The value of Polymerase Chain Reaction (PCR) in the diagnosis of leprosy

Leprosy Mailing List – April 30^th, 2011 

Ref:     The value of Polymerase Chain Reaction (PCR) in the diagnosis of leprosy
From:  Tom Gillis and David Scollard, Baton Rouge, LA, USA

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Dear Salvatore,

In response to the questions about the usefulness of PCR in the diagnosis of PB leprosy: 

Our experience with PCR for detecting M. leprae DNA in human skin biopsies has not changed appreciably since our publication (Scollard et al., Microbiol and Inf Dis 109, 1998) summarizing PCR results and patient diagnoses at the National Hansen’s Disease Programs in Baton Rouge, LA.  We found and continue to find that biopsies from lepromatous (LL) cases result in approximately 90% positivity rates and biopsies from tuberculoid (TT) cases range from 10-25% positive.  TT and indeterminate cases, where no acid-fast bacilli are seen by microscopy, are almost uniformly negative.  Accordingly, our emphasis remains on testing biopsies where AFB are seen but the diagnosis is not conclusive by clinical and histopathological assessment.  We should add that specificity for our tests as well as most tests in the literature are very close to 100%.

While gene target and primer selection are very important, we find that an often overlooked aspect is sample preparation for DNA.  Many reports characterize specificity and sensitivity using purified M. leprae DNA or bacteria from various biological sources (lepromin, mouse, armadillo, human skin biopsy).  Using defined reagents is an important aspect of assay characterization but final specificity and sensitivity for clinical applications of a given PCR test must be determined using patient materials.  Clinical materials can be unfixed biopsies, biopsies fixed in buffered formalin, embedded in paraffin or fixed in 70% ethanol.  Each of these fixed preparations can alter DNA and subsequent extractions are critical for optimal results.  Unfortunately, a well characterized, commercial test for M. leprae DNA in skin biopsies is not available and so many labs continue to report results using their own definitions of sensitivity and specificity and in most cases the results are not comparable across different clinical applications.

To understand why these technical issues are so important, consider that in some instances PCR may be done on specimens from patients classified as “paucibacillary” (PB) by counting lesions, without any support from skin biopsy or skin smear results.  It is well documented that a small percentage of patients classified as “PB” in this manner are actually mid borderline (BB) or borderline lepromatous (BL).  These would be expected to have a higher likelihood of ‘positive’ PCR results than “true PB” (borderline tuberculoid [BT] or TT) patients, but without a biopsy or skin smears they would be considered “PCR + PB”.  

Our recommendation is to link up with a laboratory that has experience in this area and has published results with clinical materials associated with sound clinical assessment of cases.  This would include experienced clinicians and a pathologist who, together, can classify patients using the Ridley-Jopling system.

Tom Gillis, Ph.D.

Chief, Laboratory Research Branch

National Hansen's Disease Programs

Baton Rouge, LA

e-mail:  Tgillis(at)hrsa.gov

David Scollard, M.D., Ph.D.

Chief, Clinical Branch

National Hansen’s Disease Programs

Baton Rouge, LA.

E-mail:  dscollard(at)hrsa.gov

Web:  www.hrsa.gov/hansens 

Skin biopsy from:	PCR positivity rates:
Lepromatous leprosy (LL)	approximately 90%
Tuberculoid leprosy (TT)	10-25%