Open Journal Systems

REVIEW ARTICLES

Received: July 30^th 2014

Accepted: September 1^st 2015

Diagnostic strategies in the Clínica de Tuberculosis of the Hospital General of the Centro Médico Nacional La Raza

Alberto Alejandro Flores-Ibarra,^a María Dolores Ochoa-Vázquez,^a Georgina Alejandra Sánchez-Tec^a

^aServicio de Neumología, Hospital General, Centro Médico Nacional La Raza, Instituto Mexicano del Seguro Social, Distrito Federal, México

Communication with: Alberto Alejandro Flores-Ibarra

Thelephone: (55) 5724 5900, extensión 23436

Email: mksapmd@yahoo.com.mx

In order to diagnose TB infection, tuberculin skin test and interferon gamma release assay are available. The tuberculin test has a sensitivity of 99 % and a specificity of 95 %. For the detection of interferon gamma in blood there are currently two tests available: TBGold QuantiFERON-In-Tube (with a sensitivity of 0.70 and a specificity of 0.90), and T-SPOT-TB (sensitivity 0.90 and specificity 0.93). To diagnose the disease, a microscopy of direct smears for acid-fast bacilli is used if the physician is facing an extensive cavitary lung disease due to M. tuberculosis (this test has a high sensitivity: 80-90 %). The most common staining techniques used are Ziehl-Neelsen and Kinyoun, and the fluorescent technique, auramine-rhodamine. The culture is the gold standard and it has a sensitivity of 80 % and a specificity over 90 %, but the results take weeks. The nucleic acid amplification test has an overall sensitivity and specificity of 0.85 and 0.97, respectively. In the presence of a pleural effusion is necessary to perform a pleural biopsy for culture with a sensitivity of 85 % if it is percutaneous and 98 % if it was taken by thoracoscopy. The adenosine deaminase can be determined in pleural fluid with a sensitivity and specificity of 95 %.

Keywords: Mycobacterium tuberculosis; Diagnosis; Tuberculosis.

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The Instituto Mexicano del Seguro Social, through evidence and experience in its tertiary center Centro Médico Nacional la Raza in the Pneumology department, has developed recommendations for the diagnosis of pulmonary tuberculosis in first, second, and third levels of care. This paper also aims to review diagnostic tests and reveal the algorithm that is used to make the diagnosis of this disease (Algorithm 1).

Tests for the diagnosis of tuberculosis infection

Inoculation with the tuberculosis mycobacterium always happens by air. Intense and extended contact with a smear-positive patient is necessary to become infected. The contagion is influenced by various factors, among which should be noted: a) the contagious patient's capacity (number of bacilli in the sputum, the intensity and frequency of coughing, and the existence of cavitation), and b) the degree of intimacy and the duration of exposure. In this sense, those who live with smear-positive patients have a higher risk of infection than people with a sporadic or casual relationship.¹

Tuberculin test

This is a test for diagnosis in countries with low economic resources, and it uses an extract obtained from the culture filtrate of tubercle bacilli, sterilized and concentrated (variant RT-23 with Tween 80 as anti-absorbent). The test is based on the fact that the mycobacterium produces a delayed immune reaction mediated by cells. 2 to 12 weeks must elapse after infection for sensitized T-lymphocytes to pass into the bloodstream and recognize the tuberculin in the epidermis.^1,2

The purified protein derivative (PPD) contains proteins that are common to Mycobacterium tuberculosis, the BCG vaccine bacillus, and some environmental mycobacteria, which lessens its specificity. The reading is done between 48 and 72 hours, but may be valid in the first seven days. This test has a sensitivity of 99% and a specificity of 95%.^1-3

The tuberculin test (TT) does not sensitize even when practiced more than once. It can even act as a stimulus in people previously infected by Mycobacterium tuberculosis, who with the passage of time have had weakened response to tuberculin. This phenomenon is known as the booster effect, and it can lead to mistaken interpretation as tuberculin conversion, which actually corresponds to the induction or restoration of response capacity.¹

TT can present false negative results in up to 25% of patients. This phenomenon is more common in the severe and disseminated forms and in various conditions that affect cellular immunity, including those found in HIV infection, malnutrition, viral infection (mumps, measles, and chicken pox), vaccination with live viruses, leukemia, lymphomas, sarcoidosis, steroid use, chronic renal failure, factors related to the tuberculin used (improper storage, improper dilution), factors related to the method of administration (subcutaneous injection or small amount of antigen administered), and factors related to the registration of the result (reader's experience).⁴

On the other hand there may be false positive results from TT in the case of environmental mycobacteria or individuals vaccinated with Bacillus Calmette-Guerin (BCG). It has been demonstrated that when a positive reaction to TT occurs secondary to BCG vaccination, this reaction remains for at least seven to 10 years after inoculation. At other times, the existence of a hematoma or abscess at the inoculation site can be interpreted as induration.^2,4

Indications for TT

TT should be used only in those whose outcome can result from therapeutic intervention (treatment of sick individuals or chemoprophylaxis of infected patients with high risk of tuberculosis); therefore, the indications of TT are limited to population groups at high risk for tuberculosis and a positivity limit of 5 mm has a positive predictive value of 99%. In countries with scarce economic resources and means, TT will only be indicated in: 1) children with symptoms suggestive of tuberculosis and contact tracing; 2) severe immune deficiencies; 3) health workers.^5,6 

A PPD reactor is defined as a person who has intradermal induration within 72 hours of applying the PPD of 5 or 10 mm depending on the risk group; this person should be treated.

Induration of 10 mm is considered positive for individuals with a moderate to high probability of infection by Mycobacterium tuberculosis, for example, having been in endemic areas within the past five years, being laboratory personnel, having medical conditions that increase probability of disease progression (including diabetes mellitus, silicosis, chronic kidney disease, lymphoma, leukemia).^2,6

In children less than five years with or without BCG, newborns, malnourished children, and immunocompromised individuals (HIV patients, transplant patients, patients receiving TNF-alpha inhibitors, those receiving drugs equivalent to 15 mg of prednisone per day for at least one month), they are considered reactors with an induration of 5 mm or more.^6,7

Due to the booster effect, in developed countries it is recommended that a second PT be practiced in people over 55 and those vaccinated with BCG. However, while less frequently, this weakness may also occur at younger ages, so it is indicated dismiss this booster in some specific groups, such as health personnel. In these cases, when the TT is negative it is advised to repeat it 7-10 days later and to take the second TT as the definitive result.^1,4 

Interferon gamma

These techniques are based on the detection of interferon gamma in blood (interferon gamma release assay [IGRA]), a key cytokine in the control of tuberculosis infection, which is released in response to in vitro stimulation of sensitized T-cells with specific M. tuberculosis antigens.¹ 

To stimulate the T-cells, use is made of antigens from genetic region RD1: early secretory antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP- 10), and the antigen from genetic region RD11: RV2654, present in M. tuberculosis complex but absent in both the BCG vaccine and most other mycobacteria (except Mycobacterium kansasii, Mycobacterium marinum, and Mycobacterium szulgai).¹

The IGRA techniques let one discriminate individuals infected with M. tuberculosis from those vaccinated by BCG and those infected by other mycobacteria, excluding those mentioned. They also incorporate controls to detect anergy and thus exclude false negatives. They can also be repeated immediately without risk of stimulating immunity, thus avoiding the booster effect. The IGRAs present additional advantages over TT, as they are objective, the determination can be repeated if necessary, the reading visit is eliminated, they are easy to standardize and apply in the laboratory, they allow the inclusion of positive controls to detect anergic patients, and they respect the individual's privacy. The main drawback of IGRAs is their greater economic cost when compared to TT.¹

There are two tests on the market: QuantiFERON-TBGold In-Tube, using ELISA techniques, and T-SPOT-TB, based on the ELISPOT technique. Both tests have operational advantages over the tuberculin and are significantly more specific in the vaccinated population.¹

The T-SPOT-TB has a sensitivity of 0.90 and specificity of 0.93, and the QuantiFERON-TBGold test has a sensitivity of 0.70 and a specificity of 0.90.³ Its use in clinical practice is still in early stages; however, some scientific societies such as in Britain, Italy, and Spain, and have already introduced them in their guidelines.¹

Diagnosis of latent tuberculosis

The diagnosis of latent tuberculosis in some risk groups is necessary to determine the need for chemoprophylaxis or anti-tuberculosis treatment.⁵

The PPD test is necessary for household contacts (five years or older) and close work or school contacts of patients with active tuberculosis. It is necessary to consider the interferon-gamma test in patients with positive PPD or those in which the PPD cannot be trusted (in those vaccinated with BCG).⁵

For immigrants to countries with high incidence of tuberculosis, children between five and 15 years can be offered the PPD test, and if this is positive, they must get the interferon gamma test. For patients 16 to 35 years of age, both the PPD test and the interferon gamma test can be offered; if these are positive, the patient should be referred to the second level to rule out the presence of active disease and consider treatment for latent tuberculosis.⁵

Diagnosis in household contacts two to five years old

In these cases, one must provide PPD for the diagnosis of latent tuberculosis; if the test is positive, one must take into account the history of BCG and send them to second level. If the PPD test is negative but the child has been in contact with a person with positive AAFB, then the interferon gamma test must be given after six weeks and the PPD repeated to increase sensitivity.⁵

Diagnosis in immunocompromised patients

Patients with HIV and CD4 count less than 200 cells/mm³ should be offered both PPD and interferon-gamma tests; if the CD4 count is between 200 and 500 cells/mm³, it is possible to offer only the interferon-gamma test or both the PPD and interferon gamma; if the result is positive, a clinical assessment must be made to exclude active tuberculosis and consider treatment for latent tuberculosis.⁵

Similarly for patients with other types of immune compromise, only the interferon gamma test or the combination of this with PPD should be offered. If the result is positive, clinical assessment must be made to determine the presence of the disease in order to initiate treatment or consider treating latent tuberculosis.⁵

Microbiological diagnosis of tuberculosis

Several tests are needed for the diagnosis of active tuberculosis, including a chest x-ray and at least three sputum samples, which must be sent initially for microscopy and culture before starting treatment, or failing this within the first seven days of starting treatment. Failing to obtain a spontaneous sputum sample and with a high suspicion of tuberculosis, sputum can be induced if this is done safely, or, otherwise, apply a bronchoscopy; morning gastric lavage can be used in the case of children.^5-7

If clinical signs and symptoms are consistent with a diagnosis of tuberculosis, treatment should be started without waiting for culture results. We must not forget that certain groups of patients with latent tuberculosis are at high risk of developing the disease. This group includes drug users, and individuals with hematologic malignancies, jejunoileal bypass, chronic renal failure, patients with hemodialysis replacement therapy with and without replacement therapy, gastrectomy, treatment with tumor necrosis factor alpha, and silicosis.^5-7

Sputum smear

The fastest, simplest, and most accessible technique for tuberculosis diagnosis is acid-alcohol-fast staining, which allows the detection of all members of the genus Mycobacterium, but to be detectable bacilli must be between 10,000 and 100,000/mL in a sputum sample of 5 to 10 mL.^1,2 This makes a variable percentage of cases of tuberculosis (between 30 and 50%) smear-negative, so a negative smear never rules out the disease. Microscopic examination of sputum for acid-alcohol-fast bacilli still shows a high sensitivity (80%) and specificity (97.5%), with a positive predictive value of 73.3 and negative of 93%.^1,2

Thus, sensitivity is high (80-90%) if one is dealing with a tuberculosis with a cavitary pattern on the chest x-ray, but it clearly decreases in tuberculosis that only has infiltrate (50-80%) and, especially, in cases that present as nodular or mass (<50%).^1,2

The most-used staining techniques are classic stains (Ziehl-Neelsen and Kinyoun) and auramine-rhodamine fluorochrome. The smear by staining with fluorochromes provides the advantage that the bacilli, when fluorescent, can be seen much better and a microscope with lower magnification can be used, allowing observation of more fields in less time. This is a time-saver that makes it cost-effective in laboratories that process more than 25 or 30 smears per technician per day.¹

Cultures

Although molecular methods are entering as part of diagnostic tools, methods based on culture are still the most valuable. The culture has a sensitivity of 80% and a specificity of over 90%, but the results take weeks and may give a false negative in a percentage ranging between 10 and 20% of cases. It is recommended to use at least two culture media (one liquid and one solid) to maximize isolation.⁷

Mycobacterium tuberculosis growth takes over 4-6 weeks for solid culture and at least 500 microorganisms/mL are needed to obtain the result of a positive culture. Because of this, 15 to 20% of cases cannot be confirmed microbiologically. Liquid medium systems significantly reduced false negative results relative to solids; they also reduced detection time 5 to 10 days.^3,4

In an attempt to resolve the main drawback of cultures, three major advances have been introduced into clinical laboratories: 1) liquid culture media; 2) biphasic culture media (MB-Septi-CheckR), and 3) techniques suitable for isolating mycobacteria from blood (blood culture).

These all have the important advantages of having a higher sensitivity than solid media and, especially, faster detection of mycobacterial growth, reducing the result by 2 to 3 weeks. However, its greatest limitations are the higher rates of contamination, difficulty in recognizing mixed cultures, and the inability to observe the colony’s morphology. There are two systems of liquid culture, radiometric (Bactec 460 TB) and non-radiometric (MGIT, ESP, MB/Bact, etc.).⁸

Nucleic acid amplification (PCR) technique

The best way to control tuberculosis requires a more accurate and rapid diagnostic test. The nucleic acid amplification test (NAAT) can give results between three and six hours; polymerase chain reaction is the most common test. There are various NAATs and each uses a different method for amplifying nucleic acid regions of the Mycobacterium tuberculosis complex.9

Several meta-analyses have evaluated the accuracy of the NAAT in pulmonary and extrapulmonary samples; most of them have reported a consistently high specificity but low sensitivity.⁹

In cases of positive smear microscopy, nucleic acid amplification techniques have a sensitivity of 90% and a specificity of 99%; on the other hand, negative smear sensitivity may be only 60%.² According to a meta-analysis, overall sensitivity and specificity were 0.85 and 0.97, respectively.⁹ 

Pleural effusion of likely tuberculous etiology

The frequency of pleural tuberculosis is highly variable and can affect up to 3.3% of tuberculosis patients. Tuberculous pleural effusion is the result of the rupture of a subpleural pulmonary caseous focus. The demonstration of bacilli in pleural fluid or biopsy of the pleura is essential to establish diagnosis. Thoracentesis can be done in secondary care by sending pleural fluid for a smear, and if diagnosis is not achieved, the patient must be sent to tertiary care for percutaneous pleural biopsy with Abrams needle to send for a culture with a sensitivity above 85%. If a diagnosis is not obtained and there is a high suspicion of tuberculosis, a thoracoscopy can be done to take a pleural biopsy, which increases the sensitivity up to 98%.¹⁰

The usefulness of various biochemical parameters in pleural fluid has been investigated, including adenosine deaminase (ADA) with a cutoff of 70 IU/L, which has a sensitivity and specificity of 95%. This study can be performed at the second-level care. However, a disadvantage of this parameter is that it can be elevated in malignant effusions (lymphomas, adenocarcinomas, and mesotheliomas), rheumatoid arthritis, parapneumonic pleural effusion, and empyemas.¹⁰

References

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2. ACCP Pulmonary Medicine Board Review: 26^th edition; 2012. DOI: 10.1378/pulm.26.0
3. Grupo de trabajo de la Guía de Práctica Clínica sobre el Diagnóstico, el Tratamiento y la Prevención de la Tuberculosis. Centro Cochrane Iberoamericano. Guía de Práctica Clínica sobre el Diagnóstico, el Tratamiento y la Prevención de la Tuberculosis. Plan de Calidad para el Sistema Nacional de Salud del Ministerio de Sanidad de Cataluña; 2009.
4. Caminero-Luna J. Guía de la Tuberculosis para Médicos Especialistas. París: Unión Internacional Contra la Tuberculosis y Enfermedades Respiratorias; 2003.
5. NICE clinical guideline 117. Tuberculosis: clinical diagnosis and management of tuberculosis, and measures for its prevention and control; 2011.
6. Secretaría de Salud. Norma Oficial Mexicana NOM-006-SSA2-1993, Para la prevención y control de la tuberculosis en la atención primaria a la salud. México: SSA; 1993.
7. World Health Organization. Treatment of tuberculosis: guidelines. 4^th ed. WHO library cataloguing Publication Data; 2011.
8. Peña J, Ferraro M, Hoffman C, Branda J. Growth Detection Failures by the Nonradiometric Bactec MGIT 960 Mycobacterial Culture System. Journal of Clinical Microbiology. 2012(6):2092-5.
9. Ling DI, Flores LL, Riley WL, Pai NN, Commercial Nucleic-Acid Amplification Tests for Diagnosis of Pulmonary Tuberculosis in Respiratory Specimens: Meta-Analysis and Meta-Regression. PLoS ONE. 2008;3(2):e1536.
10. Garrido V, Ferrer J, Hernández L, de Pablo A, Peréz E, Rodríguez F. Diagnóstico y tratamiento del derrame pleural. Arch Bronconeumol. 2006;42(7)349-72.

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