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Is the standard technique to diagnose tuberculosis infection and is one of the issues that most written in the history of medicine and that most interest and controversy has suscitado2, shows, after injection of a protein derivative state prior hypersensitivity of the body to that substance. Initially, the tuberculin of Koch was extracted from boiled culture of bacilli. Currently used the PPD (purified protein derivative) obtained after the culture filtrate of Mycobacterium tuberculosis sterilized and concentrated. The tuberculin used in Europe is the PPD RT-23. In the U.S. there are two preparations, and Tubersol Aplisol, both with response similar to the RT-23. The main drawback of PPD is that the proteins used are not specific to Mycobacterium tuberculosis, but are shared with other non-tuberculous mycobacteria, which reduces the specificity of the test.
TB skin test results Is the standard technique to diagnose tuberculosis infection and is one of the issues that most written in the history of medicine and that most interest and controversy has suscitado2, shows, after injection of a protein derivative state prior hypersensitivity of the body to that substance. Initially, the tuberculin of Koch was extracted from boiled culture of bacilli. Currently used the PPD (purified protein derivative) obtained after the culture filtrate of Mycobacterium tuberculosis sterilized and concentrated. The tuberculin used in Europe is the PPD RT- 23. In the U.S. there are two preparations, and Tubersol Aplisol, both with response similar to the RT-23. The main drawback of PPD is that the proteins used are not specific to Mycobacterium tuberculosis, but are shared with other non-tuberculous mycobacteria, which reduces the specificity of the test. Lately it has isolated the specific gene sequence of Mycobacterium tuberculosis PPD (PPD recombinant) 3 that could detect false positives in Mycobacterium tuberculosis not. This method has not yet been commercialized. Technique of tuberculin test There are two methods: Mantoux test and m?ltiples4 punctures, 5. The Mantoux technique involves the intradermal injection with a 27 gauge needle on the anterior forearm (0.1 ml) of 2 tuberculin PPD RT-23, in an area where there are no skin lesions. There must be a wheal of 6-10 mm in diameter so that the technique is correct. It is the most common method of tuberculin skin test (PT). The multiple puncture test is also performed on the forearm spiked impregnated tuberculin. Since no one knows the amount of tuberculin into the skin, it is considered improper technique. Immunological basis of tuberculin test The individual infected with the tubercle bacillus reacts to the PT with a delayed hypersensitivity response mediated by cells (especially T lymphocytes), appearing at 48-72 hours after injection, an induration in the area. This hypersensitivity response remains for life even in the elderly and in certain clinical conditions may be diminished. The fact of repetition made PT an individual is not sensitized by itself does not trigger the immune response. Reading and interpretation of the tuberculin test At 72 hours post injection is read by measuring the transverse diameter of induration along the longitudinal axis of the forearm. The result is given in millimeters. In the absence of induration but only erythema, is interpreted as 0 mm. In the diagnostic reading will be taken into account not only the size but also the individual's clinical condition: In Spain, according to the Society of Pneumology and Thoracic Surgery (SEPAR) is considered a positive induraci?n6: - In unvaccinated persons ? 5 mm. - In individuals vaccinated with BCG is the problem of discerning before a tuberculin induration, the question of tuberculosis infection or a response to antigens shared between BCG vaccine (M. bovis) and PPD, as the latter presents unique antigens of Mycobacterium tuberculosis. In this situation taking into account certain clinical conditions, considering positive PT with diameter> 5 mm as well as vaccinated are cohabiting or maintaining frequent contact with smear-positive patients, chest x-ray carriers with lesions suggestive of tuberculosis old and never treated, infected HIV and silicosis. - In the remaining vaccinated with BCG if the size of the induration is> 15 mm. PT is considered a high negative predictive value of infection when the size of the induration diameter is less than previously reported values, however, if in these cases people are vaccinated with BCG or are over 65, they should repeat the test within 7-10 days (booster effect) and that the outcome will be accepted. With respect to vaccination with BCG, despite this being a disused vaccine in our country, some people can be vaccinated tuberculin reactions similar to those produced by tuberculosis infection. In general, this reaction vaccine is not greater than 14 mm and it is considered that the larger size and more time has passed since vaccination, is more likely to be of a tuberculous infection and not a vaccine reaction (Studies in They point out that the interference of BCG on the tuberculin reaction may be negligible past 10-15 years after vaccination). The higher the probability of being infected or developing disease, such as in cases of recent contacts or infected with HIV, less to influence the history of vaccination in the interpretation of the test. In HIV patients, an induration under 5 mm, due to the state of anergy by the immunocompromised, should not exclude the diagnosis of infection. Tuberculin reactions with blistering or necrosis at the site of inoculation also considered indicative of TB infection, whatever the size of induration or vaccination history. When it comes to a study of contacts greatly simplifies the interpretation and you should not consider the vaccination history and consider an induration greater than 5 mm as indicative of tuberculosis infection. False positive and negative tuberculin test PPD comprises antigens of Mycobacterium tuberculosis nonexclusive shared by other non-tuberculous mycobacteria (M. bovis, M. avium ...), a fact that could be responsible for false positives to an individual with positive PT. There are other situations, such as inadequate technical hematoma, or infection, which in turn could alter the interpretation of the test (Table 1). There are certain circumstances dependent on the individual that can lead to false negative results of PT such as concurrent viral infection, vaccination with live virus, situations of immunosuppression or treatment with drugs that decrease the immune response, the extreme ages of life as infants in whom given the immaturity of the immune system, up to 6 months of life there is no adequate response to tuberculin; in elderly weakens the immune response and can be interpreted as false negative, the technique repeated after a week, it highlights the positive (Booster or push effect). In turn, a PPD in poor condition and inadequate Mantoux technique both in its administration and in his reading, can lead to false negative results of PT (Table 2). Booster phenomenon or thrust In some individuals a first test may be read as negative and the test repeated 7-10 days positive. This phenomenon is due to a diminished immune response in elderly patients or infected years previously vaccinated in infancy, which is evident after the second test. The final results of the test is the second reading. Can cause false negatives. Tuberculin conversion This is the situation where an individual known as PT goes negative to positive, having previously dismissed the effect Booster. Represents the acquisition of TB infection. Is defined as "convertor" one who has a tuberculin conversion or recent, is operationally defined as the individual who happens to have a tuberculin less than 5 mm to greater than or equal to 5 mm with a gap of at least 5 mm in less than 2 years. Indications for tuberculin test The PT, like any diagnostic test should be used only in those in which the outcome may result from therapeutic intervention. In tuberculosis there are only two possibilities for therapeutic intervention, the treatment of patients and of chemoprophylaxis or preventive treatment of those infected with high risk for tuberculosis2. Table 3 shows the signs of the PT as the SEPAR6. In the general population is symptomatic not recommended its use as a screening method. Attitude to the tuberculin test After reading the PT, if diagnosis of infection, rule out tuberculosis by chest x-ray, microbiological study and exploration of the individual. Once discarded, chemoprophylactic treatment is evaluated. Faced with a PT is not diagnostic of infection in unvaccinated individuals under the age of 55-65 years, and will test negative. If you are over 55-65 years, passed the test repeated 7-10 days (booster effect), and interpreted this second reading and final. If the PT is not diagnostic of infection in vaccinated individuals, the test will be repeated 7-10 days regardless of age. NEW DIAGNOSTIC TECHNIQUES "IN VITRO" OF TUBERCULOSIS Over the past 100 years, the PT has been the only method available in clinical practice to determine TB infection. This test, introduced in 1890, is the oldest diagnostic test in use, and as mentioned, measures the delayed cellular immune response on the skin after administration of PPD, which contains a mixture of antigens shared by several mycobacteria. However, in recent years have been investigated and approved new diagnostic methods based on the quantification "in vitro" cellular immune response. These methods, known generically to the literature with the acronym of IGRA (Interferon - g Release Assays) detect the release of interferon-g in response to mycobacterial antigens. Immune response to tuberculosis infection One of the most important molecules in the control of tuberculosis is the interferon-g. This cytokine, produced by T lymphocytes CD4 +, CD8 + and NK cells, activated macrophages infected with the consequent release of IL-1 and TNF-a that limit the growth and multiplication of mycobacteria. Although the production of interferon-g per se, is insufficient to control tuberculosis, their participation is essential in the protective immune response against this microorganism. Individuals with deficiencies in the receptors or in the genes of this molecule are more susceptible to mycobacterial infections more frequent and more severe. Types of IGRA There are two techniques "in vitro" for diagnosis of tuberculosis infection: the QuantiFERON-TB (Cellestis, Victoria, Australia) and T- SPOT.TB (Oxford Immunotec, Oxford, UK) 7. The first generation QuantiFERON-TB, approved by the FDA in 2001, measured the release of interferon-g in response to PPD. In 2004, the FDA approved the second generation of this diagnostic test, called QuantiFERON-TB Gold, which unlike the first generation, not used as the mycobacterial antigens PPD, but more specific antigens such as the Early Secretory Antigen Target ( ESAT-6) and culture filtrate protein 10 (CFP- 10). These two molecules, encoded by the DR-1 region of the genome of Mycobacterium tuberculosis, significantly increase the specificity with respect to PPD. These antigens are absent in all strains containing BCG vaccine and most mycobacteria (Table 4). They already work is underway with the third generation of this test, called QuantiFERON-Gold In Tube (QFT-G IT), which incorporates a third mycobacterial antigen: the TB 7.7, but has not yet been approved for use. The T-SPOT.TB is approved for use in Europe and Canada and is being evaluated for approval by the FDA. Conduct and interpretation of test 1. QuantiFERON-TB-g Gold (QFT-TB Gold): the test is performed by incubating 1 ml of peripheral blood anticoagulated with heparin, another blood thinner does not work in each of the four wells containing the antigens: saline as a negative control ; phytohemagglutinin as a positive control to measure the ability of lymphoproliferation of lymphocytes of each patient, and the antigen ESAT-6 and CFP-10. Blood must be incubated with these antigens before the next twelve hours of collection. Following a period of incubation for 16-24 h at 37 ° C, determine the concentration of interferon-g in plasma by ELISA. The results should be calculated using software provided by the manufacturer. 2. T-SPOT.TB: based on the same principle as the QFT-TB Gold, the identification of T cells producing IFN-g in response to ESAT-6 and CFP- 10. As with the QFT-TB Gold requires four wells per patient and antigens, and uses the same controls, but unlike the latter, does not use whole blood but which requires the separation of mononuclear cells prior to their stimulation, and presence of interferon-g ELISPOT is determined by ELISA in place (Fig. 1). In each well must be added an appropriate number of cells, otherwise the interpretation would be incorrect. The number of mononuclear cells per well be of 2.5 x 105. According to the manufacturer's instructions, the blood is processed in the range of eight hours after collection and should not be refrigerated or frozen whole blood samples. The results are reported as spot forming cells or spot-producing cells (Fig. 2). Each spot represents the footprint of a single T cell secreting IFN-g, and evaluation of the number of spots obtained, determines the abundance of T cells responsive to M. tuberculosis in peripheral blood. Technically, T-SPOT.TB, requires more blood, more preparation time and is more difficult than QFT-TB Gold, but it seems more sensible. The guidelines recommended by the manufacturers of these tests for their interpretation are expressed in tables 5 and 68.9. Advantage of IGRA on the Tuberculin New diagnostic techniques "in vitro" of tuberculosis offer significant advantages over PT: avoid the subjectivity of interpretation, obtaining the results is fast and can be available within 24 hours, if necessary, the test may be repeated immediately Booster effect without fear, prevents reading the visit, have a positive internal control, which provides valuable information when interpreting apparently negative test as true negative or indeterminate result of technical errors or immunosuppression. Sensitivity and specificity It is difficult, in the absence of a reference test in the diagnosis of tuberculosis infection, due to the problems of false positive and negative tuberculin test, setting the sensitivity and specificity of these new diagnostic tests. To solve the problem of sensitivity have been used three strategies: 1. evaluate patients who have active TB and therefore must be infected, 2. evaluate individuals who have been in contact with TB patients and stratifying by the degree of exposure and 3. agreement between the IGRA and the tuberculin test. a. Sensitivity using active TB as a marker of infection. In a recent meta-analysis, Menzies, et al assessed all studies published until October 2006 that have compared the T-SPOT-TB (eight studies included a total of 424 patients) and the QFT-TB-G (nine studies 393 patients) with the tuberculin skin test using a cutoff ? 5 mm (nine studies with 303 patients) in the diagnosis of tuberculosis activa10. The three tests have suboptimal sensitivity to below 90%, reaching 87% in the case of T-SPOT- TB and 80% with the QFT-TB-G, a percentage equal to that obtained with tuberculin. In the past 2 years have seen the light of three studies that have evaluated the QuantiFERON 3rd generation in this situation, and overall sensitivity is strikingly lower than the 2 nd generation QuantiFERON, reaching only 67%. These studies support the recommendations of the Centers for Disease Control indicating that for reasons of sensitivity, a QFT-TB-G negative should not be used to rule out TB activa11. b. Sensitivity using the exposure gradient as an indicator of the likelihood of tuberculosis infection and tuberculin agreement. In a contact in Denmark, a country with low TB incidence (<10/105), included 125 patients who had been in contact with a patient diagnosed with tuberculosis in a school.12. Contacts, 85 had not received the vaccine for tuberculosis. 56% of contacts with high exposure and previously unvaccinated, tuberculin had a 53% positive and one QuantiFERON-TB-Gold positive. In this group of patients the level of agreement of both tests was 93%. In vaccinated individuals, regardless of the level of exposure, tuberculin was not determined according to Danish rules. In 40 patients with low exposure and unvaccinated, 10% had a positive tuberculin compared to 5% of positive blood test registered. Kang et al evaluated in a prospective study in Korea, a country where the incidence of active pulmonary tuberculosis is intermediate (92/105) and BCG vaccination is mandatory, a total of 273 patients of whom 220 (95.7% ) had previously received vacuna13. Were divided into 4 groups according to the risk of infection: group 1 were medical students without identifiable risk factors for exposure to tuberculosis, Group 2, consisted of health workers who had casual contact with TB patients, the group 3 was comprised of individuals who had contact in your home or work with individuals smear-positive for more than 8 hours per day and group 4 included patients with active TB. Each patient underwent a PT, which was considered positive when the size of induration was ? 10 mm and a test of QFT-TB-G. The results of the skin test and the blood can be seen in Figure 3. The odds ratio for a positive result is significantly increased by 5.31 with increasing the risk of infection in each group when the assessment is performed with QFT-TB-G, while increasing only 1.52 when the assessment is performed with the PT in the vaccinated population. Richeldi et al studied 92 people who had been in contact with a parturient diagnosed with MDR TB by smear esputo14. Only 10 contacts had been vaccinated previously. When analyzing the prevalence of each positive test based on the type of contact, found that only 1 of 9 people (11%) who had shared the same room with the index case had a positive TST compared to 6 of 9 (67% ) that were positive with the T-SPOT-TB. Only 6% of patients who had direct contact with the index case, but had not been entered in the same room, both tests were positive. Therefore, according to this study, the odds ratio to present a T-SPOT-TB Positive entered in the same room was 13.8 (p = 0.001) with respect to admitted in different rooms. This author also noted that the odds ratio was increased by 1.05% for every hour that the contacts had shared with the index case. In contrast, the PT did not correlate significantly with income in the same room or the number of hours of exposure. Zellweger work done in Switzerland in which assessed a total of 92 people working in institutional residence underwent a T-SPOT-TB and PT with the aim of seeing both tests were correlated with the degree of exposici?n15. As shown in Figure 4, again as it increased the duration of exposure significantly increases the percentage of patients with a T-SPOT-positive TB, in contrast, the PT did not show this association. According to the guidelines for Switzerland, 44% of patients would have had to receive secondary prophylaxis for filing a ? 10 mm induration. Even if you increase the cutoff point ³ 15 mm chemoprophylaxis percentage would be 25% versus 15% in the T-SPOT-TB positive. In our country, is taking place in Barcelona a multicenter study to evaluate the two techniques in vitro against tuberculin 152 including contacts, grouped by degree of exposure (greater than or less than 6 hours) 16. Again, we see how the techniques in vitro correlate better with the source of the infection. Of the individuals studied in which said treatment of latent tuberculosis infection, 31.6% had a negative T- SPOT-TB and 44.4% for the QuantiFERON-TB test-G. It is therefore likely that most consistent with the degree and duration of exposure of these new techniques with respect to the PT allows a more accurate diagnosis of infection and avoid unnecessary chemoprophylaxis guidelines. Specificity The specificity of these tests can be estimated in individuals vaccinated with BCG with no risk factors for developing tuberculosis infection, and assuming that for this reason, these individuals do not have a latent infection. a. Specificity of T-SPOT-TB: the overall specificity is around 92%. Three studies involving a total of 98 individuals vaccinated against tuberculosis, showed a specificity of 100% average 17-19. In a more recent survey in South Korea, the specificity of the T-SPOT-TB in 131 patients was 92% 13. b. Specificity of quantiferonTB-Gold: Menzies et al in a meta-analysis published in 2007, calculated after analyzing six studies a specificity of 96% in this population.10. c. Specificity of tuberculin: In three studies that evaluated the specificity of the tuberculin test in the vaccinated population, is considered positive when an induration ? 10 mm, it was 56% 13,20,21. However, when increases in vaccinated cutoff 15 mm ³ to consider the positive test, according to the recommendations SEPAR22, specificity is at the 80% (Fig. 5). Analyzed studies suggest that, irrespective of the in vitro technique we are considering, the specificity of the IGRA is superior to the Tuberculin. Several studies have revealed that 10-40% of patients there is a discrepancy between the results obtained with the skin test compared to those obtained with the test in vitro23. In three cases the discrepancy was higher among vaccinated than in unvaccinated. IGRAs in immunocompromised patients Patients with impaired cellular immunity (eg HIV infection, immunosuppressive therapy, including inhibitors of TNF-g, etc ...) have an increased risk of developing disease if infected. Thus, while in the immunosuppressed population risk for TB is, from the moment they become infected, 10% over the life of the individual in HIV patients, the risk is 10% anual24 . Unfortunately in this group with such a high risk of developing tuberculosis, tuberculin test is negative when it is not very valuable. This can translate a true negative or a false negative due to anergy. Several published studies included in this group a percentage of 26 to 41% false negatives and the strategies used to try to identify them, as the administration of tuberculin with other antigens (eg. Candida), have been abandoned for lack of standardization and reproducibility. In this situation, the IGRA can have a major utility, especially considering that they have positive mitogen control helps us to know if a positive result is not a true negative or indeterminate, which translates technical errors or marked immunosuppression. Few studies have evaluated the utility of these tests in immunocompromised people. Available data suggest that the T-SPOT-TB is more sensitive than the tuberculin skin test and QuantiFERON TB-G with a smaller percentage of indeterminate studies. Piana et al compared in 138 patients with hematologic malignancies who had been in contact with a smear positive at the hospital level, the tuberculin skin test (considered positive induration ³ 5 mm) with the T-SPOT-TB25. Globamente, 44% had a T-SPOT-positive TB compared with 17.4% of positive skin test registered. Passalent, using the same methodology in 203 patients with renal failure requiring dialysis, found 35% positivity of T-SPOT-TB versus 18.2% of the tuberculina26. The percentage of indeterminate studies was 5.1%. In the only study that has directly compared T-SPOT-TB with the tuberculin test in HIV-positive patients were considered to skin test was positive when the induration was ? 10 mm, so it's probably hard to draw conclusions with obtenidos27 results. In a later work by Dheda and cabbage with this technique, the percentage of indeterminate studies was 3% and did not correlate with CD428 counting. A recently published work that has compared the QFT-TB-Gold in tube (QuantiFERON 3rd generation) with the tuberculin test in 294 patients with HIV positivos29. Defined a tuberculin test was positive when the induration ? 5 mm. The overall number of indeterminate results was 9.1%, four times greater when the CD4 cell count was <100 cells/mm3 when it is> 100 cells/mm3 (16.1% versus 3.6%). 56% of patients with TB had a positive QFT-G negative and 58% of the QFT-G had a positive skin test was negative. As the author points out, this study does not resolve the question of whether HIV + patients should either test or both simultaneously, although the results of work advocating the latter. Brooks et al in 590 HIV-positive was 24% of indeterminate studies in patients with fewer than 100 CD4 + / mm3 vs 2.8% in those with more than 100 CD4 + / mm3, 30. Overall the number of indeterminate studies was 3.4%.
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