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Tuberculosis (also referred as "TB" or "consumption"), is an airborne contagious disease caused by Mycobacterium tuberculosis or related organisms (Hecht 2011). Though curable, TB continues to be the second most common cause of death from infectious disease, following HIV/AIDS(Lawn and Zumla 2011). In 1992, the World Health Assembly (WHA) declared it as a "global public health problem"(Hecht 2011). TB also has been called a "disease of poverty" as a result of its highly prevalence in developing countries whose socioeconomic conditions lead to poverty, malnutrition, and crowded housing. In 2010, an estimated 8.8 million incident TB cases occurred worldwide, with India and China accounting for 26% and 12% of all incident TB cases respectively(Rodrigues and Vadwai 2012). This scenario is exacerbated further by an increasing prevalence of drug-resistant cases and smear-negative pulmonary and extrapulmonary TB cases, indicative of the need for highly sensitive and rapid diagnostic tools that would in turn facilitate early initiation of appropriate antitubercular treatment(Rodrigues and Vadwai 2012).
At present, diagnosis of M. tuberculosis-caused TB (MTB) is most commonly done by direct microscopy and culture methods. Acid-fast bacilli (AFB) smear microscopy plays an important role in the early diagnosis of mycobacterial infection because most mycobacteria divide and grow very slowly and culture results come out only after weeks of incubation(Rodrigues and Vadwai 2012). Bright-Field microscope (Zeihl-Neelsen staining) and Fluorescence microscope (Auramine-O staining) are used for the detection of AFB (Rahman, Munshi et al. 2011).
2 Backgrounds of Tuberculosis
TB usually occurs in the lungs and is known as pulmonary TB. Extrapulmonary TB, which occurs when tuberculosis develops outside of the lungs, may coexist with pulmonary TB. Symptoms for pulmonary TB include chest pain and a prolonged cough producing sputum(Hecht 2011). In rare cases, patients may cough up blood in small amounts and the infection may erode into the pulmonary artery, resulting in massive bleeding (Rasmussen's aneurysm)(Hecht 2011).
TB is mainly caused by Mycobacterium tuberculosis, a small, aerobic, nonmotile bacillus. M. tuberculosis divides and grows at an extremely slow rate, which is the main obstacle of early diagnosis of the disease. Another obstacle is the high lipid and mycolic acid content of its cell wall, because of which MTB either stains very weakly "Gram-positive" or does not retain dye when a Gram stain is performed(Madison 2001).
The M. tuberculosis complex (MTBC) includes four other TB causing mycobacteria: M. bovis, M. africanum, M. canetti, and M. microti(Hecht 2011). M. africanum is a major cause of tuberculosis in parts of Africa; M. bovis was largely eliminated by the introduction of pasteurized milk; M. canetti is rare and seems to be limited to the Horn of Africa; M. microti is also rare and is mostly seen in immunodeficient people, although the prevalence of this pathogen has possibly been significantly underestimated(Hecht 2011).
2.3 Pathogenesis, Transmission and Effects on Cells and Tissues
TB infection begins when the mycobacteria reach the pulmonary alveoli and invade and replicate within embosoms of alveolar macrophages(Hecht 2011). Either the upper part of the lower lobe, or the lower part of the upper lobe is the first site of infection in the lungs(Hecht 2011). Pulmonary TB may also occur via infection from the blood stream which is typically found in the top of the lung(Simon focus)(Hecht 2011). This hematogenous transmission can also spread infection to more distant sites, such as the kidneys, the brain and the bones(Hecht 2011).
The most common form of transmission of TB is by droplet nucleii created through coughing by pulmonary tuberculosis patients in a confined environment. Infected droplets remain airborne for a considerable time, and may be inhaled by susceptible persons(W.H.O 1998). As a result, scientists can identify Mycobacterium from the sputum (also called "phlegm") of TB patients using a light microscope and appropriate staining methods.
Tuberculosis is classified as one of the granulomatous inflammatory iseases. Macrophages, T lymphocytes, B lymphocytes, and fibroblasts are aggregate to form granulomas(Lawn and Zumla 2011). The granuloma prevents dissemination of the mycobacteria and provides a local environment for interaction of cells of the immune system(Lawn and Zumla 2011). Then mycobacteria inside the granuloma become dormant, causing latent infection(Lawn and Zumla 2011).
The effects of TB on cells and tissues vary widely, which are due to the organs involved. For example, destructed tissues in the lung will end up fibrosis and distortion of lung tissue followed by impairment of lung function. Destruction of tissue around the bronchi will cause bronchiectasis. If TB bacteria gain entry to the bloodstream, it will destruct blood vessel walls and cause hemorrhage as a result(Hecht 2011).
2.4 Laboratory Diagnosis
2.4.1 Microscopic Examination
Acid-fast bacilli (AFB) smear microscopy a very important method in the early diagnosis of mycobacterial tuberculosis because most mycobacteria grow slowly and culture results come out after weeks of incubation(Rodrigues and Vadwai 2012). In addition, AFB smear microscopy is fast, cheap and can be performed directly on clinical specimens. These characteristics allow the promotion of AFB smear microscopy in developing country. There are two major types, Bright-field microscopy(Ziehl-Neelsen or Kinyoun acid-fast staining) and Fluorescence microscopy(acid-fast fluorochrome staining) of microscopy used in TB diagnosis.
2.4.2 Non-microscopic Methods
Mycobacterial culture and identification of M. tuberculosis is a definitive diagnosis of TB can be obtained only by culturing clinical specimens and testing the isolates further after preliminary identification(Laszlo 1999). Traditionally, culture provides a confirmatory diagnosis of TB (Rodrigues and Vadwai 2012). Culture largely increases the sensitivity (often by 30-50%), and can detect cases earlier (often before they become infectious)(Rodrigues and Vadwai 2012). Although having higher sensitivity compared to smear microscopy, it has a long turnaround time (TAT) of approximately 4 to 8 weeks, thus resulting in further delay in initiation of appropriate antitubercular treatment(Rodrigues and Vadwai 2012).
Nucleic acid amplification test assays (NAAT)
NAAT is new way to detect small amounts of mycobacterial DNA in clinical samples using in-vitro amplification of DNA by polymerase chain reaction (PCR)(Savic, Sjobring et al. 1992). NAAT accomplished rapid diagnosis (within 1 week) and accurate identification of MTB complex directly from clinical specimens(Rodrigues and Vadwai 2012). Two major limitations in the use of these PCR assays is the high cost and the risk of cross-contamination(Rodrigues and Vadwai 2012).
3 Comparison of BF and Fluorescence Microscopy on TB Diagnosis
BF microscopy with ZN staining is the simplest method for TB diagnosis which does not require any high-level laboratory and has remained the mainstay of TB diagnosis for nearly 100 years(Kivihya-Ndugga, Cleeff et al. 2003). A number of researches had indicated that the sensitivity of BF microscopy ranges from 20% to 80%(Steingart, Henry et al. 2006). Studies had also shown that the sensitivity is significant low for the diagnosis of TB in children and does not identify smear-negative tuberculosis(Steingart, Henry et al. 2006). As a result, it is urging to explore methods to improve the sensitivity of microscopy. One method that is credited with improved sensitivity is fluorescence microscopy.
The more efficient technique of examining AFB by fluorescence microscopy on the basis of auramine staining was introduced in the mid 1940s. A systematic studies had shown that in BF microscopy, 55.3% cases were detected as AFB+ results and 44.7% cases as AFB-. On the other hand, AFB+ results were detected in by 52% and 60.7% and negative by 48% and 39.3% in conventional and LED fluorescence microscopy(Rahman, Munshi et al. 2011). This and other studies had shown that the sensitivity of fluorescence microscopy is significantly higher than that of BF with similar high specificity(Kivihya-Ndugga, Cleeff et al. 2003). In addition, a lower magnification objective (40X or 20X objective) is used to scan smears in fluorescence microscopy, thus allowing a much larger area of the smear to be seen and thus less time is taken than ZN microscopy(W.H.O 1998; Cheesbrough 2006). However, one drawback in using a low magnification is the greater probability that artifacts may be mistaken for acid-fast bacilli(W.H.O 1998).
4 Working Principles of Microscopes Used in TB Diagnosis
4.1 Bright-field microscope
The working principles of a bright-field (BF) microscope are very simple. A conventional artificial light source or reflected sunlight is used as the light source of BF microscope. The samples are stained by carbolfuchsin Ziehl-Neelsen or Kinyoun acid-fast staining.
The light path of a BF microscope consists of:
Transillumination light source, commonly a halogen lamp in the microscope stand;
Condenser lens which focuses light from the light source onto the sample;
Objective lens which collects light from the sample and magnifies the image;
Oculars and/or a camera to view the sample image(W.H.O 1998).
4.2 Fluorescence microscope
A fluorescence microscope(FM) is a kind of light microscope that uses fluorescence and phosphorescence instead of reflection and absorption of conventional light microscopes.
Fluorescence microscope requires intense and near-monochromatic illumination. Typically, fluorescence microscope uses quartz-halogen lamps, high-pressure mercury vapour lamps, lasers or high-power LEDs as light sources(W.H.O 1998).
In TB diagnosis using FM, the sputum specimens are stained with acid-fast fluorochrome staining, thus illuminated with light of a wavelength which excites fluorescence in the specimens. The fluoresced light, which is usually at a longer wavelength than the illumination, is then imaged through a microscope objective. The fluoresced light, at a longer wavelength than the illumination, is then imaged through a microscope objective. An illumination filter is used to ensures the illumination is near monochromatic and at the correct wavelength, and a second emission filter is used to ensures none of the excitation light source reaches the detector.
5 Sample Preparation for Microscopy
5.1 Specimen collection
Usually 3 sputum specimens, including 2 "spot" specimens and 1 "morning" specimen, are collected. The morning specimen is more likely to yield positive results(Laszlo 1999).
Between 2 and 5 mL of sputum should be collected every time. Specimens are received at a separate specimen delivery counter
5.2 Sputum smear preparation
The max chance of finding bacilli in unconcentrated specimens is in the solid or most dense particles part. The results of direct smear examination largely depend on the choice of these particles. The procedure for smear preparation is presented in Diagram 1.
Diagram Smear Preparation Procedures(W.H.O 1998)
5.3 Acid-Fast Staining
Mycobacteria are "acid-fast" which means they remain the primary stain after exposure to decolorising acidalcohol. A counter-stain is employed to highlight the stained organisms for easier recognition(W.H.O 1998).
5.3.1 Ziehl-Neelsen staining for Bright-field microscopy
Sulution 1: Dissolve 3.0g basic fuchsin in 100mL of 95% ethanol
Sulution 2: Dilute 5g Phenol crystals in 100mL of distilled water
Working solution: Combine 10ml of solution 1 with 90ml of solution 2 and store in an amber
bottle. Label bottle with name of reagent as well as preparation and expiry dates.
Can be stored at room temperature for six to twelve months and filter before use.
Decolourising agents: 3% acid-alcohol, 25% sulphuric acid
Counterstain: 3% Methylene blue
Procedures: Refer to Diagram 2 on Page 8.
5.3.2 Fluorochrome Staining for Fluorescence microscopy
Solution 1:10mL of Auramine O
Solution 2: 90mL of Phenol (3g Phenol crystals: 87mL Distilled water)
Mix solutions 1 and 2 and store in a tightly stoppered amber bottle away from heat and light. Store at room temperature for three months.
Diagram Ziehl-Neelsen staining Procedures(W.H.O 1998)
Decolourising solution: add 0.5mL of concentrated hydrochloric acid to 100mL of 70% ethanol
Counterstains: 5% Potassium permanganate
Procedures: Refer to Diagram 3 on Page 9.
Diagram Fluorochrome Staining Procedures (W.H.O 1998)
6 Morphological characteristics of Mycobacteria under Microscopes
M. tuberculosis is approximately 1-4 Î¼m long and appears as a non-sporing, non-capsulated slender rod(Cheesbrough 2006). Although it does not Gram stain because of its Gram positive cell wall, M. tuberculosis can be seen under microscopes using the ZN or a fluorescence staining.
When stained by ZN technique, M. tuberculosis stains red due to mycolic acids in the cell wall(Cheesbrough 2006). With fluorochrome staining(Fig 1 a), rod-shaped TB bacilli emit a bright yellow fluorescence against a pale yellow (potassium permanganate) or orange (acridine orange) background(W.H.O 1998).
Under BF microscope(Fig 1 b), ZN-stained TB bacilli appear as red rods, slightly curved, more or less granular, isolated, in pairs or in groups, standing out clearly against the blue background(W.H.O 1998).
Individual bacteria may display heavily stained areas referred to as "beads" and areas of alternating stain may produce a banded appearance(W.H.O 1998).
Fig 1 Sputum sample images of M. Tuberculosis: (a) Auramine stain TB bacilli under fluorescence microscopy(Costa, Costa et al. 2008) (b)The ZN stained TB bacilli (red) under a bright field microscope(PRASAD 2009).
Tuberculosis(TB) is a airborne disease caused by germ called Mycobacterium tuberculosis and its complex. TB affects primary the lung and can affect another sites of body.
Mycobacterium tuberculosis grows very slowly and can stay in the host of body for longtime, until the conditions of growth occur. As a result, early diagnosis is very importance for treatment and control of this disease. Methods of TB diagnosis includes direct examination of microscopy, culture identification and nucleic acid amplification test with PCR.
Numerous studies had shown that the sensitivity of fluorescence microscopy is significantly higher than that of BF microscopy. And LED fluorescence microscopy had the highest sensitivity and specificity than other microscopic methods. However, BF microscopy still has the advantage of simple procedures and low cost. The feasibility of FM microscopy in remote settings is limited because of its high cost.