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a). Brief resume of the intended work: Need for the study: Leprosy is widely prevalent in India. As on 31st March 2006, the prevalence rate was 0.84 per 10,000 population.1 Leprosy causes physical disabilities which causes social stigma.2 Early detection of leprosy is important to avoid deformities which is of obvious importance in a country like India where the majority of cases are of paucibacillary type.3 The diagnosis is confirmed by demonstration of Mycobacterium leprae (M.leprae) in tissue sections taken from the lesion.4 Ziehl Neelsen (ZN) technique which had been traditionally used is now replaced by modified Fite-Faraco method as lepra bacilli are not as acid fast as tubercle bacilli. As this method is tidious and less sensitive 4, 5, there is need for simple yet sensitive procedure. Auramine staining is clearly shown to be superior to conventional Ziehl-Neelsen staining both in sensitivity and quality of staining.7 Also in paucibacillary cases it can be used as a supplementary tool when modified Fite- Faraco method fail to detect the bacilli in tissue sections.8 Hence the present study will be done to evaluate the diagnostic utility of fluorescent microscopy in the diagnosis of leprosy in tissue sections of clinically diagnosed leprosy patients in this institute. Review of literature: 6 Kaiserling in 1917 was the first to observe spontaneous whitish – blue fluorescence of a suspension of tubercle bacilli. Bachmann and Finke in 1939 were the first to apply fluorescence microscopy for the detection of acid fast bacilli in tissue sections. Jariwala and Kelkar 9 observed that fluorescence microscopy was superior to modified Fite – Faraco method for detecting acid fast microorganisms in paraffin sections of cases of leprosy by studying 50 cases of leprosy. 22 biopsies showed acid fast organisms in fluorescence microscopy and 20 in the fite – faraco method. The ease and speed of fluorescence microscopy appeared to be a great advantage. 8 Bhatia et al in 1987 at Chengalpattu, Tamilnadu carried out their study on 65 skin biopsies taken from treated leprosy patients which were negative by modified Fite – Faraco. They found that 64 cases were positive for M.leprae by fluorescence microscopy . Sunil Nayak et al 4 studied 56 skin biopsies from patients having leprosy, particularly the paucibacillary type and compared the sensitivity of fluorescent method with that of the modified Fite – Faraco method and found that the positivity rate with the former was 52% as compared to 20% with latter for indeterminate type of leprosy. In 1988, Bhatia et al 7 screened 84 smears of which 75 were positive for M.leprae with the auramine staining and only 57 smears positive by Ziehl-Neelsen staining. Also they showed that Bacteriological Index in smears stained by Ziehl-Neelsen method was lower than in auramine staining in 31 smears. They also proved that interobserver variation was also less with auramine stain. Further they opined that the procedure may be valuable in cases where negativity of smears is to be certified. Objectives of the study: 1. To compare the efficacy of auramine rhodamine with Ziehl- Neelsen and modified Fite-Faraco staining in the diagnosis of leprosy in tissue sections. 2. To assess the role of auramine rhodamine staining method in grading of Hansen’s disease. b). Materials and methods: Source of data: Skin biopsies from patients clinically diagnosed as leprosy received in the department of pathology, B.L.D.E.A’s Shri B.M.Patil Medical College Hospital and Research Centre, Bijapur from August-2005 to July-2009. Method of collection of data: All cases clinically diagnosed as leprosy received in the department of pathology from August-2005 to July- 2009 will be studied. Informed consent will be taken from all study patients. The area to be biopsied will be cleaned with spirit and a punch biopsy will be taken from the active edge using 3mm disposable punch after infiltrating the area with 2% xylocaine. The biopsied area will be tightly packed and dressed. The specimen will be fixed in 10% formalin. Paraffin blocks will be made and sections will be cut at 4- 5µ thickness. The slides stained by Haematoxylin & eosin,Ziehl-Neelsen & modified Fite – Faraco stains will be observed under light microscope and those stained by auramine rhodamine stain will be observed under fluorescent microscope . Sample size: Based on the study by Nayak SV et al, lepra bacilli were seen in 66% by the fluorescent method and 42% by the modified Fite-Faraco method. So Po=66% and P1 = 42%. n=2(Zα + Zβ)2 p (1-p) (P0-P1)2 Therefore the calculated sample size, n=57 approx. 60. Cases taken from August-2005 to July-2009 will be included in the study to arrive at the expected sample size. This includes two years retrospective and two years of prospective study. Statistical analysis: Statistical data will be shown by tabulations and diagrammatic presentation wherever necessary. Sensitivity, specificity and positive predictive value will be analyzed. Data collected will be analyzed using chi-square test. Inclusion criteria: All cases clinically diagnosed as leprosy will be included in the study. Exclusion criteria: Does the study require any investigation or intervention to be conducted on patients or other humans or animals? If so describe briefly. This study requires skin biopsy to be taken from patients clinically diagnosed as leprosy for which informed consent will be taken from the patient. Has ethical clearance been obtained from your institution ? Yes. Ethical clearance has been obtained on 13/11/2007 from our institution. c). List of references: 1. Park K.Park’s text book of preventive and social medicine. 19th ed. Jabalpur: M/S Banarasidas Bhanot; 2007. p. 264-278. 2. Ganapati R, Pai VV, Kinsley S. Disability prevention and management in leprosy : a field experience. Ind J Dermatol Venerol Lepr 2003;63:369-74. 3. Ramu G, Kartikeyan S, Balakrishnan S. Histopathological and immunological correlation of suspected leprosy patients. Indian J Lepr 1986;68:153. 4. Nayak SV, Shivarudrappa AS, Mukkamil AS. Role of fluorescent microscopy in detecting Mycobacterium leprae in tissue section. Annals of Diagnostic Pathology 2003 April; 7(2): 78-81. 5. Culling CFA, Allison RT, Barr WT, editors. Cellular pathology technique. 4th ed. London: Butterworth and Co.Ltd; 1985.p.331-347. 6. Klaus WF, Teng KP. Demonstration of acid-fast bacilli in tissue sections by fluorescence microscopy. Canadian Medical Association Journal 1962 Oct 20;87(16):837-841. 7. Bhatia VN, Cherial E, Harikhrishna S. Auramine staining in detecting small number of bacilli in skin smears. Indian J Lepr 1988 Jan;60(1):13-16. 8. Bhatia VN, Rao S, Saraswathi G. Auramine staining in histopathology sections. Indian J Lepr 1987;59:386- 388. 9. Jariwala HJ, Kelkar SS. Fluorescent microscopy for detection of M.leprae in tissue sections. Int J Lepr 1979;47:33-36.
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