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Tuberculosis (TB) is a infectious disease and causes a global health problem as it is one of the leading agent of death from a single infectious agent after human immunodeficiency virus. It is one of the leading cause of mortality in India, killing two persons every three minutes, almost thousand every day. This infectious disease most frequently infects the lung, and the condition is known as Pulmonary Tuberculosis (PTB). The symptoms of PTB patients are fever, loss of appetite, weight loss, chest pain or dyspnea (TB report, 2019; Bhalla et al., 2015).
TB is caused by Mycobacterium tuberculosis which is contagious and spreads through air. Throughout the world, examination of sputum smear stains by microscopy has remained the cornerstone for PTB diagnosis and screening (WHO Tuberculosis Fact, 2007). Manual screening is labor intensive and has a high false negative rate. The manual procedure is time consuming, inaccurate and inefficient process. It takes about 40 minutes to even 3 hours to analyze a slide which atmost depends on the level of infection (Sotaquira et al., 2009). In low and middle-income countries to assure early detection of the disease TB diagnosis is centered on the microscope, the use of which is fast, cheap and a repeatable method.
Different test modalities prevail to diagnose PTB, but microscopic examination of sputum smears remains the most widely used investigation in clinical practice, especially in developing and countries with high prevalence of TB. Many underdeveloped countries depend on sputum smear microscopy to diagnose tuberculosis since it is fast, inexpensive, and with appropriate training guarantee concise results. Direct sputum smear is still the first-choice tool for screening of tuberculosis worldwide. Sputum smear microscopy represents one of the five pillars for tuberculosis control in the directly observed treatment short course strategy (Díaz-Huerta et al., 2019; Rao, 2009; Coronel et al., 2019).
The recent guidelines for diagnosis of TB are primarily based on the demonstration of acid-fast bacilli on sputum smear microscopy. Among the diagnostic techniques of TB, culture of Mycobacterium tuberculosis is the gold standard. However, culture is a slow process and requires specialised laboratories with high skilled technician. It takes about six to eight weeks to declare the severity of the disease. Serological tests are currently not reliable enough for the diagnosis of tuberculosis (Javed et al., 2015; Goyal and Kumar, 2013). Chest radiograph finds a conclusive result only at a later stage of the disease. Though computed tomography is frequently used in the diagnosis and follow-up of TB, it is not advised in the national and international guidelines. The use of ultrasound and magnetic resonance imaging is lacking in the literature for TB patients. Thus India being a large burden of TB, it is important to establish imaging criteria and recommendations for the diagnosis (WHO Tuberculosis Fact, 2007).
The International Union Against Tuberculosis and Lung Disease and World Health Organization specify that the essential step in the investigation of patients who are suspected of having pulmonary tuberculosis is the microscopic examination of their sputum samples. Thus sputum smear microscopy has been an integral part of global strategy for the control of TB (Desikan, 2013).
Microscopic examination of sputum smears include two staining procedures such as Ziehl-Neelson (ZN) and auramine stained specimens. ZN is the most extensively used procedure for identifiying Mycobacterium tuberculosis in smear. The ZN staining procedure require basic fuchsin, phenol, absolute alcohol, sulphuric acid and methylene blue. Oil immersion objective of microscopy reveals Mycobacterium as red bacilli. ZN stain method is less sensitive relative to fluorescent stain as it takes more time to scan the view fields under the microscope (Javed et al., 2015).