The present cross-sectional study was carried out on patients clinically diagnosed with Leprosy, attending dermatology OPD in AVBRH, Sawangi, Wardha, India in a period of two years from September 2017 to August 2019, after obtaining Institutional Ethics Committee clearance {DMIMS (DU) IEC /2017-18/6710}.

Sample size formula for studies with binary test outcome was used.

n=Z²_α/2 p(1-p)/d²

where, ‘p’ is predetermined value of sensitivity or specificity as ascertained by previously published data and for α=0.05 Z_α/2 is inserted by 1.96.

Total 88 patients who gave informed consent to participate in study were enrolled and their clinical features were recorded including number, size and location of lesions and loss of sensation. All cases diagnosed clinically as leprosy were enrolled in the study whereas patients not willing to participate in the study or investigate the disease were excluded from the study. Any patients with cutaneous manifestations of leprosy or peripheral nerve trunk enlargement were noted in enrolled patients and were further classified according to the Ridley DS and Jopling WH Classification [10].

SSS were taken from six sites (two earlobes, two from forehead just above the medial aspect of eyebrows and two from lesions) from a patient. A 5 mm thick smear was made. After air-drying the smears, they were stained by carbol Fuchsin for 10 minutes followed by wash and decolourising with 1% acid alcohol and then counterstained with Methylene blue [2]. Slit skin smear was interpreted as positive if M. leprae were detected, stained in red in a light blue background and negative if no bacteria were seen on examination of the complete smear.

After performing punch skin biopsy, the samples were fixed in 10% formalin for about 8-12 hours, following which the samples underwent routine processing after which the paraffin embedded sections of 4-5 μm thickness were stained with haematoxylin and eosin for microscopic examination of the morphology and modified Fite Faraco’s staining for identifying Mycobacterium leprae in the tissue.

The paraffinized sections were placed in Xylene and peanut oil mixture with two changes for 30 minutes followed by staining with strong commercially available Carbol fuchsin for 25 minutes followed by washing in warm running tap water for 3 minutes. Sections were then decolourised in 5% sulfuric acid in 25% alcohol to changes of 90 seconds each till sections turn into pale pink colour followed by washing in warm running tap water for 5 minutes. Counter-staining was done in working Methylene blue {working methylene blue 5 mL stock (methylene blue 1.4 gm in 100 mL of 95% alcohol) add in 45 mL tap water} by one quick dip till sections turn into pale blue. Sections were then blotted followed by drying in 50-55°C oven for 5 minutes. Once dry, the slides underwent a quick dip in xylene. Then the sections were mounted with permanent mountant. The stained sections with the above modifications were observed under oil immersion using 100X objectives. The entire dermis was observed for acid-fast bacilli which looked bright red and the nuclei and other tissue elements looked pale blue [7].

After studying the histopathological features and noting the bacteriological status, the diagnosis of leprosy was confirmed and classified according to Ridley and Jopling classification and a report was created including diagnosis and AFB status after which clinico-pathological correlation was done.

Statistical analysis was done by Statistical Package for Social Sciences (SPSS) version 21.0. Continuous variables were presented as mean±SD and median. Categorical variables were presented as number and percentage (%). Normality of data was tested by Kolmogorov-Smirnov test.

Eighty eight (n=88) clinically diagnosed leprosy cases were enrolled in this study out of which 2, 17, 15, 6, 20, and 25 cases were diagnosed as indeterminate, TT, BT, BB, BL, and LL, respectively. SSS was done in these patients [Table/Fig-1]. Also, punch biopsies were taken from these patients and processed for histopathological studies [Table/Fig-2,3].

[Table/Fig-1]:

Demonstrating AFB on SSS in clumps/globi (100X).

[Table/Fig-2]:

Photomicrograph showing many Virchow cells-laden with plenty of Acid-fast Bacilli arranged in globi. (100X; Fite-Faraco stain).

[Table/Fig-3]:

Photomicrograph showing numerous Acid-fast Bacilli. (100X; Fite Faraco stain).

Considering clinical diagnosis as a reference, the concordance of different techniques were as follows: SSS could detect AFB in 19 cases (21.59%), H&E staining suggested 68 cases (77.27%) as positive with an indirect evidence of features suggestive of M. leprae infection [Table/Fig-4]. Fite Faraco (FF) staining showed a total of 10 cases (11.36%) positive for the presence of AFB in the tissue section as shown in [Table/Fig-5]. There were 10 (11.36%) cases in whom AFB were detected by SSS but not by Fite Faraco, while in 7 (7.95%) cases AFB were detected by Fite Faraco but not by SSS. In 9 (10.23%) cases, AFB was detected by both the methods SSS as well as Fite Faraco. The difference in the two methods of detection was found statistically significant as shown in [Table/Fig-5].

The “true” diagnostic performance of various techniques is shown in [Table/Fig-6] with histopathology kept as gold standard. A higher sensitivity i.e., 26.47% in SSS and higher specificity i.e., 100% in Fite Faraco staining was observed.

Early diagnosis is of fundamental importance to all the aspects of Hansen’s disease like epidemiology, management and prevention of deformities. Both SSS followed by Zeihl Nelson (ZN) staining and Fite Faraco staining detect AFB in tissues. SSS detects AFB in smear taken from dermis whereas Fite Faraco method detects AFB in tissue obtained by skin biopsy taken from clinically suspected lesions. In the present study, sensitivity of SSS was found to be 26.47% which was comparable with values mentioned in the study by Gautam M and Jaiswal A but a specificity of 95% was observed in the present study while it was 100% as compared by the same perivious study, which could be explained due to the presence of non-pathogenic AFB positive bacilli in the environment [11]. Sensitivity of Fite faraco was observed lower ie. 23.53% in comparison to other studies by Cabice et al and Reja AHH et al with 40-70% and 74.6% respectively [7,8]. This could be due to undetected dead bacilli. A statistically significant difference was found between the two diagnostic modalities i.e., fite faraco staining and SSS staining, with p-value 0.002. In the study by Kumaran SM et al., done in India, 100% skin biopsy specimens demonstrated AFB in their sections by modified Fite Faraco staining tissue specimens in whom SSS was found positive [9]. The reason for SSS positive cases diagnosed as Fite Faraco negative could be that the bacillary load in the biopsy sample was less as it was taken from a single site whereas SSS smears were taken from multiple sites. In the present study, according to [Table/Fig-4], detection rates in the diagnostic modalities SSS and Fite Faraco stain on tissues were compared with detection rate of 21.59% of SSS while the detection rate by Fite Faraco staining on biopsy tissue with was found to be 18.18%. In a study by Reja AHH et al., detection rate of Fite Faraco was found to be 60% and SSS detected least with 40.6% detection rate [7]. The detection rates in the present study and the study by Reja AHH et al., was not comparable. Low detection of Fite Faraco and SSS could be explained by lack of AFB in tissues either due to effect of treatment or killing mediated by immune responses [7].

The bacillary index on slit skin smear and bacillary index of granuloma was not calculated.

It was evident from the findings in the present study that Slit Skin Smear (SSS) is more sensitive diagnostic modality whereas Fite Faraco was more specific. The modalities of diagnosis; SSS and Fite Faraco may have some additive value in diagnosing leprosy but cannot diagnose substantial amount of cases when used alone. Hence, the conclusion that there is a need for elaborate research for feasible, sensitive and specific modalities of diagnosis for leprosy in today’s time.