Mycobacterium Tuberculosis in New Biologic Era

Mycobacterium tuberculosis in new Biologic Era.

Claudia Diniz Lopes Marques, Angela Luzia Branco Pinto Duarte, Fernando de Souza Cavalcanti.

This chapter places tuberculosis and latent tuberculosis infection (LTBI) in a world context; recounts the origins of the two main tests for detecting LTBI and ant-TNF; evaluates their effectiveness as seen today bearing in mind the incidence of active TB in any given country; and makes proposals for their optimum use including when IGRAs might be used most appropriately.

INTRODUCTION

In 2010 there were 8.8 million new cases of tuberculosis (TB) in the world (range, 8.5 to 9.2 million), 1.1 million deaths among people not infected with virus HIV (range, 0.9 to 1.2 million), and 350,000 deaths (range, 0.32–0.39 million) from HIV-associated TB. Most of these cases (80%) are in developing and emerging countries, where TB is considered a major public health problem. In Brazil, a total of 70.997 new cases were reported in 2010, and the annual incidence reported was 43 cases per 100,000 populations. It is estimated that 50 million Brazilians are infected with the tuberculosis bacillus (1). In this high burden countries, TB is more common in men, from 15 to 54 years old, persons deprived of liberty (prisoners), the homeless, the poorest and the least educated. On the other hand, it is the elderly, ethnic minorities and immigrants who are the most affected in First World countries (2). Although, the priority in disease control is to treat infected people, identifying and treating latent tuberculosis infection (LTBI) is considered essential to prevent the spread of the disease, with a significantly higher percentage of available resources in First World countries being expended on prevention in comparison to treatment (3).

In most people, infection with M. Tuberculosis is initially contained by host defenses, and the infection becomes latent. However, LTBI has the potential to become an active infection at any time, and thus a source of infection (4). About a third of the world population is carriers of LTBI, and in immunocompetent patients, active infection only occurs in 10% of cases (1).

The use of inhibitors of tumor necrosis factor (anti-TNF) for treating inflammatory joint diseases, particularly rheumatoid arthritis (RA), was approved little more than ten years ago. Soon after they began to be used in clinical practice, several cases of active lung and extra-pulmonary TB were reported, even in countries with a low prevalence of TB, which led to creating mandatory screening for LTBI before initiating treatment with anti-TNFs in all countries (5, 6).

DIAGNOSIS OF LATENT TUBERCULOSIS INFECTION

The purified protein derivative (PPD) test is the test of choice for diagnosing LTBI, but it has limited sensitivity and specificity, especially in countries with a high incidence and prevalence of TB and high immunization coverage (7, 8). Furthermore, patients with autoimmune diseases such as RA have a low frequency of positive skin test compared to patients who do not (9). RA patients do not produce an adequate response to PPD, even in those infected with M. tuberculosis, which makes PPD unsuitable for detecting LTBI (10, 11). Therefore, tests using interferon-gamma (INF-γ) release assays (IGRAs) have been carried out with a view to their use in assisting to diagnose LTBI (12-15).

The best strategy for increasing the accuracy of diagnosis accuracy would be to submit RA patients to both PPD and IGRA tests prior to using anti-TNF (16, 17). This seems to be a reasonable strategy in countries with a low prevalence of TB. However, in high prevalence countries, what would be the role of these screening tests for LTBI? Given their high cost and the difficulty of accessing them in emerging and developing countries, this issue should be discussed in-depth by a wide range of the experts involved, with a view to drawing up specific guidelines for health authorities in these countries. Diagnosis of latent tuberculosis infection in Brazil is made when the PPD is positive but excluding active TB (the bacteriological analysis of induced sputum is negative) and there being no clinical or radiographic evidence of active tuberculosis (18).

In an LTBI patient, there are a small number of viable dormant bacilli that are contained within the granulomas, and the rate of replication of bacilli is slow. Therefore, active infection does not occur. However, when it does, morbidity and mortality are quite high (19). Almost 1.7 billion people, a third of the world’s population, are infected with M. tuberculosis (20). Of this total, 80% of cases are in countries where the ​​prevalence of TB is high. However, First World countries are not shielded from the TB epidemic that is expanding in developing countries. This is demonstrated by the fact that 50% of new TB cases in the United States occur in people who areforeign-bornresidents (21). In countries with a high ​​prevalence of TB, the cases of LTBI are even higher. In Brazil, a country that is among the 22 with the highest prevalence and incidence of TB, LTBI is mainly associated with males and the incidence varies depending on the city where the study was conducted (22).

Currently two tests are used in clinical practice which are designed to measure the adaptive immune response mediated by cells in asymptomatic hosts exposed to M. tuberculosis, namely the tuberculin skin test (PPD) and assays based on the release of interferon-gamma (or Interferon Gamma Release Assays - IGRAs) (25).

Among groups such as prisoners and health professionals, who studies show to have to have greater contact with people with infectious tuberculosis, the prevalence of LTBI increases considerably, and ranges between 47 and 73% (23, 24). Therefore people in such groups should be tested for LTBI even when they show no signs or do not report symptoms of infection, if the patient belongs to any one of the groups listed in Table 1 below (7, 25, 26, and 27).

Table 1: People with increased risk of tuberculosis infection that should be tested for LTBI (25)

Those who are at increased risk of exposure to cases of active disease / ·  Contact intra-household with TB cases
·  Health workers who deal directly with infected patients
Those who are at increased risk of tuberculosis infection / ·  Foreign-born residents from countries with a high incidence of TB
·  The homeless
·  People who lives or work in a crowded place
Those who are at increased risk of active disease once infection has occurred / ·  HIV infection
·  Those who have been recently infected with TB*
·  Drug abusers
·  Chronic kidney disease patients
·  Silicosis
·  Diabetes mellitus
·  Lymphoproliferative disease
·  Malnutrition
·  Patients receiving immunosuppressive therapy
·  Patients who have undergone ileojejunal intestinal bypass surgery or gastrectomy
* Recently-infected people include children under four years of age and those whose tuberculin test conversion shows an increase in induration of at least 10mm over a period of 2 years

TUBERCULIN SKIN TEST

Until 2001, PPD was the only method available for diagnosing LTBI (28, 29). The tuberculin test was first described by Robert Koch in 1891 as a preparation that used cultures of M. tuberculosis injected subcutaneously. In 1908, Charles Mantoux, a French physician who worked with Koch presented his first study of intradermal injections to the French Academy of Sciences, entitled “Intradermo-reaction de la tuberculine”. From this date on the tuberculin skin test (TST) replaced the subcutaneous one and was called the Mantoux test (30).

The TST, which uses the standard preparation of Purified Protein Derivative (PPD), has been used since 1930 to determine who is infected with M. Tuberculosis (Figure 1.A). It contains a mixture of antigens that induce a delayed hypersensitivity reaction and reflects cellular immunity directed against the bacillus and, despite its known limitations with regard to sensitivity and specificity, is still used as a criterion gold standard for diagnosing LTBI. Although occasionally used in the diagnosis of symptomatic infection, its main use remains that of detecting LTBI (31). However, the practice of conducting a screening test for TB using PPD is somewhat disappointing due to the low specificity, since both vaccination with BCG, and exposure to tuberculous mycobacteria do not produce a response similar to that induced by infection by M. tuberculosis (32).

The measurement of reaction to PPD is used to classify individuals according to the probability of whether or not they are M tuberculosis positive. Among its limitations is that, as measured on a continuous scale, there is no inherent value to indicate the presence or absence of infection (Figure 1.B). Thus, criteria were established using a cut-off point above which the PPD is considered positive (4, 33) (Table 2). In patients with inflammatory rheumatic diseases, the PPD is considered positive when its value is above 5 mm. using this cut-off, rather than 10 mm for positive testing minimizes interference in the treatment of RA and suppresses hypersensitivity (34).

Table 2 – Cut-off point to evaluate the PPD result
Induration / Reading / Clinical situation
0-4 mm / Non-reactive / Uninfected by M. Tuberculosis/reduced hypersensitivity.
5-9 mm / Weak reactor / Individual vaccinated with BCG/ infected with M. tuberculosis and other mycobacteria
10mm or more / Strong reactor / Infected with M. tuberculosis, may or may not be ill, and individuals vaccinated with BCG in the past two years

A major complication in diagnosing LTBI in RA patients is the cellular immune function abnormality observed in these patients (35). There is a reduction in the responsiveness of peripheral mononuclear cells, leading to a loss for delayed hypersensitivity to the recognition of antigens, such as PPD (35, 36). The mechanism for this change is not exactly known, but the change has been shown to be cause by a deficiency of IL-2 (10) or chronic exposure to Tumor Necrosis Factor (TNF) (37).

The regulatory T cells (Treg), which play a key role in preventing auto-immunity, have a reduced number and function in RA (11) and as demonstrated by Martin et al (38), the number of T cells is directly related to the magnitude of the response to PPD. The Mantoux test evaluates the in vivo cellular immune response against the PPD of M. tuberculosis, resulting in a classical reaction of delayed hypersensitivity, T cells migration dependent, thus producing INFγ to the site of injection of the antigen (Figure 2). RA patients are unable to produce an adequate response to PPD, even in individuals infected with M. tuberculosis, thus making it unsuitable as a test that recognizes latent forms in these patients (10). Some experts also recommend that RA patients who are PPD negative, but are at great clinical or epidemiological risk with regard to TB infection should be empirically treated for LTBI before initiating therapy with a biological drug (31).

Several studies show that the frequency of PPD positivity in RA patients is lower than in that of the general population. These studies were conducted not only in countries with a high prevalence of TB, such as Peru, Brazil and Turkey, but also in countries with a low prevalence of the disease, such as Italy. Table 3 summarizes the main results of some of these studies.

Table 3 - Main results of studies conducted to assess the frequency of positivity in RA patients
Country / Population / Results / Reference
Kuwait / RA patients compared with those active tuberculosis infection and healthy
patients / Responsiveness to skin test
was lower in the RA group
associated with the DR7 haplotype / 39
Peru / RA patients compared with immunocompetents ones matched by age and sex / PPD positive 71% in immunocompetent group versus 29% in RA patients / 9
Brazil / RA compared with immunocompetent patients / Stronger PPD positivity in the control group (33.3%) than in the RA group (14.6%). / 40
Turkish / RA patients compared with those with ankylosing spondylitis, gout and osteoarthritis / Low PPD positivity in RA (29.8%) compared with ankylosing spondylitis (65.9%), gouty arthritis (68.8%) and osteoarthritis (63%). / 41
Italy / Patients with chronic inflammatory diseases / Radiographic changes compatible with LTBI, but PPD was positive in only 8m7% / 26
Brazil / Patients undergoing treatment with infliximab, compared with patients with chronic illness in research for LTBI / PPD positivity in only 8.7% in RA patients, while in the group with chronic diseases was 22%. / 26

INTERFERON-GAMMA RELEASE ASSAYS (IGRAS)

The identification of regions of the genome of M. Tuberculosis that are absent in BCG and other environmental mycobacteria enabled new diagnostic tools for LTBI to be developed, which did not have the specific problems presented by the PPD. The RD1 region, present in various species of mycobacteria, is deleted primarily during the processing of Mycobacterium bovis (M. bovis) to BCG, and hence so are the genes it decodes. The RD1 comprises the genes Rv3879c Rv3871, which includes genes for the early-secreted antigen target of 6 kDa (ESAT-6) and protein L45 homologous to culture-filtered protein 10 (CFP-10). Both proteins encoded by these regions, ESAT-6 and CFP-10, are used as specific antigens due to their absence in the BCG vaccine and other mycobacteria. These induce a strong immune response of T cells in experimental models, leading to the production of interferon gamma (IFN-γ), which is measured by the Interferon Gamma Release Assay (IGRA) test (33, 43-45). Early versions of these tests (QuantiFERON ®-TB - Cellestis Limited, Carnegie, Australia), approved by the US Food and Drug Administration (FDA) in 2001, used PPD as an antigen stimulation, which led to the same problems seen in specificity skin tests. Therefore, this test has been replaced by another, Quantiferon-TB-Gold ®, which uses ESAT-6 and CFP-10 instead of PPD. The Quantiferon-TBGold whole blood test is used to quantify the presence of IFN-γ using the technique of (44) ELISA (Enzyme-Linked-Immnosorbent Assay). The T-SPOT.TB ® (Immunotec Oxford, Oxford, England) uses an ELISPOT assay (enzyme-linked immunospot-assay) with peripheral blood mononuclear cells that produce IFN-γ in response to stimulation with ESAT-6 and-CFP 10 (44).