Use of PCR-mediated amplification of Mycobacterium leprae DNA in different types of clinical samples for the diagnosis of leprosy

Abstract

DNA of Mycobacterium leprae, obtained by a highly efficient nucleic acid extraction procedure, was used for standardisation of the amplification of an M. lepraespecific repetitive sequence by use of the polymerase chain reaction (PCR). With pure DNA, M. leprae-specific amplification was obtained with as low as 100 ag (1 ag = 10-l' g) of target DNA, a quantity equal to about one-tenth of the bacterial genome. Optimal processing of different types of clinical samples such as biopsy material, blood and lymph fluid, from multibacillary leprosy patients, was studied. Simple freezing-boiling cycles in the presence of Triton X 100, with some additional sample-specific modifications such as pre-treatment with NaOH to eliminate PCR inhibitors, was found to be sufficient to yield amplification of bacterial DNA in samples from paucibacillary patients. Clinical samples from 27 untreated leprosy patients, covering the various clinical forms of the disease, and with a bacterial index ranging from 5 + to 0, were collected and processed for PCR analysis. After hybridisation of the amplified material with a specific sequence, 25 of 27 patients analysed gave positive results for M. leprae in at least one of the samples. The potential of PCR for the diagnosis of leprosy is discussed.

Detection of Mycobacterium leprae DNA by polymerase chain reaction in the blood and nasal secretion of Brazilian household contacts

“…But, this is not likely since our method for M. leprae detection in healthy contacts, was able to detect the bacterial DNA in at least one PB contact. Whatever the relationship between positivity of PCR and development of the disease, PCR is much more sensitive than microscopic examination for direct detection of the bacilli (Santos et al 1993). In matter of fact, using the same PCR methodology, M. leprae DNA could be detected in blood, skin hair bulbs and nasal secretion or lymph after the completion of treatment (6 to 8 years; Santos et al 2001).…”

“…All negative samples were reconstituted with 100 pg of purified M. leprae DNA and submitted to another amplification to exclude the possibility of inhibition. After the exclusion of inhibited samples, all other PCR products were submitted to a southern hybridization using a 32 Plabeled oligonucleotide, as described before (Santos et al 1993).…”

“…PCR reactions were performed as described before by Santos et al (1993). The set of primers used was sense 5'-GCACGTAAGCCTGTCGGTGG-3' and antisense 5'-CGGCCGGATCCTCGATGCAC-3' according to Woods and Cole (1989).…”

Search for Mycobacterium leprae in wild mammals

“…5 Woods and Cole (1989), based their PCR on the specific repetitive element of M. leprae (RLEP) demonstrated by visualization of a 372 bp product. 6 This marker was later used for DNA hybridization by Santos et al (1993). 7 The search for the pathogen in wild and domestic animals is practically unexplored.…”

“…6 This marker was later used for DNA hybridization by Santos et al (1993). 7 The search for the pathogen in wild and domestic animals is practically unexplored. Except for non-human primates, the unique animal group in which M. leprae grows successfully is in armadillos, especially the nine-banded D. novemcinctus.…”