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Role of modified rapid AFB method in histopathological sections of hansen's disease
S V Nayak
H No. 4, "Prubhu Nilaya", 3rd Cross, Subbanna Gerden, Vijaynagar, Banglore - 560 040, Karnataka
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Nayak S V, Shivarudrappa A S, Nagarajappa A H, Sacchidanand S, Ahmed S M. Role of modified rapid AFB method in histopathological sections of hansen's disease. Indian J Dermatol Venereol Leprol 2003;69:173-174
AbstractLeprosy is a chronic debilitating disease. A reliable diagnosis hinges around a good histopothological diagnosis and demonstration of the bacilli in the histopathological section. The usual method performed Modified Fite Faraco Method is time consuming, laborious and less sensitive. It has been our endeavor to devise a more rapid and sensitive method for the diagnosis and bacillary load detection in histopathological sections. The Modified Rapid AFB devised by us is sensitive and time saving which is essential for the pathologist and for the treatment by the Dermatologist. We hove studied about 53 cases of different types of Hansens disease and compared with both Modified Fite Faraco method and Modified Rapid AFB method. The results were found to be very encouraging with the Modified Rapid AFB method.
Modifed Fite Faraco is the oldest method used to detect Mycobacterium leprae in tissues., Pararosanaline stain has been used in some studies to detect Mycobacterium leprae and was found to be sensitive in few studies., It was our endeavor to devise a new method to increase the sensitivity of detecting Mycobacterium leprae in tissue sections by using pararosanaline stain and periodic acid especially for paucibacillary cases. We describe our experience with Modified Fite Faraco and Modified Rapid AFB Method in histopathological sections.
Materials and Methods
Fifty-three leprosy cases attending Victoria Hospital and Bowring and Lady Curzon Hospital from Mar 2001 to Sept 2002, Bangalore were involved in the study. Punch biopsies were taken from the active lesion and classified according to clinical features, slit-skin smear study, histopathological features and Modified Fite Faraco method into: Indeterminate leprosy, tuberculoid leprosy, borderline tuberculoid leprosy, mid borderline leprosy, borderline lepromatous leprosy and lepromatous leprosy.
The biopsies were routinely processed. Haematoxylin eosin stained sections were studied. Ribbons containing 10 serial sections, 5 pm thick were taken, 5 each for Modified Fite Faraco method and 5 for Modified Rapid AFB method.
Modified Rapid AFB method
Ribbons containing 5 serial sections, 5 Nm thick were taken on a clean glass slide with egg albumin.
1. Deparaffinization was done by 1:3 peanut oil Xylene.
2. Blot dry
3. Dip slide in 10% periodic acid for 30 minutes.
4. Dip it in water for 2-3 times
5. Blot dry
6. Flood the slide with pararosanaline stain and keep it in the incubator at 70 Degrees for 8-10 minutes.
7. Wash in running tap water.
8. Blot dry.
9. Decolorise with 1 % hydrocloric acid in 70% ethanol until faint pink.
10. Wash it in running tap water.
11. Blot dry.
12. Counterstain with 0.1% malachite green or 1 methylene blue for 30 seconds to 1 minute
13. Wash it in running tap water.
14. Blot dry.
15. Dehydrate by dipping in absolute alcohol (1-2 dips).
16. Blot dry immediately.
17. Dip in xylene and mount in DPX immediately.
The slides were then screened with 1 Ox, 40x and 100x objective and findings noted.
Modified Fite Faraco method
Ribbons containing 5 serial sections were taken on a clean glass slide with egg albumin and stained conventionally, and the findings noted.
The conventional Fite Faraco method is the most commonest method used for tissue sections in detecting Mycobacterium leprae and has stood the test of time. In our study the combination of periodic acid and pararosanaline stain was found to be very effective. The sensitivity with Modified Rapid AFB method was found to be 54.7%.
The com-bination of pe-riodic acid and pararosanaline stain can be used as a supplement or can be used per se in paucibacillary cases.
We faced few technical problems during this procedure such as sections getting detached from the slide while staining. This was overcome by hav-ing thin sections.
The sections should be absolutely thin and complete deparaffinisation of the section should be done otherwise it effects the process of decolorisation.
Clinical correlation was carried out on all cases. Out of 54 cases diagnosed histopathologicaly, in 39 cases clinical and histopathological correlation was established. The remaining 14 cases mostly belonging to paucibacillary type, no histopathological or clinical correlation could be established. It is in these cases that modified rapid AFB method plays a role in typing of leprosy.
The coincidence of classification by histopathological and clinicial examination as percentage for each mode is shown in [Table - 2]. Maximum clinical correlation was seen in lepromatous leprosy (100%) and midborderline (100%) were as clinical cor-relation was found to be lacking in border-line tuberculoid leprosy (76.92%), tubercu-loid leprosy (66.66%). The clinical correla-tion was least in indeterminate leprosy (63.64%). The overall coincidence of di-agnosis of classification was seen in 39 cases (73.55%) study.
We agree with Prakash Kumar et al that maxium clinical correlation can be established in multibacillary type of leprosy than in paucibacillary type of leprosy.
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