAT-6 and MTB-CPF-10. Microplates are put in a wet chamber at 37C in an incubator with 5% CO2 atmosphere for the time indicated by each manufacturer [91]. The specificity in HIV-infected individuals is 90%, while the specificity of the intradermal reaction is 57% [92].

QuantiFERON Gold In tube (QFT) was specifically designed to LTBI. QFT-GIT is produced by QIAGEN. The QFT-GIT assay is an ELISA-based method. The test uses whole blood samples

#### 7.2. Immunoprecipitation in cellulose acetate strips

A usual format for rapid TB diagnosis is to use cellulose acetate strips with one line for the test to which MTB antigens have been prefixed and another line as a positive control having predefined MTB antigens and human antibodies anti-MTB antigens. At the end of the strip, a section is left where the patient's blood or serum sample is placed, plus a suitable volume of some saline buffer, and then, a conjugate of anti-human antibodies labeled with colloidal gold or some chromophore is placed. A positive reaction is observed when the antibodies from the patient and the conjugate migrate on the cellulose acetate strip, and immunoprecipitation occurs, and consequently, binding of the conjugate on the line with the preset MTB antigens. On the other hand, the human chromophore/anti-antibody conjugate also binds to the positive control line with the MTB antibody and the preselected human anti-MTB anti-antibody. These tests take only a few minutes. Due to its usefulness and simplicity, it is currently considered a high priority to rigorously evaluate these tests to take advantage of them with safety and advantage as soon as possible. These tests are conducted by the National TB Programs of the United States of America [86]. Even though immunoprecipitation in cellulose acetate strips is considered as a promising tool, the WHO disapproves the use of such immunological methods for the diagnosis of TB, especially when it is intended to replace the search for AFB with microscopy [88].

#### 7.3. Enzyme Linkage Immuno Assay (ELISA)

ELISA serves as a presumptive test in cases of suspected TB. The ELISA plates are sensitized with a complete soluble extract of M. bovis, strain BCG. The test serum is diluted, and after addition of the immunoenzymatic conjugate and its substrate, the spectrophotometric reading is made and it is determined whether it is positive or negative, according to the cutoff value determined in 100 sera from apparent healthy subjects coming from an endemic area of TB. The result is reported as positive or negative. Given the individual differences in response and cross-reactions with antibodies induced by other mycobacteria, the negative result should be interpreted with caution as it does not necessarily rule out TB [55]. Therefore, commercial ELISA tests are considered to have limited sensitivity and inconsistent specificity for the diagnosis of TB. Nevertheless, this method is the basis for interferon-gamma release assays (IGRAs), which are useful to detect LTBI (see below), using interferon-γ detection, as we discuss below.

## 7.4. Interferon (INF)-γ

the aspects to be considered in the evaluation of commercial methods or the design of new methods based on the detection of anti-TB antibodies is that the profile of antigenic MTB proteins that are recognized by the antibodies generated in the immune response of the host. A possible limitation of these methods consists of that the set of antibodies used as biomarkers varies according to the progression of TB [87]. Therefore, a reliable diagnostic tool based on an adequate combination of antigens is still required. Currently, there are about 40 commercial serological tests for rapid TB diagnosis. These systems use various antigen compositions to detect patients with anti-MTB antibodies. However, there are insufficient data on its reliability

A usual format for rapid TB diagnosis is to use cellulose acetate strips with one line for the test to which MTB antigens have been prefixed and another line as a positive control having predefined MTB antigens and human antibodies anti-MTB antigens. At the end of the strip, a section is left where the patient's blood or serum sample is placed, plus a suitable volume of some saline buffer, and then, a conjugate of anti-human antibodies labeled with colloidal gold or some chromophore is placed. A positive reaction is observed when the antibodies from the patient and the conjugate migrate on the cellulose acetate strip, and immunoprecipitation occurs, and consequently, binding of the conjugate on the line with the preset MTB antigens. On the other hand, the human chromophore/anti-antibody conjugate also binds to the positive control line with the MTB antibody and the preselected human anti-MTB anti-antibody. These tests take only a few minutes. Due to its usefulness and simplicity, it is currently considered a high priority to rigorously evaluate these tests to take advantage of them with safety and advantage as soon as possible. These tests are conducted by the National TB Programs of the United States of America [86]. Even though immunoprecipitation in cellulose acetate strips is considered as a promising tool, the WHO disapproves the use of such immunological methods for the diagnosis of TB, especially when it is

ELISA serves as a presumptive test in cases of suspected TB. The ELISA plates are sensitized with a complete soluble extract of M. bovis, strain BCG. The test serum is diluted, and after addition of the immunoenzymatic conjugate and its substrate, the spectrophotometric reading is made and it is determined whether it is positive or negative, according to the cutoff value determined in 100 sera from apparent healthy subjects coming from an endemic area of TB. The result is reported as positive or negative. Given the individual differences in response and cross-reactions with antibodies induced by other mycobacteria, the negative result should be interpreted with caution as it does not necessarily rule out TB [55]. Therefore, commercial ELISA tests are considered to have limited sensitivity and inconsistent specificity for the diagnosis of TB. Nevertheless, this method is the basis for interferon-gamma release assays (IGRAs), which are useful to detect LTBI (see below), using interferon-γ detection, as we

in patients with HIV/AIDS and uninfected persons.

88 Mycobacterium - Research and Development

7.2. Immunoprecipitation in cellulose acetate strips

intended to replace the search for AFB with microscopy [88].

7.3. Enzyme Linkage Immuno Assay (ELISA)

discuss below.

The basic utility of IFN-γ is the diagnosis of LTBI, although IFN-γ detection is also used intensively for the diagnosis of active TB. Tests based on the measurement of IFN-γ release for the diagnosis of TB are generically called IGRAs. The infection usually ends by infiltration into the lung tissue of CD4+ T lymphocytes, which release IFN-γ. In turn, IFN-γ activates macrophages [89]. Identification of LTBI is a difficult issue. Therefore, the current diagnostic methods are based on markers of infection. Because of this, the intradermal reaction described above has long been used. Currently, kits are available to measure the release of IFN-γ as an immunodiagnosis alternative.

## 7.5. Interferon-gamma release assays (IGRAs)

IGRAs are designed to diagnose LTBI. These detect a cellular immune response to MTB. A remarkable characteristic of IGRAs consists in that results produced by these methods are not affected by the status of vaccination with BCG. Thus, in contrast with Mantoux method, IGRAs are useful for evaluation of LTBI in BCG-vaccinated individuals, particularly in settings where BCG vaccination was administered after infancy or multiple times. On the other hand, the IGRA's limitations are as follows: (1) These are unable to distinguish between an LTBI and an active-TB disease. (2) A positive IGRA result may not necessarily indicate an MTB infection, because it can also be caused by a non-tuberculous mycobacteria infection. (3) A negative IGRA does not rule out an active TB disease; many researchers have shown that up to a quarter of patients with active TB have negative IGRA results.

The IGRAs mainly used in the world are base ELISPOT and Quanti-FERON Gold In-tube.

## 7.6. Enzyme-linked immunospot (ELISPOT) assay

One of the best-known IGRA is ELISPOT, produced by Abcam. This system is used to determine the number of IFN-γ-producing T cells. ELISPOT employs monoclonal or polyclonal antibodies preferably monoclonal antibodies because of their greater specificity than polyclonal antibodies. The antibodies are immobilized under aseptic conditions in the bottom of polyvinylidene fluoride (PVDF) microplates. The microplates are blocked, usually with serum albumin, which does not react with any antibody in the assay. Patient's cells are deposited on the microtiter plates at different densities in the presence of an antigen (in this case, the ESAT-6 MTB protein or the CPF10 protein filtrate). Both targets are strongly specific for TH1 cells in an MTB infection and are absent in BCG [90], whereas M. kansasii, M. szulgai, M.marinum, M. flavensens and M. gastric react with MTB-ESAT-6 and MTB-CPF-10. Microplates are put in a wet chamber at 37C in an incubator with 5% CO2 atmosphere for the time indicated by each manufacturer [91]. The specificity in HIV-infected individuals is 90%, while the specificity of the intradermal reaction is 57% [92].

## 7.7. Quanti-FERON Gold In-tubeTM (QFT-GIT)

QuantiFERON Gold In tube (QFT) was specifically designed to LTBI. QFT-GIT is produced by QIAGEN. The QFT-GIT assay is an ELISA-based method. The test uses whole blood samples from LTBI-suspicious patients and peptides from three TB antigens (ESAT-6, CFP-10 and TB7.7) in an in-tube format. The result is reported as quantification of IFN-γ in international units (IU)/ mL of blood. An individual is considered positive for LTBI if the concentration of IFN-gamma concentration secreted by the responsive cells to TB antigens is higher than the test cut-off. These estimations are made after subtracting the IFN-γ concentration found in the negative control [93]. Several authors have reported that QFT-GIT has a sensitivity of 64–93% with an average of 70% [94]. Diel et al. found an average of 88% sensitivity of QFT-GIT with a range of 85–90% [95].

[8] WHO. Early Detection of Tuberculosis. An Overview of Approaches, Guidelines and Tools. Geneva, Switzerland. 2011. Available from: http://apps.who.int/iris/bitstream/10665/70824/

Conventional and Molecular Diagnosis of Drug-Sensitive and Drug-Resistant Pulmonary Tuberculosis

http://dx.doi.org/10.5772/intechopen.75004

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## Author details

Yazmin Berenice Martínez-Martínez, Herminia Guadalupe Martínez-Rodríguez and Salvador Luis Said-Fernández\*

\*Address all correspondence to<EMAIL_ADDRESS>
Department of Biochemistry and Molecular, Medicine School, Autonomous University of Nuevo Leon (UANL), Monterrey, Nuevo León, Mexico

## References

[8] WHO. Early Detection of Tuberculosis. An Overview of Approaches, Guidelines and Tools. Geneva, Switzerland. 2011. Available from: http://apps.who.int/iris/bitstream/10665/70824/ 1/WHO\_HTM\_STB\_PSI\_2011.21\_eng.pdf?ua=1 [Accessed: Feb 13, 2017]

from LTBI-suspicious patients and peptides from three TB antigens (ESAT-6, CFP-10 and TB7.7) in an in-tube format. The result is reported as quantification of IFN-γ in international units (IU)/ mL of blood. An individual is considered positive for LTBI if the concentration of IFN-gamma concentration secreted by the responsive cells to TB antigens is higher than the test cut-off. These estimations are made after subtracting the IFN-γ concentration found in the negative control [93]. Several authors have reported that QFT-GIT has a sensitivity of 64–93% with an average of 70% [94]. Diel et al. found an average of 88% sensitivity of QFT-GIT with a range of 85–90% [95].

Yazmin Berenice Martínez-Martínez, Herminia Guadalupe Martínez-Rodríguez and

Department of Biochemistry and Molecular, Medicine School, Autonomous University of

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cca.2012.01.036

**Chapter 5**

**Provisional chapter**

**Diagnosis of Tuberculosis among Children and**

**Diagnosis of Tuberculosis among Children and**

DOI: 10.5772/intechopen.69227

The authors discuss the challenging aspects of the diagnosis of tuberculosis in children and adolescents, since there is no gold standard for its diagnosis. The different clinical and radiological presentations and the low bacteriological positivity of tuberculosis in childhood are grounds for confrontation to the present. Immunological tests called inter‐ feron gamma release assays (IGRAs) failed to overcome the tuberculin skin test in prac‐ tice. Advances with nucleic acid amplification tests, on the other hand, have contributed to the diagnosis of tuberculosis among adolescents. Standardized systems for diagnosis can be useful as tools for screening or for decision‐making in childhood tuberculosis.

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

It is estimated that one‐third of the world population is infected by *Mycobacterium tuberculosis* and that each year about nine million people develop the disease, out of which 11% are chil‐ dren. This percentage can be higher in countries with high burden of tuberculosis (TB). At least one million children are sick with TB every year. In 2015, as many as 210,000 children died

It is believed that genetic predisposition influences the resistance of certain individuals who, even in contact with patients with baciliferous TB, are not infected with *M. tuberculosis* [3].

In childhood, the distinction between infection and disease is often difficult. Some authors avoid the term latent TB (or latent TB infection) in children, preferring to use the term TB

**Adolescents**

**Adolescents**

Clemax Couto Sant'Anna,

Rafaela Baroni Aurílio

**Abstract**

**1. Introduction**

Maria de Fátima B. Pombo March and

http://dx.doi.org/10.5772/intechopen.69227

Additional information is available at the end of the chapter

**Keywords:** tuberculosis, diagnosis, child, adolescents

from TB, out of which 40,000 were patients coinfected with HIV [1, 2].

Additional information is available at the end of the chapter

Clemax Couto Sant'Anna, Maria de Fátima B. Pombo March and Rafaela Baroni Aurílio

#### **Diagnosis of Tuberculosis among Children and Adolescents Diagnosis of Tuberculosis among Children and Adolescents**

DOI: 10.5772/intechopen.69227

Clemax Couto Sant'Anna, Maria de Fátima B. Pombo March and Rafaela Baroni Aurílio Clemax Couto Sant'Anna, Maria de Fátima B. Pombo March and Rafaela Baroni Aurílio Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.69227

#### **Abstract**

The authors discuss the challenging aspects of the diagnosis of tuberculosis in children and adolescents, since there is no gold standard for its diagnosis. The different clinical and radiological presentations and the low bacteriological positivity of tuberculosis in childhood are grounds for confrontation to the present. Immunological tests called inter‐ feron gamma release assays (IGRAs) failed to overcome the tuberculin skin test in prac‐ tice. Advances with nucleic acid amplification tests, on the other hand, have contributed to the diagnosis of tuberculosis among adolescents. Standardized systems for diagnosis can be useful as tools for screening or for decision‐making in childhood tuberculosis.

**Keywords:** tuberculosis, diagnosis, child, adolescents

## **1. Introduction**

It is estimated that one‐third of the world population is infected by *Mycobacterium tuberculosis* and that each year about nine million people develop the disease, out of which 11% are chil‐ dren. This percentage can be higher in countries with high burden of tuberculosis (TB). At least one million children are sick with TB every year. In 2015, as many as 210,000 children died from TB, out of which 40,000 were patients coinfected with HIV [1, 2].

It is believed that genetic predisposition influences the resistance of certain individuals who, even in contact with patients with baciliferous TB, are not infected with *M. tuberculosis* [3].

In childhood, the distinction between infection and disease is often difficult. Some authors avoid the term latent TB (or latent TB infection) in children, preferring to use the term TB

infection. The progression of infection to disease can be subtle and go unnoticed. This dif‐ ficulty becomes more remarkable, often in extrapulmonary TB [2, 3].

small number of bacilli. However, a study with commercially available serum tests in 2011 supported the WHO position of not recommending them for routine use. Thus, serological

In adolescents, the diagnosis of TB may be bacteriologically proven in most cases. Patients from this age group with suspected TB are, most of the times, able to expectorate and are bacilliferous [5, 6, 9]. Soon, sputum smear microscopy can be a useful method, associated with

Signs and symptoms: persistent fever, cough >2 weeks,

Diagnosis of Tuberculosis among Children and Adolescents

http://dx.doi.org/10.5772/intechopen.69227

Radiography: infiltration in the upper third, excavations,

night sweats, weight loss, blood spitting

Some risk factors for active TB in childhood continue to be valued for the diagnosis of active

The clinical manifestations of TB, especially in young children, are subtle and may go unno‐ ticed. Discrete changes such as weight loss or lack of weight gain, fatigue, and decreased interest in playing and decreased physical activity can be frequent and sometimes are not noticed by the family. Unexplained fever, irritability, or nystagmus can be subtle findings in the early forms of meningoencephalitis caused by TB, for example. TB meningitis is a case of

methods are no longer used as a diagnostic tool in children [3].

**3. Basis for the clinico‐radiological diagnosis**

**Table 1.** Differences in pulmonary tuberculosis in children and adolescents.

• Intradomiciliary contact with a bacilliferous case recently diagnosed

TB [4]:

• Under 3 years of age

• Severe malnutrition

• HIV infection

*\*Gene‐Xpert MTB/RIF.*

**Table 1** shows differences in pulmonary TB in children and adolescents.

Slow evolution pneumonia Slow evolution pneumonia Reactive tuberculin skin test Reactive tuberculin skin test

Positive Xpert \* in a few cases (∼10%) Positive Xpert in most cases

**Children (primary TB) Adolescents (≥10 years old) (adult‐type TB)**

Negative bacteriological tests Positive bacteriological tests (AFB and culture)

Contact with adult with TB There may be no history of contact with people with TB

pneumonia

clinical and radiological findings.

Signs and symptoms: persistent fever, weight loss, interruption or loss in height and weight percentile,

Radiography: enlarged mediastinal lymph nodes, miliary pattern, pneumonia with no response to antibiotics

cough> 2 weeks, fatigue, night sweats

From a practical point of view, the diagnosis of TB infection occurs when the child is asymp‐ tomatic with normal chest radiography and a reactive TB skin test (TST) or interferon gamma release assays (IGRA). On the other hand, the diagnosis of TB disease or active TB is a chal‐ lenge. It occurs when the child is symptomatic (with symptoms consistent with TB) and chest or other X‐ray is abnormal, compatible with TB. TST or IGRA can be reactive or not.

## **2. Differences between pulmonary tuberculosis in children and adolescents**

The WHO has adopted standards for TB management in childhood that emphasize the impor‐ tance of clinical, radiological, and epidemiological data consistent with TB and bacteriologi‐ cal confirmation, where possible, to diagnose the disease. Anti‐HIV serology is also useful, especially in endemic areas of both diseases [4]. Even today these elements are essential for the diagnosis, since bacteriological confirmation is difficult in general, in children. Children mostly develop primary TB and are non‐bacilliferous or paucibacillary (in 80% of cases the diagnosis is made without bacteriological confirmation). Up to 7 or 8 years of age, children do not know how to expectorate properly to take samples for examination and, in addition, TB in this age group is usually paucibacillary, that is, with sputum smear negative. Recently, the incorporation of molecular diagnosis of TB allowed positive results in a higher percentage than those observed with the smear. Yet TB in children differs fundamentally from the disease in adults, as in childhood forms of negative TB prevail [4].

It is important to divide TB in childhood into two aspects. TB in children up to 10 years of age (TB in children) and from 10 years of age (TB in adolescents). In the first case, most of the forms are *primary TB*. In adolescents, most are *adult‐type TB*. That is, it is similar in clinical and radiological presentation to the pattern seen in adults. Adolescents are often bacilliferous and may transmit TB, develop excavated pulmonary forms, and, moreover, are able to cough and eliminate bacilli, similar to what occurs in adults [5, 6]. To collect specimens for bacteriological examination of children is only possible, most of the times, with the use of gastric lavage and induced sputum, since children under 5 or 6 years of age are almost unable to expectorate. The sputum smear or other specimen is processed by the traditional Ziehl‐Neelsen method. The culture can be per‐ formed by Loewstein‐Jensen method or liquid media—Ogawa or Middlebrook. More recently, molecular methods of nucleic acid amplification (NAATs) have been advocated. Among these methods, the *Gene‐Xpert MTB/RIF* method (Cepheid, California) was recommended by the WHO in 2013 to diagnose TB in childhood [5, 7, 8].

Note that in childhood, compared to a case with high clinical suspicion, although the bacterio‐ logical and molecular tests are negative, the diagnosis of TB can and should be done.

Serological diagnosis of childhood TB was insistently sought, as it does not depend on the bacterial burden in the lesions. This is an advantage in patients whose disease progresses with small number of bacilli. However, a study with commercially available serum tests in 2011 supported the WHO position of not recommending them for routine use. Thus, serological methods are no longer used as a diagnostic tool in children [3].

In adolescents, the diagnosis of TB may be bacteriologically proven in most cases. Patients from this age group with suspected TB are, most of the times, able to expectorate and are bacilliferous [5, 6, 9]. Soon, sputum smear microscopy can be a useful method, associated with clinical and radiological findings.

**Table 1** shows differences in pulmonary TB in children and adolescents.

**Table 1.** Differences in pulmonary tuberculosis in children and adolescents.

## **3. Basis for the clinico‐radiological diagnosis**

Some risk factors for active TB in childhood continue to be valued for the diagnosis of active TB [4]:

infection. The progression of infection to disease can be subtle and go unnoticed. This dif‐

From a practical point of view, the diagnosis of TB infection occurs when the child is asymp‐ tomatic with normal chest radiography and a reactive TB skin test (TST) or interferon gamma release assays (IGRA). On the other hand, the diagnosis of TB disease or active TB is a chal‐ lenge. It occurs when the child is symptomatic (with symptoms consistent with TB) and chest

The WHO has adopted standards for TB management in childhood that emphasize the impor‐ tance of clinical, radiological, and epidemiological data consistent with TB and bacteriologi‐ cal confirmation, where possible, to diagnose the disease. Anti‐HIV serology is also useful, especially in endemic areas of both diseases [4]. Even today these elements are essential for the diagnosis, since bacteriological confirmation is difficult in general, in children. Children mostly develop primary TB and are non‐bacilliferous or paucibacillary (in 80% of cases the diagnosis is made without bacteriological confirmation). Up to 7 or 8 years of age, children do not know how to expectorate properly to take samples for examination and, in addition, TB in this age group is usually paucibacillary, that is, with sputum smear negative. Recently, the incorporation of molecular diagnosis of TB allowed positive results in a higher percentage than those observed with the smear. Yet TB in children differs fundamentally from the disease

It is important to divide TB in childhood into two aspects. TB in children up to 10 years of age (TB in children) and from 10 years of age (TB in adolescents). In the first case, most of the forms are *primary TB*. In adolescents, most are *adult‐type TB*. That is, it is similar in clinical and radiological presentation to the pattern seen in adults. Adolescents are often bacilliferous and may transmit TB, develop excavated pulmonary forms, and, moreover, are able to cough and eliminate bacilli, similar to what occurs in adults [5, 6]. To collect specimens for bacteriological examination of children is only possible, most of the times, with the use of gastric lavage and induced sputum, since children under 5 or 6 years of age are almost unable to expectorate. The sputum smear or other specimen is processed by the traditional Ziehl‐Neelsen method. The culture can be per‐ formed by Loewstein‐Jensen method or liquid media—Ogawa or Middlebrook. More recently, molecular methods of nucleic acid amplification (NAATs) have been advocated. Among these methods, the *Gene‐Xpert MTB/RIF* method (Cepheid, California) was recommended by the

Note that in childhood, compared to a case with high clinical suspicion, although the bacterio‐

Serological diagnosis of childhood TB was insistently sought, as it does not depend on the bacterial burden in the lesions. This is an advantage in patients whose disease progresses with

logical and molecular tests are negative, the diagnosis of TB can and should be done.

or other X‐ray is abnormal, compatible with TB. TST or IGRA can be reactive or not.

**2. Differences between pulmonary tuberculosis in children and**

ficulty becomes more remarkable, often in extrapulmonary TB [2, 3].

in adults, as in childhood forms of negative TB prevail [4].

WHO in 2013 to diagnose TB in childhood [5, 7, 8].

**adolescents**

100 Mycobacterium - Research and Development

• Severe malnutrition

The clinical manifestations of TB, especially in young children, are subtle and may go unno‐ ticed. Discrete changes such as weight loss or lack of weight gain, fatigue, and decreased interest in playing and decreased physical activity can be frequent and sometimes are not noticed by the family. Unexplained fever, irritability, or nystagmus can be subtle findings in the early forms of meningoencephalitis caused by TB, for example. TB meningitis is a case of clear cerebrospinal fluid (CSF), the laboratory test of which shows a predominance of mono‐ cytes in the early stages and lymphocyte stage, in evolution, lower glucose blood sugar, and high levels of protein [4].

Household contact is more important than an external or sporadic one. However, in certain locations, the difference between household and external contact is subtle. In poor communi‐ ties, it is common for people to live together in neighboring houses; children are cared for by people in these communities and sleep in their relatives' houses. So it is worth asking if the child has contact with other people supposedly with TB (cough> 3 weeks) in or out of his/her home. In nurseries or schools of endemic regions of TB, cases are sometimes discovered where employees or teachers with TB who were diagnosed late and may have infected children who interacted with them. A situation of extreme gravity is the occurrence of TB cases in pediatric intensive care units (PICU) or nurseries. Newborns in condition of extreme vulnerability can be infected by *M. tuberculosis* and can develop the disease, the diagnosis of which is often difficult. It is known that the period of greatest risk of developing TB is the first 2 years following the primary infection by *M. tuberculosis*. This interval of 2 years is the most appropriate time to initiate preventive measures such as treatment of latent TB infection (isoniazid preventive

Diagnosis of Tuberculosis among Children and Adolescents

http://dx.doi.org/10.5772/intechopen.69227

The most common radiological patterns of pulmonary TB in children are hilar lymphadenop‐ athy identified on front X‐ray and more visible in profile incidence (**Figure 1a** and **1b**); miliary

**Figure 1.** (a and b) Enlarged hilar lymph glands: front and profile incidences (Primary TB).

therapy, IPT) [10].

**4. Radiological diagnosis**

Pulmonary TB is taken into account in some situations, such as slow evolution pneumonia. Cases where the child is being treated with antibiotics for acute pneumonia and shows no good performance should be valued as suggestive of TB. Most relevant clinical data such as evening fever, weight loss, profuse night sweats, and coughing or combinations of these signs and symp‐ toms may facilitate suspected diagnoses of TB, especially if it has remained for over two weeks.

Some aspects remain as challenges for the diagnosis of TB in children. Young infants may present a subtle case of TB, with signs and symptoms that are confused with other infectious and noninfectious diseases. In such cases the diagnosis may be delayed by a longer or shorter time, depending on the diagnostic suspicion capacity of the health team, and its consequences can be disastrous. TB externalized as severe and acute pneumonia (patients admitted to ICU on ventilatory failure) is rare and difficult to diagnose. The suspected clinical appearance of TB in HIV‐infected patients or with severe malnutrition or the discovery of HIV in patients with TB almost always confuses doctors who are attending such patients. An extensive differ‐ ential diagnosis of diseases related to HIV immunosuppression, which are confused with TB is necessary. It is no less difficult to diagnose multidrug‐resistant TB (MDR‐TB) in children. In general, contact with adults with MDR‐TB is the key to establish such a diagnostic suspi‐ cion [2, 10]. The performance of susceptibility testing of patient specimens with suspected MDR‐TB is of limited use in childhood, because most cases are negative (no bacillus). When bacteriological examinations of the child cannot be done, but information is available on the sensitivity to adult drugs with which the child comes into contact, it can be assumed that his/her treatment is done with the same regimen of adult MDR‐TB. When the child's clinical specimen is available and it is possible to perform Xpert, rifampicin resistance may be a proxy resistance to isoniazid. That is, there is high probability of being a case of MDR‐TB. More recently, *Line‐probe assays* have become available, which can simultaneously detect resistance to rifampicin and isoniazid. Another promising method is *MTBDR‐sl*, which detects resis‐ tance to fluoroquinolones, aminoglycosides, and ethambutol simultaneously. These latest tests are rarely used in childhood (Heemskerk, Guidance).

Although there is more radiological and clinical data suggestive of TB than others, the epide‐ miological context and the clinical picture have to be taken into account to achieve the diagno‐ sis of TB. Since there is no gold standard for the diagnosis of TB in childhood, it is necessary to consider the epidemiological reality of the region where the work is being done. Is it an endemic area for TB? Is the patient followed up in a basic health unit? Or in a reference center? Has the child with suspected TB had contact with an adult with TB or suspected TB? Was this a sporadic or lasting contact? Has the child suspected of intrathoracic TB been treated with antibiotics for common germs recently, with no successful result? All these questions may help contextualize the highest probability or not of diagnosing the disease in the child.

The possible contact of the child with an adult (especially if bacilliferous) is of great interest especially in young children. Infants and preschool children tend to have closer contact with adults than children of school age and adolescents. Thus, the history of contact with a per‐ son with TB can be epidemiologically valuable data on suspected TB diagnosis in childhood. Household contact is more important than an external or sporadic one. However, in certain locations, the difference between household and external contact is subtle. In poor communi‐ ties, it is common for people to live together in neighboring houses; children are cared for by people in these communities and sleep in their relatives' houses. So it is worth asking if the child has contact with other people supposedly with TB (cough> 3 weeks) in or out of his/her home. In nurseries or schools of endemic regions of TB, cases are sometimes discovered where employees or teachers with TB who were diagnosed late and may have infected children who interacted with them. A situation of extreme gravity is the occurrence of TB cases in pediatric intensive care units (PICU) or nurseries. Newborns in condition of extreme vulnerability can be infected by *M. tuberculosis* and can develop the disease, the diagnosis of which is often difficult.

It is known that the period of greatest risk of developing TB is the first 2 years following the primary infection by *M. tuberculosis*. This interval of 2 years is the most appropriate time to initiate preventive measures such as treatment of latent TB infection (isoniazid preventive therapy, IPT) [10].

## **4. Radiological diagnosis**

clear cerebrospinal fluid (CSF), the laboratory test of which shows a predominance of mono‐ cytes in the early stages and lymphocyte stage, in evolution, lower glucose blood sugar, and

Pulmonary TB is taken into account in some situations, such as slow evolution pneumonia. Cases where the child is being treated with antibiotics for acute pneumonia and shows no good performance should be valued as suggestive of TB. Most relevant clinical data such as evening fever, weight loss, profuse night sweats, and coughing or combinations of these signs and symp‐ toms may facilitate suspected diagnoses of TB, especially if it has remained for over two weeks. Some aspects remain as challenges for the diagnosis of TB in children. Young infants may present a subtle case of TB, with signs and symptoms that are confused with other infectious and noninfectious diseases. In such cases the diagnosis may be delayed by a longer or shorter time, depending on the diagnostic suspicion capacity of the health team, and its consequences can be disastrous. TB externalized as severe and acute pneumonia (patients admitted to ICU on ventilatory failure) is rare and difficult to diagnose. The suspected clinical appearance of TB in HIV‐infected patients or with severe malnutrition or the discovery of HIV in patients with TB almost always confuses doctors who are attending such patients. An extensive differ‐ ential diagnosis of diseases related to HIV immunosuppression, which are confused with TB is necessary. It is no less difficult to diagnose multidrug‐resistant TB (MDR‐TB) in children. In general, contact with adults with MDR‐TB is the key to establish such a diagnostic suspi‐ cion [2, 10]. The performance of susceptibility testing of patient specimens with suspected MDR‐TB is of limited use in childhood, because most cases are negative (no bacillus). When bacteriological examinations of the child cannot be done, but information is available on the sensitivity to adult drugs with which the child comes into contact, it can be assumed that his/her treatment is done with the same regimen of adult MDR‐TB. When the child's clinical specimen is available and it is possible to perform Xpert, rifampicin resistance may be a proxy resistance to isoniazid. That is, there is high probability of being a case of MDR‐TB. More recently, *Line‐probe assays* have become available, which can simultaneously detect resistance to rifampicin and isoniazid. Another promising method is *MTBDR‐sl*, which detects resis‐ tance to fluoroquinolones, aminoglycosides, and ethambutol simultaneously. These latest

Although there is more radiological and clinical data suggestive of TB than others, the epide‐ miological context and the clinical picture have to be taken into account to achieve the diagno‐ sis of TB. Since there is no gold standard for the diagnosis of TB in childhood, it is necessary to consider the epidemiological reality of the region where the work is being done. Is it an endemic area for TB? Is the patient followed up in a basic health unit? Or in a reference center? Has the child with suspected TB had contact with an adult with TB or suspected TB? Was this a sporadic or lasting contact? Has the child suspected of intrathoracic TB been treated with antibiotics for common germs recently, with no successful result? All these questions may help contextualize the highest probability or not of diagnosing the disease in the child.

The possible contact of the child with an adult (especially if bacilliferous) is of great interest especially in young children. Infants and preschool children tend to have closer contact with adults than children of school age and adolescents. Thus, the history of contact with a per‐ son with TB can be epidemiologically valuable data on suspected TB diagnosis in childhood.

tests are rarely used in childhood (Heemskerk, Guidance).

high levels of protein [4].

102 Mycobacterium - Research and Development

The most common radiological patterns of pulmonary TB in children are hilar lymphadenop‐ athy identified on front X‐ray and more visible in profile incidence (**Figure 1a** and **1b**); miliary

**Figure 1.** (a and b) Enlarged hilar lymph glands: front and profile incidences (Primary TB).

aspect—diffuse micronodular pattern in both hemithoraces, corresponding to hematogenous spread of TB (**Figure 2**) and appearance of alveolar pneumonia, that is, pattern of bacterial pneumonia caused by common germs, like pneumococcus, which may only be a case of TB when it becomes a case of slow evolution, not responsive to antibiotics (**Figure 3**) [4, 10]. It is often emphasized that many times we observe clinical and radiological dissociation in TB in children. The clinical evaluation of the child is disproportionately favorable in relation to the extent of radiation injury. That is, the radiological image suggests a serious pneumonia but the patient does not show signs of severity that would be expected in a pneumonia caused by common germs.

Some of these radiological findings in suspected cases of TB in children always require some differential diagnoses. Thus, the miliary pattern may appear on acute histoplasmosis or lung metastases of neoplasm (**Figure 4**); the unilateral or bilateral adenomegalies may arise in the course of Hodgkin's disease or sarcoidosis, and slow progression of pneumonia may also occur in atypical pneumonia (for Mycoplasma, for example), pneumonia caused by common germs, actinomycosis, nocardiosis, and other noninfectious agents in immunocompromised patients. In adolescents, radiological aspects are identical to the pattern seen in adults: adult‐type TB

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**Figure 4.** Micronodules in both lungs. Aspect of miliary TB. In fact, it was a thyroid carcinoma with metastases.

**Figure 5.** Pulmonary infiltrate in upper zone of the right lung. Aspect of TB *adult type*.

(**Figure 5**).

**Figure 2.** Micronodules in both lungs; miliary TB.

**Figure 3.** Slow evolution pneumonia; radiological aspect of a Pneumococcus pneumonia. Infiltrate on the left lung.

Some of these radiological findings in suspected cases of TB in children always require some differential diagnoses. Thus, the miliary pattern may appear on acute histoplasmosis or lung metastases of neoplasm (**Figure 4**); the unilateral or bilateral adenomegalies may arise in the course of Hodgkin's disease or sarcoidosis, and slow progression of pneumonia may also occur in atypical pneumonia (for Mycoplasma, for example), pneumonia caused by common germs, actinomycosis, nocardiosis, and other noninfectious agents in immunocompromised patients.

aspect—diffuse micronodular pattern in both hemithoraces, corresponding to hematogenous spread of TB (**Figure 2**) and appearance of alveolar pneumonia, that is, pattern of bacterial pneumonia caused by common germs, like pneumococcus, which may only be a case of TB when it becomes a case of slow evolution, not responsive to antibiotics (**Figure 3**) [4, 10]. It is often emphasized that many times we observe clinical and radiological dissociation in TB in children. The clinical evaluation of the child is disproportionately favorable in relation to the extent of radiation injury. That is, the radiological image suggests a serious pneumonia but the patient does not show signs of severity that would be expected in a pneumonia caused by

**Figure 3.** Slow evolution pneumonia; radiological aspect of a Pneumococcus pneumonia. Infiltrate on the left lung.

common germs.

104 Mycobacterium - Research and Development

**Figure 2.** Micronodules in both lungs; miliary TB.

In adolescents, radiological aspects are identical to the pattern seen in adults: adult‐type TB (**Figure 5**).

**Figure 4.** Micronodules in both lungs. Aspect of miliary TB. In fact, it was a thyroid carcinoma with metastases.

**Figure 5.** Pulmonary infiltrate in upper zone of the right lung. Aspect of TB *adult type*.

## **5. Immunological diagnosis**

The main methods of immunological diagnosis of TB are the tuberculin skin test (TST) and *interferon gamma release assay* or IGRA.

radiological picture will allow diagnosis. Thus, the negative result of these tests does not exclude TB disease, nor does its positive result confirm it. Both tests have limitations inher‐ ent in specific methodologies and rationales. In addition, IGRAs are still poorly studied in children living in regions with high TB prevalence or HIV‐infected children [7]. Some studies suggest that in children above 5 years of age vaccinated with BCG, IGRAs may be preferred to the TST, whereas in children who have not been vaccinated with BCG, the IGRAs and TST are equally indicated. In Brazil, to consider a child to be infected with TB, the cutoff point of the TST ≥ 5mm was adopted in children vaccinated more than 2 years or in immunosuppressed children; in children vaccinated with BCG less than 2 years, the cutoff point is ≥ 10 mm. These cutoff points were decided as above, since during the 2 years after the application of BCG vac‐

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IGRAs and TST alone do not allow the diagnosis of TB disease; its role is to indicate TB infec‐ tion. Both tests have a similar sensitivity for detecting infection by *M. tuberculosis.* More stud‐ ies are needed in children, because IGRAs still yield indeterminate results, which are justified by the low capacity of producing immunoglobulins in children in early childhood. In patients infected with HIV, the sensitivity of IGRAs is low, similar to what occurs with the TST [11].

The cost of IGRAs is much higher and is also technically more complex than the TST. Furthermore, it involves collecting blood, which is a limitation in the case of children. On the other hand, the TST requires two visits to the clinic in order to get the result within 48–72 h. The trend of international standards is to keep the TST as a standard for identifying cases of TB infection, at the expense of IGRAs, due to the advantages of the TST in terms of cost, more

No No

Yes Only rare species \*

Yes Unknown

reliable results in small children, and lack of need for laboratory resources [4].

Antigens used Many, PPD 3 (QFT) or 2 (T‐SPOT)

**Table 2** lists the main differences between IGRAs and TST.

**Characteristic TST IGRA**

Patient visits required 2 1

Cross‐reactivity with BCG Yes No

Differing positive values by risk Yes (5‐10‐15) No Causes boosting Yes No Subject to boosting by previous TST Yes Possible

Difficulties with test reproducibility Yes Yes Relative cost Lower Higher Location of need for trained staff "Bedside" Laboratory

Distinguish between Latent TB and

Durability over time (stays positive with or without treatment)

Cross‐reactivity with non‐TB

TB disease

mycobacteria

Sample Intradermal injection Blood draw

cine, the TST reaction will gradually decline [6].

The TST with PPD, conducted by the Mantoux technique, introduced in 1907, is the intrader‐ mal injection of 5 tuberculin units (TU) of purified protein derivative PPD‐S (PPD) or 2 TU PPD RT23 (are equivalent). In the event of cellular immunity to such tuberculin antigens, a delayed hypersensitivity reaction will occur within 48–72 h [11].

The reaction will cause skin induration at the injection site, and its transverse diameter must be measured (induration mm) by a trained individual and interpreted using stratified cutoffs: 0–4 mm = no reaction; 5 mm or more = reaction.

Immunity measured by TST may also reflect exposure to other environmental mycobacteria, vaccination by BCG (Bacillus Calmette–Guérin‐*Mycobacterium bovis*), or a previous TB infec‐ tion. This type of reactivity can lead to false‐positive results. On the other hand, false negative results may occur especially in immunosuppressed people, for example, with advanced HIV infection or using immunosuppressive medication [3].

The IGRA is an *in vitro* test which detects the production of IFN‐γ in peripheral blood by T cells of the host infected by *M. tuberculosis.* The identification of immunogenic proteins of the mycobacteria, antigens ESAT‐6, CFP‐10, and TB 7, which are specifically expressed by pathogenic strains of *M. tuberculosis* complex, allowed the development of IGRA. These antigens are encoded in the region of difference 1 (RD1) of the genome of *M. tuberculosis*. RD1 contains genes encoding the secretive system of the bacillus, known as ESX‐1. Such antigens are very specific of *M. tuberculosis* although there is some evidence of cross‐reactiv‐ ity with *Mycobacterium leprae*, not yet proven; positive results in individuals infected with *Mycobacterium marinum* and *Mycobacterium kansasii* have also been evidenced [3, 11].

There are two types of IGRA. The test conducted by QuantiFERON TB Gold methodology allows the measurement of *in vitro* level of IFN‐γ produced by T cells that have been stimulated by the said antigens. The result is reported as number of IFN‐γ in international units (IU) per milliliter. An individual is considered positive if the amount of IFN‐γ is above the cutoff point of the test (considering the negative control). Another method may be used: T‐SPOT.TB. It is an immunosorbent assay (ELISPOT) performed in separate and counted mononuclear cells from peripheral blood which are incubated with ESAT‐6 and CFP‐10 antigens. The result is reported as the number of T‐cells producing IFN‐γ (spot‐forming cells). An individual is considered pos‐ itive if the points count in the antigens exceeds the negative control points. Indeterminate IGRA results may occur due to a low IFN‐γ response of the positive control (mitogen) or a high IFN‐γ response of the negative control. Further studies are needed on this test in children [7, 11].

## **6. IGRA and TST in tuberculosis diagnosis in children**

The purpose of carrying out the TST or IGRA in children is to determine whether there is or not *M. tuberculosis* infection while other factors such as contact history and clinical and radiological picture will allow diagnosis. Thus, the negative result of these tests does not exclude TB disease, nor does its positive result confirm it. Both tests have limitations inher‐ ent in specific methodologies and rationales. In addition, IGRAs are still poorly studied in children living in regions with high TB prevalence or HIV‐infected children [7]. Some studies suggest that in children above 5 years of age vaccinated with BCG, IGRAs may be preferred to the TST, whereas in children who have not been vaccinated with BCG, the IGRAs and TST are equally indicated. In Brazil, to consider a child to be infected with TB, the cutoff point of the TST ≥ 5mm was adopted in children vaccinated more than 2 years or in immunosuppressed children; in children vaccinated with BCG less than 2 years, the cutoff point is ≥ 10 mm. These cutoff points were decided as above, since during the 2 years after the application of BCG vac‐ cine, the TST reaction will gradually decline [6].

IGRAs and TST alone do not allow the diagnosis of TB disease; its role is to indicate TB infec‐ tion. Both tests have a similar sensitivity for detecting infection by *M. tuberculosis.* More stud‐ ies are needed in children, because IGRAs still yield indeterminate results, which are justified by the low capacity of producing immunoglobulins in children in early childhood. In patients infected with HIV, the sensitivity of IGRAs is low, similar to what occurs with the TST [11].

The cost of IGRAs is much higher and is also technically more complex than the TST. Furthermore, it involves collecting blood, which is a limitation in the case of children. On the other hand, the TST requires two visits to the clinic in order to get the result within 48–72 h. The trend of international standards is to keep the TST as a standard for identifying cases of TB infection, at the expense of IGRAs, due to the advantages of the TST in terms of cost, more reliable results in small children, and lack of need for laboratory resources [4].

**Table 2** lists the main differences between IGRAs and TST.

**5. Immunological diagnosis**

106 Mycobacterium - Research and Development

*interferon gamma release assay* or IGRA.

0–4 mm = no reaction; 5 mm or more = reaction.

infection or using immunosuppressive medication [3].

delayed hypersensitivity reaction will occur within 48–72 h [11].

The main methods of immunological diagnosis of TB are the tuberculin skin test (TST) and

The TST with PPD, conducted by the Mantoux technique, introduced in 1907, is the intrader‐ mal injection of 5 tuberculin units (TU) of purified protein derivative PPD‐S (PPD) or 2 TU PPD RT23 (are equivalent). In the event of cellular immunity to such tuberculin antigens, a

The reaction will cause skin induration at the injection site, and its transverse diameter must be measured (induration mm) by a trained individual and interpreted using stratified cutoffs:

Immunity measured by TST may also reflect exposure to other environmental mycobacteria, vaccination by BCG (Bacillus Calmette–Guérin‐*Mycobacterium bovis*), or a previous TB infec‐ tion. This type of reactivity can lead to false‐positive results. On the other hand, false negative results may occur especially in immunosuppressed people, for example, with advanced HIV

The IGRA is an *in vitro* test which detects the production of IFN‐γ in peripheral blood by T cells of the host infected by *M. tuberculosis.* The identification of immunogenic proteins of the mycobacteria, antigens ESAT‐6, CFP‐10, and TB 7, which are specifically expressed by pathogenic strains of *M. tuberculosis* complex, allowed the development of IGRA. These antigens are encoded in the region of difference 1 (RD1) of the genome of *M. tuberculosis*. RD1 contains genes encoding the secretive system of the bacillus, known as ESX‐1. Such antigens are very specific of *M. tuberculosis* although there is some evidence of cross‐reactiv‐ ity with *Mycobacterium leprae*, not yet proven; positive results in individuals infected with

*Mycobacterium marinum* and *Mycobacterium kansasii* have also been evidenced [3, 11].

**6. IGRA and TST in tuberculosis diagnosis in children**

There are two types of IGRA. The test conducted by QuantiFERON TB Gold methodology allows the measurement of *in vitro* level of IFN‐γ produced by T cells that have been stimulated by the said antigens. The result is reported as number of IFN‐γ in international units (IU) per milliliter. An individual is considered positive if the amount of IFN‐γ is above the cutoff point of the test (considering the negative control). Another method may be used: T‐SPOT.TB. It is an immunosorbent assay (ELISPOT) performed in separate and counted mononuclear cells from peripheral blood which are incubated with ESAT‐6 and CFP‐10 antigens. The result is reported as the number of T‐cells producing IFN‐γ (spot‐forming cells). An individual is considered pos‐ itive if the points count in the antigens exceeds the negative control points. Indeterminate IGRA results may occur due to a low IFN‐γ response of the positive control (mitogen) or a high IFN‐γ response of the negative control. Further studies are needed on this test in children [7, 11].

The purpose of carrying out the TST or IGRA in children is to determine whether there is or not *M. tuberculosis* infection while other factors such as contact history and clinical and

The following samples should not be processed: those composed exclusively of saliva; con‐ taining food particles; consisting solely of pus (no mucus and greenish); consisting exclu‐ sively of blood; with quantity less than 1 ml in the case of pulmonary and extrapulmonary

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The sensitivity and specificity values for a single Xpert in adult sputum approach 88% (95% CI: 83–92) and 98% (95% CI: 97–99), respectively. Among those with positive sputum smear, the sensitivity is around 98% (95% CI: 97–99) and among those with negative sputum smears,

Xpert has still limited use in childhood, since its excellent performance is seen in bacteriologi‐ cally confirmed TB, which is the minority of cases in children [7]. It has been shown that, by using culture as the gold standard, the sensitivity of Xpert on a single test in children under 15 years of age is from 42.9 to 90%, both in sputum and in induced sputum [13, 15, 16]. For the gastric lavage samples, sensitivity is 68.8% [15] and for nasopharyngeal aspirate it is 48% [17]. Specificity is similar to the adult (over 98%). A second test in children with negative sputum

The prospect of broadening the diagnosis of TB in children with the use of Xpert was the subject of some studies carried out in South Africa and other African countries. There was higher posi‐ tivity of Xpert in cases of PT in relation to culture for *M. tuberculosis* on specimens, such as gastric lavage, nasopharyngeal aspirate, and sputum induction. It became clear that Xpert contributes to increased diagnostic capacity of pulmonary TB in microbiologically confirmed TB [12, 17].

In Detjen and colleagues meta‐analysis with studies using Xpert for the diagnosis of pulmo‐ nary TB in children and adolescents, the pooled sensitivities and specificities of Xpert for TB detection, when compared to culture: 62% (IC 95%: 51–73) and 98% (IC954%: 97–99) in sputum and induced sputum, respectively, and for samples from gastric lavage: 62% (95% CI: 51–73) and 98% (95% CI: 96–99), respectively. When compared with microscopy, the Xpert

In New Delhi, India, in a study investigating suspected cases of pediatric TB in public ser‐ vices, it was observed that Xpert was able to detect twice as many cases of pulmonary TB compared to smear and to increase the capacity to detect resistance to RIF, which is a quick

The clinical examination does not always contribute significantly to the diagnosis of TB in children. It is recommended to evaluate the child's weight and development and to identify possible growth retardation attributable to the chronic nature of TB. In adolescents the clini‐ cal examination can be more informative: weight loss, poor general condition and presence of cough, and sometimes, blood spitting, call the doctor's attention to the possibility of the disease. Physical examination changes in the respiratory tract, such as bronchial or *cavernous breath*, have been reported for decades in patients with excavation (caverns) in the lungs, but they are currently little researched or recognized. Occasionally, wheezing in the chest can be

samples, except cerebrospinal fluid (CSF); CSF samples amounting to less than 0.1 ml.

it is around 68% (95% CI: 59–75) [14].

sensitivity was better [18].

and promising test for pediatric patients [19].

**8. The role of clinical examination**

smears may increase the sensitivity of Xpert in 27.8% [13].

Source: Ref. [11].

**Table 2.** Comparison of the TST and IGRA.

## **7. Molecular diagnosis**

The Xpert MTB‐Rif system (Xpert) is a test based on nucleic acid amplification used to detect *M. tuberculosis* and for the screening of drug‐resistant strains. It uses real‐time polymerase chain reaction (PCR) technique, on the Gene Xpert platform, which allows to integrate three processes: purification, concentration, and amplification of nucleic acids by polymerase chain reaction (PCR) and detection of nucleic acid sequences in the genome of *M. tuberculosis*, specifically in *rpoβ* gene. This technique does not require mycobacterial DNA manipulation after amplification and thus reduces the complexity and risk of cross reactivity by DNA product amplification [12].

Not only does it allow, in a period of 2 h in the laboratory, to identify *M. tuberculosis* but also to detect bacterial resistance to rifampicin (RIF) by amplification, using PCR, of five overlap‐ ping probes which are supplemental to the region determining RIF resistance, consisting of 81 base pairs of rpoβ gene *in M. tuberculosis*. Then, this region is examined in order to identify mutations associated with resistance [3].

It is a quick, highly automated test and does not depend upon the machine operator. The only manual step involves adding the correct dose of the reagent into the specimen under analysis, and it is subsequently homogenized for 15 min and transferred to the Xpert cartridge.

The system consists of a Gene Xpert instrument, a computer, a barcode reader, and a prein‐ stalled software to perform tests on samples taken and to view the results generated on the screen and to report as negative or positive *M. tuberculosis*, as well as to report whether sen‐ sitive or not to RIF. However, a positive result does not necessarily indicate the presence of viable bacilli, since the method can identify the DNA of live or dead microorganisms [7, 12].