Tuberculosis (TB) is a chronic infectious disease that usually affects the lungs, it is mainly caused by the Mycobacterium tuberculosis (Mt). This infection is transmitted directly without contact, through coughing by micro-droplets of saliva. According to the World Health Organization (WHO), TB currently affects more than 9 million people worldwide and causes around 1.5 million deaths per year. Mt is an acid-fast bacillus, a characteristic conferred to the bacterium by the structure of its bacterial wall. Virulence factors avoid the destruction of bacteria and results in the development of latent tuberculosis with a risk of 5–10% for progression to active tuberculosis, this risk increases substantially in immunocompromised patients. Accurate diagnosis of TB must combine clinical and laboratory elements in order to confirm the clinical suspicion. Diagnostic laboratory techniques can be divided into microbiological tests (direct test seeking the bacteria) and indirect tests (seeking bacterial products or immune response generated for Mt). This chapter discusses the main features of Mt, as well as pulmonary tuberculosis and microbiological diagnosis of this pathogen.
Chapter 11
Mycobacterium tuberculosis: clinical and microbiological aspects
R.Y. Ramírez-Rueda Pedagogical and Technological University of Colombia, Faculty of Health Sciences, School of Nursing, Tunja, Colombia
Abstract
Keywords
latent tuberculosis
active tuberculosis
mycobacterium tuberculosis
chronic respiratory infections
sputum smear
mycobacterium tuberculosis culture
susceptibility testing antimycobacterial drugs
tuberculin test
interferon gamma release assays
1. Introduction
Tuberculosis (TB) is a chronic infectious disease that usually affects the lungs. It is mainly caused by Mycobacterium tuberculosis (Mt). This infection is transmitted directly without contact, through coughing by micro-droplets of saliva (flügge) that transport the bacterium, which are found inside the airways of people with active tuberculosis.
The bacterial wall of Mt contains arabinogalactan, esterified with fatty acids of heavy molecular weight termed mycolic acids, which turns it into in an acid-fast bacillus (AFB), a particular feature of Mycobacterium genus. Virulence factors of Mt are responsible for its survival inside of the human body and also for many of the deleterious effects on the host. Such effects are especially enhanced in immunosuppressed hosts. However, M. tuberculosis infection is usually asymptomatic in healthy people because their immune system acts to develop a barrier (tuberculous granuloma) around the bacterium. Ordinarily, TB appears as a pulmonary syndrome called pulmonary TB. After beating the immune system, Mt replicates slowly but continuously in the lungs until the occurrence of the first symptoms, which marks the beginning of active tuberculosis.1
The oldest evidence of a TB case dates back to 9000 BC. This proof of coevolution with the bacterium was found through studies of paleopathology in the remains of an approximately 1-year-old child found near the Mediterranean Sea.2 Numerous studies have been reported demonstrating the existence of TB through human evolution and its cosmopolitan distribution. Another archeological evidence for the presence of the disease is the demonstration of spinal tuberculosis, or Pott’s disease, in ancient Egyptian mummies over 5500 years ago.3
According to the World Health Organization, TB currently affects more than 9 million people worldwide and causes around 1.5 million deaths per year.4 Morbidity and mortality in TB are associated with coinfection of primary syndromes which affects the immune system, and acquired immune deficiency syndrome (AIDS), is the principal associated cause of TB. AIDS infected people carry a greater risk of developing severe diseases because it accelerates the course of HIV disease by activating viral replication and increased depletion rate of CD4+ T cells. Progress in scaling up interventions to deal with the AIDS/TB coepidemic continues. However, the increase of morbidity is greater than the efforts made at this level.5 These efforts, and those made in the treatment of TB, consist of improving and introducing new diagnostic techniques, drugs, vaccine research, and surveillance, not only in TB but in the diseases associated with this disease.
2. Description of causal microorganism
M. tuberculosis is an AFB, a characteristic conferred to the bacterium by the structure of its bacterial wall, which in addition to the peptidoglycan (common component of bacterial cell wall), has arabinogalactan. The distal ends of the arabinogalactan are esterified with fatty acids of heavy molecular weight, termed mycolic acids, which have a size and a unique structure for mycobacteria. The disease caused by the bacilli is determined by the immune status of the host and factors of virulence within the bacteria. Upon entering the body, Mt is able to survive the attack of macrophages, blocking the acidification of the phagosome (through the production of ammonia), which is essential in the formation of the phagolysosome and the activation of the bactericidal factors released during the merger. An alternative bacterial mechanism to avoid its destruction within the phagolysosome consists of blocking the oxidative phosphorylation process and suppressing the production of reactive Oxygen and Nitrogen species by means of a sulfated glycolipid from the bacterial wall called sulfolipid 1.6
There is no doubt that much of the virulence of the Mt is in its envelope (wall and cell membrane), so that apart from the lipids (present in large number and variety) there are also important proteins such as the Exported repetitive protein (Erp). The absence of Erp in mutant strains decreases the replication of the bacteria within macrophages.7 The mycocerosic acid synthase (mas) is essential for the synthesis of mycocerosic acids and their derivatives. When there is deficiency of one of these, such as phtioceroldimycocerasic acid, strains with attenuated growth in macrophages in vitro and murine models are observed.8 Lipids such as phtioceroldimycocerosate (PDIM) have been also nominated as virulent factors, showing growth reduction in vivo and in murine models when Mt strains defective in PDIM or in fad26/28 (fatty-acid-Coa synthase) were inoculated.9 In addition to the production of PDIM, its transport and localization in the cell membrane of Mt by transporting proteins such as mmpl7 are essential for the virulence. For this reason, in Mt mutant strains for the gene encoding this protein, similar effects were observed as those described above. The importance of Mt growth inside phagocytes is highlighted by the enzyme fibronectin binding protein mycolytransferase and its essential part in the synthesis of mycolic acid, as attenuated growth phenotypes were observed in human monocytes and murine macrophages when inoculated with gene defective strains coding for this enzyme.10 Other enzymes, such as methoxymycolic acid synthase 4 (mmaA4) and mycolic acid cyclopropane synthase (pcaA), are important in the cell permeability and their absence produces phenotypes with decreased growth in vivo and a reduction of mortality in mice.11,12
Mt also has virulent factors such as lipoarabinomannane (LAM) which is able to inmunomodulate the host response decreasing the production of interferon-γ, scavenging the Oxygen free radicals and blocking the production of protein-kinase C.13 Moreover, extrapulmonary dissemination could be partially due to the presence of proteins such as heparin-binding hemaglutin (HbhA) which decreases the bacterial phagocytosis in the lung.14
The bacterial metabolism plays an important role in Mt virulence and adaptation to different substrates within the host. This microorganism has several enzymes as isozitratelyase (Icl),15 lipase/esterase LipF, polyketide synthase (FasD33),16 a great number of phospholipases C (Rv2351c, Rv2350c, Rv2349c, Rv1755c, plcA, plcB, plcC, plcD),17 pantothenatesynthetases and aspartate-1-decarboxylase (Rv3602c, Rv3601c, pan C, panD)18; which help metabolize several lipids required for its development and replication, unlike mutant strains without them.
The biosynthesis of aminoacids and purines is essential for the development and expression of virulence in Mt, just like lipid synthesis. The bacterium uses enzymes such as isopropylmalate isomerase (LeuD), anthranilate phosphoribosiltransferase (TrpD), pyrroline-5-carboxlate reductase (ProC) and 1-phosphoribosylaminoimidazole-succinocarboxamide synthase (PurC) for the biosynthesis of amino acids and purines.19–21 Magnesium and Iron are essential for the life of various pathogens (including Mt), this is the reason why some transporters and acceptors are required of these metals. In Mt the presence of Mg2+ transport P-type ATPase, ABC transporters such as Iron ABC transporter (coded by mbtB) and Iron-dependent regulatory proteins (IrtAB and ideR) are important for the synthesis of siderophores, the micobactina and the carboximicobactina are the most important to Mt. These are essential in the biosynthesis of cytochromes and hemoproteins, acting also as cofactors of proteins involved in the synthesis of amino acids, pyrimidines and bacterial DNA.22 These siderophores are developed by the bacterium in response to the low concentration of Iron inside the macrophages of mammals (approximately 1000 times less than the concentration required for normal bacterium growth) and, in their absence, Mt has an attenuated growth in macrophages and lungs of murine models.23
Adaptation to different Oxygen concentrations inside the host, in different stages of the disease, is also a significant feature in bacterial virulence. Late stages of TB such as granulomas, cause microaerophilic microenvironments with the concomitant production of Oxygen free radicals (oxidative stress). Enzymes such as alkyl hydroperoxidereductase and catalase-peroxidase enzyme, which catabolize hydrogen peroxide and other organic peroxides, are important in the elimination of Oxygen free radicals. In the absence of such enzymes, Mt and Mycobacterium bovis show a growth decrease in animal models.24 With the same purpose, Mt also exhibits enzymes superoxide dismutase-type which detoxify the bacterium of radicals of Oxygen; also proposing that such enzymes could inhibit the redox signaling necessary for the initiation of the immune response generated by the macrophage.25
Bacterial adaptability to express certain genes in response to external stimuli is also considered as a virulence factor. It determines bacterial survival when subjected to microenvironments such as inside the human body. Sigma type transcriptional factors found in Mt are A, C, D, E, F, G, H, and L; which are responsible in allowing the expression of a diverse repertoire of virulence factors. These serve, among other things, in the transcription of housekeeping genes to activate the latency, withstand increased temperatures, and act as a buffer in the treatment with detergents such as sodium dodecyl sulfate (SDS) or the action of oxidizing agents such as diamides.26–28
Another way bacteria respond to environmental changes is the expression of regulatory factors such as the two-component systems, the regulators of response, and kinases sensors. In Mt, 11 two-component systems have been described until now, without which the bacterium usually loses or minimizes its ability to replicate intracellularly in vitro and in animal models. One of the most important two-component system in Mt is the PhoP-Phor system, which is related to the regulation of lipid metabolism and cellular respiration processes.29 Other outstanding two-component systems in Mt are SenX3-RegX3 (which regulates 100 genes involved in the maintenance of the cell envelope, some regulatory functions, and energy metabolism),30 DosR/S/T (responsible of the latent stage)31, and MprA/AprB (which responds to the damage in the cell envelop caused by alkaline pH, detergents, and antibiotics).32
To control the expression of large groups of genes, Mt uses other transcriptional regulators such as hspR, which act as transcriptional repressors of the hsp70 heat-shock genes, whose repression starts at 37°C and is suppressed at 45°C. The absence of these transcriptional repressors is reflected in a colony forming units (CFU) reduction in animal model organs.33 Sporulation is an important phenomenon in the survival of bacteria in unfavorable environments for their growth. Mt is unable of sporulation, but it remains latent; for this reason ortholog genes of the WhiB family described originally in S. coelicolor have been studied; as well as genes coding for the WhiD protein, which interrupts the sporulation and septation in this bacterium.34 Studies conducted in a homologous gene to WhiB2 (WhmD) in M. smegmatis have demonstrated that this gene is required for cell division35; whereas in Mt suppression of WhiB2 does not affect the growth of the bacterium in murine models and Guinea pigs; however, it affects its survival. The half-life of Mt was found to be increased in the mutant strains for the gene WhiB3 (350 days), compared with mice infected with wild strains (225 days). Recently, it has been shown that the function of whiB2 in Mt is the control of cell division (Rv3260c gene) as well as in M. smegmatis.36
3. Respiratory disease caused by M. tuberculosis
Exposure to M. tuberculosis often results in the development of latent tuberculosis (LTB) with a risk of 5–10% for progression to active tuberculosis (ATB). Most TB cases occur in the first 2 years after the establishment of the bacteria in the lungs.37
3.1. Latent tuberculosis
Mt primary infection begins when the bacilli are able to reach the alveoli, where they are phagocytized by alveolar macrophages that transport them across the lymphatic system to regional lymph nodes (hilar, mediastinal and sometimes supraclavicular or retroperitoneal), producing a nonspecific inflammatory response which is usually asymptomatic. This immune response results in LTB, classically defined as an immunological sensitization to bacteria in the absence of transmission and manifestations of active disease (fever, chills, night sweats, weight lost, cough, hemoptysis, or opacities in the chest X-ray) due to the absence of bacterial replication. A granuloma is formed at the site of inoculation of Mt, which becomes necrotic and finally calcifies. In most cases (in immunocompetent hosts) the bacilli are destroyed, and then the only evidence of infection is a positive tuberculin hypersensitivity skin test; but sometimes bacilli can survive, leading to the LTB.38
The immunological reaction to Mt is evaluated using the tuberculin skin test (TST), also called Mantoux test or through Interferon gamma release assays (IGRAs). A positive result for any of the methods mentioned above is indicative of LTB, whereas microbiological test such as smear microscopy and culture for Mt should be negative, as well as the individual’s ability to transmit the infection.39 The homogeneity in the definition of LTB is currently being discussed because it does not consider how long the focus has been present, which could vary depending on the virulence of the Mt strain and the individual host susceptibility. A fact that reinforces the heterogeneity theory in LTB is the evidence of replication of Mt inside the granuloma of patients suffering LTB; supported by decreased risk of progression to ATB when treatment is performed with isoniazid, an antibiotic that inhibits bacterial cell wall synthesis (a process that occurs only when bacterial replication is active).40
3.2. Active tuberculosis
About 2–10 weeks after the inoculation of Mt, an injury appears demonstrable by X-ray in cases of ATB, which is caused by the necrosis of the primary focus after the accumulation of antigens in that focus. The accumulation of these antigens in sufficient quantities causes cell-mediated hypersensitivity, resulting in necrosis with calcification radiologically visible that could be detected through a tuberculin test or purified protein derivative (PPD). The combination of a peripheral lung injury and a calcified parahilar node is known as Ghon complex. TB disease or ATB develops when this immune response fails and the bacterium is not contained. TB disease or ATB is caused either by reactivation of the primary focus or by progression of a primary infection. The development of ATB occurs (on an average) within 2 years since the Mt primary infection has occurred. ATB is also produced by reinfection, which means a new infection that goes beyond the holding capacity of the immune system. In these cases the host’s immune reaction results in a pathological lesion typically localized, and it often presents extensive tissue destruction and cavitation.41 The risk of ATB is high in immunocompromised situations, such as extremes of age or people with debilitating diseases such as cancer or diabetes.42 When the infection is not contained at that level, bacilli may reach to the bloodstream and spread. Most of the pulmonary lesions or those disseminated lesions scar, turning into foci of potential future reactivation for TB. If such dissemination occurs, it may result in meningeal or miliary tuberculosis, which is particularly fatal in immunocompromised individuals.
4. Diagnosis of pulmonary tuberculosis
Pulmonary TB presents a broad spectrum of signs and symptoms in patients, which forces clinicians to establish a differential diagnosis with other lung infectious diseases such as bacterial or viral pneumonia, pneumocystosis, histoplasmosis, acquired bronchiectasis, or lung abscess; or with noninfectious conditions such as asthma, congestive heart failure, nonacquired bronchiectasis, lung cancer, or chronic obstructive pulmonary disease. Accurate diagnosis of TB must combine clinical and laboratory elements in order to confirm the clinical suspicion.
4.1. Clinical diagnosis of pulmonary tuberculosis
Pulmonary tuberculosis has nonspecific clinical manifestations. Usually, the affected individual has clinical manifestations over several weeks with cough, expectoration (sometimes hemoptoic), chest pain and nonspecific symptoms (fever or low grade fever, sweating, asthenia, anorexia, and weight loss). The results after the chest examination may be unremarkable, but localized rales or adventitious sounds with asymmetry in breathing sounds can be auscultated. It is essential to suspect the presence of disease in all patients with respiratory symptoms lasting more than 2–3 weeks and in all patients with hemoptysis, independent of the duration of respiratory symptoms. In addition to the clinical diagnosis, the epidemiological suspicion can be added when patients have an underlying disease causing immunosuppression (AIDS, cancer, etc.), are under 5 or over 60 years old, are smokers, alcoholics or drug addicts; and patients who had TB in the past or maintain an intense and prolonged contact with people suffering ATB.43
4.2. Laboratory diagnostic of pulmonary tuberculosis
Clinical suspicion of TB requires confirmation by laboratory techniques, which can range from simple microscopic observations to determinations requiring molecular biology and other cutting edge techniques.
4.2.1. Microbiological tests
Microbiological testing has been and remains the mainstay of the diagnosis of TB. The parameters that contribute to the diagnosis of pulmonary TB can range from simple observation to the confirmation of bacillus species by molecular biological methods. These tests are explained in the following sections.
Sputum smear
The sputum smear test consists of the microscopic examination of AFB in an extended lung secretion from patients with presumptive symptoms of TB. The smear is stained with Ziehl Neelsen stain (ZN), in which carbolfuchsin is retained by the mycobacterium (due to the presence of mycolic acids and waxes in its cell wall) after discoloration process with acid alcohol. The mycobacterium is observed as red bacilli on a blue background provided by the methylene blue, which acts as contrast dye. The ZN stain sputum allows the detection of all members of Mycobacterium genus, but the presence of AFB is not a sufficient parameter for confirm the presence of Mt in the samples evaluated by this technique.44 Other limitations of this testing include the requirement of 5.000 to 10.000 AFB/mL in the sample (optimally 100.000) in order to be detected. This, along with the fact that approximately a 30–50% of TB patients are smear sputum negative, decreases the sensitivity of smear microscopy, and makes it impossible to consider a negative result as a unique parameter to rule out the disease. The diagnostic sensitivity of smear microscopy is estimated between 22–43% in a single sample and 50–70% with two to three samples examined in different days. Therefore, the recommendation in the diagnosis of pulmonary TB is to examine three sputum samples through ZN staining on three different days, preferably in the morning samples.45 The specificity of the smear sputum microscopy depends on several factors, including the quality of coloration and the expertise of the examiner in order to distinguish AFB from acid-fast elements such as food particles, precipitated dye, environmental AFB, normal microbiota, atypical mycobacteria, or Nocardia species; fibers (cotton, wool, etc.), pollen granules; and even defects on the glass slide where the smear was made. It is also important to consider that false negatives could occur due to deficiencies in the dying of the sample, reading of the test, sputum collection (saliva collection or nasopharyngeal secretions), and the selection of the portion of the sample used for the test.45–47
Fluorescent dyes
The fluorescent stains for Mt as auramine O, rhodamine, or a combination of these, represent an alternative to the ZN staining. With these stains the AFB appear fluorescent. With auramine yellow to orange, with rhodamine red, and with auramine-rhodamine are reddish yellow on a dark background when using potassium permanganate as contrast dye.48 An advantage of using fluorochromes in the observation of AFB is that these allow its detection in a shorter time, compared to regular ZN staining. Thus, the bacilli can be observed with low power lenses, and then confirming its morphology with the oil immersion technique. Another feature of the technique is that it allows subsequent staining of smears with ZN in order to study the morphology of the bacilli and its acid-fast properties. The sensitivity of fluorescent stains in the diagnosis of pulmonary TB is greater than in ZN, such sensitivity increases in samples with low quantity of bacilli. The specificity of these two techniques is similar, and some studies suggest that the difference in sensitivity and specificity using different fluorescent compounds is not significant.49 It is important to mention that this technique, as well as the ZN stain, could lead to false positive results due to the presence of artifacts in the preparation50; or false negatives due to poor sampling preparation or poor staining technique, particularly considering the fact that fluorescence is stable for only 3 days even when the glass slide is protected from light.51
Culture
This technique is considered to be the gold standard in the diagnosis of TB because it confirms the positive results of the microscopic test. The culture has much a greater sensitivity than the sputum smear staining, being able to detect between 10 and 100 AFB/mL of sputum; allowing the isolation of the mycobacterium for identification and determination of antibiotic susceptibility. The biggest disadvantage of cultures (using traditional methods) is the long waiting time for the results, which ranges from 4 to 8 weeks on solid media as Ogawa Kudoh (OK) or Lowestein Jensen (LJ); a fact that is determined by the metabolic characteristics of Mt.44 Some techniques have been developed to minimize this incubation period that involve the use of liquid media incorporated into automated systems that provide results in a significantly shorter time.
Cultures in solid medium
Solid consistency of these kind of media can be provided by compounds such as egg (Ogawa Kudoh, Lowenstein Jensen and Coletsos) or agar (7H10 and 7H11 of Middlebrook). The egg-based media are enriched with the addition of an inhibitor called malachite green. These media allow the recovery of most of the mycobacterial species due to their components, inhibitory power of the contaminant flora, and their buffering capability that allows the neutralization of multiple toxic products present in the clinical samples. Furthermore their half-life is long (6 months at a temperature between 2 and 8°C), and the phenotypic characteristics of the isolated strains represent a good degree of fidelity. Among the disadvantages are the slowness with which Mt develops in the media and the difficulties of preparation (coagulation method), leading to variations between batches.52 The agar-based media allows a rapid detection of colony growth of Mt (10–12 days), and its recovery for susceptibility testing. The Middlebrook 7H11 media containing 0.1% of casein hydrolisate favors the recovery of isoniazid-resistant Mt strains. The disadvantages with respect to the egg-based media, are the short half-life (1 month at a temperature between 2 and 8°C) and a higher rate of contamination.53–54
An alternative technique developed for the identification and susceptibility testing of Mt is the mycobacteriophage system. Phage-based assays for Mt detection have high specificity (ranging 0.83–1.00), but modest and variable sensitivity (ranging 0.21–0.88). This method is more useful for detecting rifampicin resistance directly from sputum smear-positive samples or indirectly from culture. The test has a manual format and the results are read visually. Results are available within 2 days after of inoculation of mycobacteriophages, and use basic microbiological equipment available in most laboratories.55
Cultures in liquid media
This test uses cultures of pulmonary secretions in liquid media into automated systems for finding Mt, which is the most effective recovery of this bacterium and also the fastest method. Consequently, this test is always recommended for the primary isolation of clinical samples. Liquid media systems are the basis for identification and susceptibility testing in automated incubation and reading systems. The disadvantage of these media is the inability to distinguish colonies and therefore possible contamination, as well as its high cost. However, its performance and adaptation to the new technologies of identification and systematization make them ideal for fast and effective detection of Mt.56 These media can be classified into manual reading media and automated reading media. The manual reading culture media contains mycobacterial growth indicators without apparent presence of bacterium. Such indicators may be tetrazolium salts present in culture media as MB Redox medium or fluorescent compounds such as Ruthenium pentahydrate component of Mycobacterial Growth Indicator Tube (MGIT).57 On the other hand, semiautomated systems such as BACTEC 460 TB use a radiometric marker (palmitic acid labeled with 14C) as an indicator for the detection of mycobacteria, and can be used for the presumptive identification of Mt and susceptibility testing. Unfortunately, due to a lack of integration with an automated reading system, it may have problems of cross-contamination, and there may be a possible production of aerosols during handling and reading. The evolution of automated system makes it possible to produce systems that combine incubation and reading, such as the BACTEC MGIT 960 which uses MGIT media detecting O2 consumption by fluorometric sensors with the ability to perform susceptibility testing for first-line antimycobacterial drugs (including pyrazinamide).58 There are alternatives such as ESP Culture System I, whose detection system uses pressure sensors detecting the O2 consumption of the mycobacteria. The basic medium of this system is a Middlebrook 7H9 media with enriching supplements and inhibition of contaminant agents.59 This system is able to perform susceptibility testing for isoniazid, rifampicin, and Ethambutol.60 There are also automated methods based on colorimetric methods such as MB/BacT ALERT 3D, which uses production of CO2 as indicator of bacterial growth, and Middlebrook 7H9 media with growth and inhibition factors for samples that come from human compartments that are normally nonsterile. Once inoculated, cultures remain in the “incubator-reader” system without additional handling until the equipment notifies the results. Following a positive result, susceptibility testing can be performed for first-line antimycobacterial drugs (excluding pyrazinamide).61
4.2.2. Molecular tests
Several methods for detecting DNA of Mt directly from samples or positive cultures have been developed. These tests take a great value today due to the increasingly frequent occurrence of multi-drug resistance Mt strains. Some of the most used tests for MDR detection are the molecular line probe assays, which use multiplex polymerase chain reaction (PCR) amplification and reverse hybridization to identify Mt complex and gene mutations associated with rifampicin and isoniazid resistance. Line probe assays are highly sensitive and specific for detection of rifampicin resistance (≥97% and ≥99%) and isoniazid resistance (≥90% and ≥99%) on culture isolates and smear-positive sputum.62 Amplification and detection of Mt DNA is one of the fastest and most sensitive ways to detect tuberculosis, and may also allow the detection of genetic mutations associated with drug resistance. Some of these methods are complicated and require specialized training, and can only be performed on material that has been subjected to processing and DNA extraction. Between the automatic systems of detection stands the GeneXpert system (Cepheid), which is a closed, self-contained, fully-integrated and automated platform that allows a relatively untrained operator to perform sample processing, DNA amplification, and detection of a large variety of microorganisms that cause infectious diseases in less than 2 h.63 In the TB field, GeneXpert systems has developed a semi-nested RT-PCR assay for Mt that simultaneously detects rifampicin resistance called Xpert MTB/RIF, this assay has shown excellent performance in a multicentre study, where in a unique direct MTB/RIF testing detected 92.2% of cases of pulmonary tuberculosis, including 72.5% of those with smear-negative disease, which was equivalent to that reported for solid culture.64 A striking molecular method is a loop-mediated isothermal amplification assay, which is a simple DNA amplification method that does not require a thermocycler or detection system and reportedly allows visual detection of amplification.65
4.2.3. Indirect tests
These are assays based on detection of organism’s effects on the host. These can be classified into antibody detection test (humoral response) and tests for cellular response detection, either by direct cell production and its effect in vivo or by the production of some molecules released by leukocytes sensitized with a TB antigen.
Serodiagnostic tests
Unlike many infectious diseases whose diagnosis can be supported strongly in the application of serodiagnostic tests, it has not been possible to develop a test for TB with high sensitivity and specificity. This is most probably because the primary immune response to Mt is cell-mediated, instead of being a humoral response. Evidence suggests that the humoral response to the bacilli is heterogeneous.66 Currently, the International Standards for TB Care discourage about the use of serological tests in routine practice and no international guidelines recommend their use; however, many commercial serological tests for TB diagnosis are offered for sale in many countries around the world. WHO is emphatic in their policy statements to recommend strongly that these commercial tests not be used for the diagnosis of pulmonary and extrapulmonary TB. Results from studies performed in several countries of the most widely used tests showed sensitivity at 76% and 59%, and specificity at 92% and 91% in smear-positive and smear-negative patients, respectively. In conclusion, currently available commercial serodiagnostic tests provide inconsistent and imprecise results.67 Independent studies, such as one made in India where 1.259 subjects were included in the study, using three different enzyme immunoassays, also conclude that the commercial serological test evaluated showed poor sensitivity and specificity, and suggests no utility for the detection of pulmonary tuberculosis.68
Immunological tests
The TST is a traditional method used as a screening method for the diagnosis of TB, which is also called PPD or Mantoux test. Tuberculin is a method based on a delayed-type hypersensitivity reaction, developed in the skin, after intradermal inoculation with PPD. The Mantoux test is typically used to determine immunity to TB in humans and positive reactions develop not only in individuals previously exposed to Mt but also in those previously immunized with the Bacillus of Calmette and Guérin (BCG) vaccine. After 24–72 h later the tuberculin (PPD-RT23) is inoculated in the forearm, in a dose of 0.1 mL; an induration that is larger in diameter than a certain size (ranging from 5 to 15 mm) is considered a positive result and indicates infection with Mt.69 Despite its importance, the Mantoux test is falsely negative in 10–47% of patients with active disease.70 The PPD test has lower sensitivity in populations of immunocompromised patients, recently infected individuals, and very young children. Specificity is low because the PPD contains various antigens widely shared among different species of mycobacteria.71 In the presence of coinfection with HIV, a far higher proportion of patients with active disease will have a false-negative test result. The degree of immunosuppression determines the rate of false negatives, being 30% for patients with a CD4 T-lymphocyte cell count of more than 500/mL, compared with almost a 100% of patients with a CD4 T-lymphocyte cell count of less than 200/mL.72 The Mantoux test will be replaced in the future by specific antigens only found in Mt. Recombinant dimer ESAT-6 (rdESAT-6) has been successfully tested for this purpose. Improvements of this new skin test continue to be developed using other antigens such as CFP-10.73
To improve the performance of Mantoux test, new tests such as T-cell-based IGRA have been designed. IGRAs are in vitro blood tests based on interferon-gamma release after stimulation by antigens such as Early Secreted Antigenic Target-6 (ESAT-6) and Culture Filtrate Protein-10 (CFP-10). These antigens are not shared with any of BCG vaccine strains or certain species of nontuberculosis mycobacteria. Indeterminate results are likely in immunocompromised individuals with low CD4+ cell counts.74 There are currently two commercial IGRAs: the QuantiFERON-TB Gold In-Tube (QFT) and the two-stage T-SPOT.TB assay. There is strong evidence that IGRAs, especially QFT, have excellent specificity unaffected by BCG vaccination and appear to correlate well with markers of TB exposure. Regarding the sensitivity, T-SPOT.TB appears to be more sensitive than QFT. This could be partly because the cut-off for T-SPOT.TB is designed to maximize sensitivity, while the cut-off for QFT is designed to maximize specificity. Resuming, the estimated sensitivity of IGRAs in patients with active TB is between 75% and 95%, decreasing in immunocompromised individuals (60–80%), and estimated specificity in healthy persons with no TB disease or exposure is between 95% and 100%.75
4.3. New perspectives in TB diagnostic
In recent years, new alternatives for TB diagnostic have been developed as a solution to the problems that occur with current techniques. Tests such as breathalyser screening test, which use an instrument for detection of volatile organic compounds of Mt, is performed and a readout is obtained in under 10 min. The instrument is fully portable and runs off rechargeable AA batteries.76 The detection of Mt molecules in urine is a test that is based on detection of lipoarabinomannan (LAM) excreted in the urine of TB patients. Urinary antigen detection may be of particular value in diagnosing TB in HIV-coinfected patients. It may prove valuable for rapid and simple diagnosis of TB, particularly in developing countries at peripheral levels.77 An increase of the sensitivity of sputum samples is being searched for through the use of techniques of concentrating sputum, such as sputum filtration. In this technique, sputum is liquefied and passed through a filter, which is then stained or cultured by standard techniques. Filtration considerably concentrates mycobacteria, increasing sensitivity. Another advantage of using concentrating sputum is the reduction in time spent on sputum examination.78 TB diagnosis using immunodominant secreted antigens of Mt is being developed. In this method, skin patches delivering MPT64, ESAT6, or CFP-10 (specific proteins of Mt) are placed on the patient’s skin and in those with active infectious TB a localized immune response consisting of erythema and/or vesiculation appears 3–4 days after application to the skin.79
5. Conclusion and future perspectives
Infectious diseases have accompanied people since the beginning of the time. Likewise, because humans were affected by disease, they have always looked for cures. In the case of TB the story is the same and, as in most infectious diseases, despite the significant progress made to fight against it the disease succeeds to the present day. However, at present, the health situation is much better than it was. This is caused in part by the actions implemented against infectious diseases. The development of antibiotics against Mt, implementation in diagnostic methods, prophylaxis through vaccination, and improving the conditions of poverty, have been masterpieces in reducing TB.
The development of molecular biology and the other genomic technologies has aided the diagnosis of TB. For example, it has allowed the fast detection of TB. In comparison with traditional techniques, current methods provide much more information in a shorter time.
A more effective TB vaccine is expected to be introduced over the next few decades. In addition, we also expect the introduction of antibiotics that can be used against MDR-Mt, and fast and accurate diagnostic techniques that are more affordable for developing countries. The ultimate goal is the eradication of TB. This should be parallel to the reduction in cases of AIDS, which is one of the main risk factors for acquiring TB.
Fundamental and applied research on TB and the maintenance and proper implementation of public health policies should be the cornerstone on which must be founded the fight against one of the most important and ancient infectious diseases in the history of humanity.
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