By Usman Waheed, Department of Molecular Biology, Quaid-i-Azam University, Islamabad
Tuberculosis or TB, it seems, has always been with us. Tubercular decay has been found in the spines of Egyptian mummies in British museum (3000–2400 BC) and skeletal remains from a Neolithic Settlement in the Eastern Mediterranean also showed TB in pre-historic humans (7000 BC). The study of tuberculosis dates back to Ibn Sina (Avicenna) in the 1020s. He was the first physician to identify pulmonary TB as a contagious disease and developed the method of quarantine to limit the spread of TB in his famous book “The Canon of Medicine”. In the past, tuberculosis has been called Consumption, Phthisis (Greek term for consumption) and Koch’s disease, after the German scientist Robert Koch who first isolated the tubercle bacilli in 1882 (received the Nobel Prize in 1905).
TB is still a leading killer of young adults worldwide and is highly contagious. It is estimated that TB kills 2 million people each year – on average one person every 15 seconds. Of these deaths, about 98% are in developing countries. In the last 100 years, 200 million people have died of TB. According to WHO, new infections occur at a rate of about one per second and about 9 million people develop active TB disease every year. 22 countries accounting for approximately 80% of all new TB cases each year are included in High Burden Countries (HBC) for Tuberculosis and Pakistan is ranked 8th in order of total number of TB cases per country which is very alarming. TB has significant development and economic implications. More than 75% of TB-related disease and death occurs among people between the ages of 15 and 54 – the most economically active segment of the population. That is why TB is one of the three primary diseases of poverty along with AIDS and malaria.
The treatment for tuberculosis includes combination of different antibiotics for six to eight months. It is very important to complete the course of the treatment because partial treatment for TB is worse than no treatment at all as tubercle bacilli are likely to resist anti-tuberculosis drugs in future which lead to TB drug resistance. TB drug resistance is a man-made amplification of spontaneous mutations in the genes of TB bacilli. Multidrug-resistant tuberculosis (MDR TB) refers to resistance to two or more anti-TB drugs which can be acquired, primary or natural. According to a survey conducted by WHO, up to 4% of all TB cases worldwide are resistant to more than one anti-tuberculosis drug.
TB is spread by airborne particles, called droplet nuclei (5 um in diameter and contains tubercle bacilli). A single cough can produce 3000 droplet nuclei which are enough for a person to become infected. Direct sunlight kills the bacilli in 5 minutes but it can survive in dark for longer periods. TB can infect any part of the body, but usually (70-80%) infects the lungs (pulmonary TB). Extra-pulmonary tuberculosis refers to disease outside the lungs. The disease can be; a) Latent TB (dormant) in which patient has no symptoms, no sickness, can’t spread TB to others and tubercle bacilli are present inactively; or b) Active TB where the tubercle bacilli become active, patient becomes sick, can transmit the bacilli and shows the symptoms of coughing, chest pain, loss of appetite, weight loss, night sweats, and fever. Active TB disease is fuelled by the vast reservoir of latent infection. Various factors increase the risk for progression from latent TB infection to active TB disease and HIV is the strongest risk factor for developing active TB disease among those who are infected with latent TB. This lethal combination has boosted the resurgence of tuberculosis in developed and developing world. WHO estimates 11.4 million people worldwide are infected with both Mycobacterium tuberculosis and HIV. The primary cause of death in those infected with both microbes is from TB, not AIDS.
Early detection and treatment helps stop the disease from spreading, but current diagnostic tests have many shortcomings. The most widely used test, called sputum smear microscopy (SSM) allows visual identification of the TB bacilli but misses more than half of TB cases. A more sensitive test involves growing bacterial cultures. It can spot drug resistance but may take up to 6 weeks to get results. Both tests require assessment by trained staff. The Mantoux tuberculin or PPD test is another diagnostic tool used for latent tuberculosis in non-immunized person but has certain limitations in those previously vaccinated for TB due to cross reaction with BCG. It has poor sensitivity particularly in immune-compromised groups. To overcome these problems, interferon-gamma release assays (IGRAs) are used in the diagnosis of TB particularly latent TB as SSM, culture, X-rays or even PCR cannot be helpful in latent tuberculosis diagnosis.
T-SPOT.TB is a type of IGRA used for latent and active tuberculosis diagnosis. It is an in vitro diagnostic test approved by FDA for the detection of effector T cells that respond to stimulation by M. tuberculosis antigens (ESAT-6, CFP-10) by expressing interferon (INF) gamma. It is used for diagnosis of latent and active TB disease of both pulmonary and extrapulmonary regions. The T-SPOT.TB assay is a simplified enzyme-linked immunospot (ELISPOT) method which is more sensitive than ELISA. The reason T-SPOT.TB is considered best for diagnosis of latent tuberculosis and also for patients suspected of having tuberculosis is because it is not relying on production of a reliable antibody response and the TB specific antigens (ESAT-6, CFP-10) also eliminate cross-reactivity to BCG.
For this purpose, peripheral blood mononuclear cells (PBMCs) are separated from whole blood, washed and counted before being used in the test. After PBMCs overnight incubation with the TB antigens in the wells (pre-coated with antibodies to INF-gamma), the secreted INF-gamma is released by the activated T cells. After washing, a secondary antibody (conjugated with alkaline phosphatase) is added. A substrate is added after washing, reaction is stopped, and a coloured spot of insoluble precipitate is formed at reaction site indicating individual INF-gamma-secreting T cell. By counting the number of spots, measurement of M. tuberculosis effector T cells in the blood can be made.
Over 170 studies have been conducted in various countries on the effectiveness of T-SPOT.TB. In a study done at Germany, 70 out of 72 patients with confirmed TB infection were T-SPOT.TB positive, indicating a sensitivity of 97.2% (Meier T et al 2005). A study in Tokyo, Japan indicated 98.1% specificity of T-SPOT.TB in low-risk group and for patients with TB infection; sensitivity was 89 % (Mori T et al 2004). Another study (Lee JY et al 2006) showed T-SPOT.TB sensitivity 96.6% in subjects with culture confirmed active TB and 100% in 29 of these subjects who were immunosuppressed. In Netherlands, T-SPOT.TB test ruled out active disease in 4 suspects allowing clinicians to focus on alternative diagnoses (van Leeuwen et al 2007). From Italy, Piana F et al in 2006 suggested that the T-SPOT.TB test maintains its sensitivity and performance in immunocompromised patients, identifying a large number of truly infected patients anergic to the Mantoux test. T-SPOT.TB test was positive in 94% of subjects with confirmed and probably extra-pulmonary TB (Kim SH et al 2007).
A tentative conclusion to draw from various research studies already conducted might be that the greater sensitivity and unmatched specificity of the T-SPOT.TB test makes it the world’s most accurate test to rule out latent TB and active disease in even immunocompromised populations. It is very essential to have in-country research studies to compare the two tests (PPD and T-SPOT.TB) available. We are way behind in publishing and reporting our results internationally.
Effective TB control is achieved only if latent TB can be diagnosed effectively and here T-SPOT.TB is a significant advantage over other TB detection methods, which are complex, labor-intensive, and technically challenging. It will ensure the detection of TB in a single clinic visit and perhaps begin treatment immediately. It has been used successfully across the world and also listed in CDC guidelines and National TB guidelines of many developed countries. It is likely that the higher sensitivity and specificity of T-SPOT.TB will provide for better healthcare and will make a major contribution to the control and eventual elimination of tuberculosis.
“Tuberculosis control and elimination strategies must aim at diminishing the incidence and prevalence of latent infection to reduce the pool of those with tuberculosis infection from which future cases of tuberculosis will emanate.”
(WHO)
T-SPOT.TB is a diagnostic test manufactured by Oxford Immunotec Ltd. in the UK (www.oxfordimmunotec.com) and distributed by Future scientific in Pakistan (www.futurescientific.com.pk).
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