Comparison of Single Sputum Smear (Home Collection) by Fluorescent Microscopy Vs Two Smears by Ziehl Neelsen Method (Routine RNTCP)

Geethalakshmi, S (2013) Comparison of Single Sputum Smear (Home Collection) by Fluorescent Microscopy Vs Two Smears by Ziehl Neelsen Method (Routine RNTCP). Masters thesis, Stanley Medical College, Chennai.


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INTRODUCTION: Tuberculosis (TB) is one of the oldest known diseases worldwide. This disease has been known with different names, since ancient times such as Phthisis, Consumption, Scrofula, White Plague, etc. It is an important public health problem in India. In 1993, World Health Organization (WHO) announced it as an international emergency. As per the WHO Global TB Report 2011, in total there were 9 lakh cases of TB worldwide in 2010. Inspite of India ranking the second country in the world in terms of population , India has more number of new TB patients every year than any other country in the world. Almost 20% of the global incident TB cases are from India. Every year approximately 2 million people acquire TB in India, out of which around 9 lakh are infectious. Every year around 3 lakh Indians die due to TB. Tuberculosis is a chronic infectious disease caused by mycobacterium tuberculosis, a highly obligate aerobe transmitted by droplet infection. The lungs are commonly affected, though any organ in the body can be affected. The common clinical features include cough with expectoration, evening rise of temperature, chest pain, weight loss, haemoptysis and night sweats. The risk factors increasing the development of TB disease are HIV infection, Diabetes mellitus, overcrowding, smoking, etc. Tuberculosis is a potentially curable disease, provided it is diagnosed early. The cornerstone of success of Revised National Tuberculosis Control Programme is case detection as enabled by sputum smear microscopy. It is routinely done by Ziehl Neelsen (ZN) technique used for detecting acid fast bacilli (AFB). Microscopy is a very useful diagnostic tool in highly endemic countries like India. Microscopy can also be done in other pathological materials like lymph node aspirates and body fluids including Cerebro Spinal Fluid. Sputum microscopy is also useful to assess whether the patient is responding to treatment, and to establish whether the patient is cured or to detect treatment failure when the treatment is completed. Although inexpensive and highly specific, it has low sensitivity (40-60%) when compared to culture and is also time consuming. The sensitivity of conventional light microscopy is affected by many factors, such as the prevalence of the disease, its severity, the kind of specimen used for diagnosis, the quality of the specimen collected, the number of mycobacteria in the specimen, the processing method that is used (direct or concentrated), the method of centrifuging the specimen, the staining technique, and the quality of the examination. So, when microscopy is done accurately, a lot of time is consumed. The Ziehl Neelsen (ZN) method used at present is a significant modification of Robert Koch’s original method using alkaline methylene blue. Fluorescent microscopy has been used for detection of acid-fast bacilli since Auramine O based method was introduced by Hagemann and Richard et al. It is easy to identify fluorescent bacilli against a dark background. Thus using the fluorescent microscopy (auramine /rhodamine), the examiner can go through the slide at a lesser magnification and observe a large area than that is seen with Ziehl Neelsen -stained smears. The above factors decrease the time required for screening a slide and lead to an increase in sensitivity. Thus, it is widely accepted that the fluorochrome method must be preferred over the Ziehl Neelsen (ZN) method. There are several reports indicating that fluorescent microscopy (either auramine phenol or auramine rhodamine) of smears significantly increases the sensitivity of direct microscopy. Although many researchers have investigated the significant difference in sensitivity between ZN and FM staining methods, the technical and procedural factors can influence the sensitivity of each staining method. At present, in India under Revised National Tuberculosis Control programme (RNTCP), the use of fluorescence microscopy is linked to the culture and Drug Sensitivity Testing (DST) activities at the level of the Intermediate Reference Laboratories (IRL). In this study, the sensitivity, specificity, efficacy and other advantages of the conventional gold standard ZN method and Flourescent microscopy are compared with each other in the detection of acid fast bacilli in sputum and the results analysed. AIMS AND OBJECTIVES: 1. To assess the value of fluorescence microscopy in diagnosing sputum smear positivity among chest symptomatics when compared to conventional light microscopy by Ziehl Neelsen method for diagnosing cases of pulmonary tuberculosis. 2. To assess the value of fluorescent microscopy in picking up additional smear positive cases among smear negative chest symptomatics. 3. Whether the objectives one and two can be achieved with the single home collection sample compared with routine two smear (Ziehl Neelsen) RNTCP smear microscopy. 4. Age and sex distribution of patients with smear positive pulmonary tuberculosis 5. Additional parameters are also compared such as the incidence of smear positive pulmonary tuberculosis in a. Diabetics, b. Smokers. MATERIALS AND METHODS: This study was conducted at Govt. Stanley Hospital. For this study, chest symptomatics referred from various departments to RNTCP cell of Govt. Stanley medical college for sputum AFB smear microscopy were selected. Patients were selected based on their history and physical examination. All the patients were enrolled in the study after fulfillment of inclusion and exclusion Criteria and getting appropriate consent. Study Period : 6 months from April 2012 to September 2012 The patients were advised routine two sputum smear samples for (Ziehl Neelsen) microscopy as per RNTCP guidelines. From the home collection specimen, two smears were prepared, one for Ziehl Neelsen microscopy and the other for fluorescence microscopy. The technicians were not allowed to cross check the results to avoid bias. Standard stastical methods were used to assess the correlation between the variables. Inclusion Criteria: All chest symptomatics referred for sputum smear Acid Fast Bacilli as per RNTCP criteria 1. Chronic cough >2weeks. 2. Evening rise of temperature. 3. Unexplained weight loss. Exclusion Criteria: 1. ATT treatment failure patients. 2. Relapse of pulmonary tuberculosis. 3. ATT defaulters. 4. Age < 13 years. CONCLUSION: This study found that fluorescent microscpy is more sensitive than Ziehl Neelsen method in identifying acid fast bacilli.The fluorescent method was relatively easy to perform and can be done quickly. The mycobacteria could be detected even under lower magnification. Because of this, many fields could be screened in a short time span. The percentage of false negatives was higher when Ziehl Neelsen method was used. Also Fluorescent Microscopy had a greater sensitivity when single home collection specimen was used when compared with Ziehl Neelsen method done in 2 samples as per routine RNTCP. Hence Fluorescent Microscopy fares as a better screening test when compared with Ziehl Neelsen method. Hence these two approaches, a) Collecting a single early morning sample and b) Screening it using Fluorescent Microscopy could result in rapid as well as accurate diagnosis of Pulmonary Tuberculosis. The major limiting factor in using a fluorescent microscope is installation and maintenance problems associated with the equipment. The cost of the equipment and also the fluorochrome dyes are also high. To overcome all this problems, LED Fluorescent Microscopes have been introduced. There are several studies demonstrating a higher sensitivity while using LED Fluorescent Microscopes when compared to the conventional fluorescent and light microscopy. It is also found that fluorescent microscopy (45%) has a higher sensitivity than the conventional microscopy (29%) in patients co infected with HIV and TB, as fluorescent microscopy identifies more number of pauci bacillary cases , as HIV patients are more likely to be paucibacillary. Fluorescent microscopy is a rapid, useful and a sensitive tool for screening various specimens for mycobacterium tuberculosis. At present it is used only at the level of Intermediate Reference Laboratories. With the advent of LED fluorescent microscopes and demonstration of its superiority over the other techniques in terms of cost effectiveness as well as sensitivity, it should replace the conventional light microscopes in all DOTS centers.

Item Type: Thesis (Masters)
Uncontrolled Keywords: Single Sputum Smear (Home Collection) ; Fluorescent Microscopy ; Two Smears by Ziehl Neelsen Method (Routine RNTCP).
Subjects: MEDICAL > General Medicine
Depositing User: Subramani R
Date Deposited: 25 Mar 2018 08:18
Last Modified: 28 Mar 2018 02:08

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