Molecular detection of Mycobacterium leprae using RLEP-PCR in post elimination era of leprosy.

Leprosy is considered as a contagious disease and is still a health problem in several countries including India. Diagnosis of leprosy is based either on clinical findings or on acid fast bacilli staining. Due to low sensitivity of acid fast bacilli staining most of the leprosy cases were remained undetected. The present study aims to assess the efficacy of RLEP-PCR in the field condition where majority of the patients are acid fast bacilli negative and have early disease. A total of 80 suspected leprosy cases were recruited. Slit skin smear samples were taken for microscopy and molecular experimentation. DNA was extracted and RLEP-PCR was executed for all the 80 samples. To establish the statistical correlation χ2 test and Fisher's exact test were made. To elucidate the sensitivity of the test Receiver Operating Characteristic (ROC) was drawn. These 80 leprosy patients comprised of 38 paucibacillary and 42 multibacillary leprosy cases. Of 80 leprosy patients 18 (22.5%) were AFB positive while 53 (66.25%) leprosy cases were RLEP-PCR positive. The results of test of significance (P=0.0001) and Cohen's kappa coefficient (κ) (0.614) indicated that the RLEP-PCR is a better diagnostic tool over AFB microscopy in case detection of leprosy. From the findings we concluded that RLEP-PCR could be used for the definitive detection of leprosy cases in accordance with the clinical findings in the field condition in the post elimination era of leprosy.

Multiple strain infection of Mycobacterium leprae in a family having 4 patients: A study employing short tandem repeats.

BACKGROUND: Leprosy is a slow, chronic disorder caused by Mycobacterium leprae. India has achieved elimination of leprosy in December 2005 but new cases are being detected and continue to occur in some endemic pockets. The possible ways of transmission of leprosy is not fully understood and is believed that leprosy is transmitted from person to person in long term contact. Studying the transmission dynamics is further complicated by inability to grow M. leprae in culture medium and lack of animal models. More than one family members were found to be affected by leprosy in some highly endemic pockets. This study reported the transmission pattern of leprosy in a family having 4 patients. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the transmission of leprosy in a single family having 4 patients using microsatellite typing. DNA was isolated from slit skin smear samples taken from the patients and the isolated DNA were amplified using microsatellite loci TA11CA3. The amplified products were sequenced using Sanger's sequencing methods and the copy number variation in the microsatellite loci between strains were elucidated by multiple sequence alignment. The result showed that all the 4 members of the family acquired infection from 3 different strains of M. leprae from 3 different sources. The elder and middle daughters were infected by same types of strains having the repeat unit TA13CA3 and could have acquired the infection from social contacts of leprosy cases while the father and younger daughter were infected by strains with the repeat unit TA12CA3 and TA11CA3 and could have acquired infection from social contacts. CONCLUSIONS/SIGNIFICANCE: The study suggested that three family members viz, elder daughter, father and younger daughter could be infected by M. leprae from 3 different sources and the history of the disease and genetic analysis showed that the middle daughter acquired infection from her elder sister in due course of contact. This study implies that the transmission of leprosy not only occurred amongst the house hold members but also has been transmitted from social and neighborhood contacts in long term association with the them.

Predominance of three copies of tandem repeats in rpoT gene of Mycobacterium leprae from Northern India.

This study has been carried out to get understanding of the origin among the strains of Mycobacterium leprae in patients from Northern India by using number of tandem repeats in rpoT gene as marker. Biopsies were collected from hundred leprosy cases (paucibacillary (PB) as well as multibacillary (MB)) across the spectrum from patients attending clinic at JALMA or diagnosed in Field Unit at Ghatampur (Kanpur). These biopsies were homogenized and DNA was extracted by a physiochemical procedure. rpoT region was amplified by using the primers and conditions earlier published. Among 100 strains from Northern Indian patients, 89% exhibited the presence of three copies of the 6bp tandem repeat in the rpoT gene, while 11% contained four copies. These profiles along with other genotyping data may help in studying the historical spread of leprosy by strains of M. leprae disseminated by various human races that migrated to Northern India from other places of Asian continent.

In situ hybridization in the histological diagnosis of early and clinically suspect leprosy.

The present study tests the utility of the in situ hybridization procedure for M. leprae rRNA in the histological diagnosis of early leprosy and clinically suspect leprosy, both diagnostically demanding situations. The histological confirmation obtained with routine histopathology (Haematoxylin-Eosin staining for studying morphologic alterations and Fite-Faraco staining for demonstration of acid-fast bacilli) were 32% for early leprosy and 25% for clinically suspect leprosy. With performance of the in situ hybridization on the histologically unconfirmed cases, the positivity rates obtained were 58.8% and 55%, respectively. The results of the study confirm the utility of the procedure in the diagnostically difficult situations of early and suspect leprosy, and it is proposed that the procedure be employed in situations of clinical doubt.