This study was conducted in an endemic population in the district of Bankura (prevalence rate greater than 2 per 10,000 populations) in West Bengal, India. After taking formal consent, a total no of 234 paucibacillary (PB) and 205 multibacillary cases (MB) attending the Public Health Centers were assessed by AFB (Acid Fast Bacilli) smear examination as well as multiplex-PCR (M-PCR) to assess the diagnostic efficiency of the later. Out of 234 PB cases 140 were tuberculoid (TT) and 94 were borderline tuberculoid (BT). Similarly, of 205 MB cases 53 were borderline lepromatous (BL) and 152 were lepromatous leprosy (LL)
Patients were grouped in the following categories:
(i) Patients without treatment, (ii) Patients on treatment not more than two months, (iii) Patients complaining of hypoesthesia but showing no clinical symptoms of leprosy - considered as Indeterminate type, and (iv) Patients released from treatment (RFT) and later developed a new active lesion/i.e. relapsed cases.
Slit skin smear (SSS) for acid -fast bacilli (AFB) staining were obtained from all patients for determination of Bacterial Index (BI). All diagnosed cases were given MDT as per the national leprosy control programmed guidelines . Competent health care workers followed up household contacts of these patients. A total of 182 persons of which 110 were MB contacts and 72 were PB contacts, participated in this study voluntarily. Nasal swabs/slit skin smear specimens were obtained from all contacts after obtaining their necessary consent. The contacts were followed up for two years for observing the development of clinical leprosy.
Ethical approval was taken from the Ethical Committee of the Institute (Office of the Director, Instt of Post Graduate Medical Education & Research, Kolkata, Govt. Of West Bengal). Ref No. Inst/IEC/1835 dated 2.8.05 as a part of the project entitled "Development of Multiplex PCR for Early Diagnosis and Strain Differentiation of M.leprae" and since then reviewed periodically.
Slit Skin Smear: SSS were obtained from each patient (from 3 to 6 sites, depending on the type of leprosy) for determination of bacterial index (BI). 4 mm punch biopsy/SSS from three to six sites for each patient were obtained. Half of the biopsy samples from each patient was used for paraffin embedded sectioning and the other portions were stored at -20°C until PCR was performed. BI (bacteriological index) was also determined microscopically from paraffin section of biopsy specimens.
Collection of Nasal Swabs: The surface of the nasal septum either side of each patient were swabbed with sterilized wet cotton swabs, frozen in buffered saline containing 0.05% Tween80, which released the sample from cotton swabs. The aliquot was centrifuged at 10,000X g. The sediment was processed for DNA extraction as described bellow.
DNA Extraction from clinical samples
Extraction of DNA
a) From Skin Tissues: The Frozen section of tissues/skin biopsy specimens were cut to small pieces with sterile scissors. These samples were homogenized in a manual homogenizer with 1 ml sterile distilled water. It was then incubated in lyses buffer containing 300 μl of 100 mM Tris-HCl, pH8.5 (containing 0.05% Tween 20 and 60 μg of proteinase K per ml) for 18hrs at 60°C. Paraffin oil (40 μl) was layered on top of the buffer to prevent evaporation. Thereafter, the samples were incubated at 97°C for 15 minutes , followed by heating for inactivation of proteinase K. Equal volume of phenol-chloroform-isoamyl alcohol (25:24:1) was later layered on the lysed homogenated product. The tube was shaken vigorously for 1 minute. After centrifugation of the material for 8 minutes, the aqueous phase was collected and again mixed with an equal volume of chloroform-isoamyl alcohol. This was followed up by another brief centrifugation. Then, the uppermost phase was collected and boiled for 10 minutes to destroy DNase, followed by precipitation of DNA with ethanol. The precipitated DNA was resuspended in 100 μl of distilled water and used for M-PCR.
b)Extraction of DNA from Nasal swab: Frozen samples were quickly thawed and centrifuged at 10,000X g for 20 minutes The sediment was subjected to DNA extraction following the same procedure as mentioned above.
A M-PCR was developed in our laboratory  based on:
(a) Primers amplifying the 372 bp of the repetitive sequence of M.leprae, known to be specific for M.leprae and is not present in twenty other mycobacterial species .
(b) A pair of primers was designed to amplify 201 bp flanking entire21 TTC repeats .
Sequences for (a): 5'-CGG CCG GAT CCT CGA TGC AC-3' (primerR1)
5'-GCA CGT AAG CTT GTC GGT GG-3' (primerR2)
For (b): 5'-GGA CCT AAA CCA TCC CGT TT-3' (TTC-A)
5'-CTA CAG GGG GCA CTT AGC TC-3' (TTC-B)
Reaction mixtures, conditions of reactions and cycling conditions were optimized as follows:
The reaction mixture contains 50 ul of 10 mMTris-HCL(pH 8.3), 50 mMKCL, 1.5 MgCl2, 0.01% (wt/Vol) gelatin, 200 uM each dATP, dGTP, dCTPand dTTP, 1U of Taq polymerase(Perkin -Elmer Cetus, Norwalk, Conn)0.5 μm each primer and DNA extracted from biopsy samples.
PCR Condition: PCR is carried out in a thermocycler for 35 cycles consisting of denaturation at 94°C for 1 min, annealing at 60°C for 2 mins and primer extension at 72°C for 3 mins. After purified DNA is added to the PCR mix, triple distilled water is used as negative control. The tubes are kept for at least 10 mins at room temperature. After amplification is finished, a 20 μl portion of the reaction mixture is run in a 2% agarose gel. After electrophoresis, the gel is stained with ethidium bromide, and the 372 bp and 201 bp bands examined under UV illumination.
Sensitivity of smear examination for AFB and M-PCR of skin biopsy samples is calculated considering clinically diagnosed cases as true gold standard of positivity.