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Molecular and Cellular Probes (1992) 6, 401-410

Detection of Mycobacterium leprae and the potential for


monitoring antileprosy drug therapy directly from skin
biopsies by PCR

Diana L . Williams,'* Thomas P . Gillis,' Paolo Fiallo, 1 ' 3 Charles K . Job,'


Robert H . Gelber,1 ' 2 Carlotta Hill 4 and Shinzo Izumi 5

'G . W. Long Hansen's Disease Center, LSU SVM, PO Box 25072, Baton Rouge, LA 70894,
USA, 2National Ambulatory Hansen's Disease Program, San Francisco, CA, USA, 3 C .I .R .
LEP. University of Genoa, Genoa, Italy, 'National Ambulatory Hansen's Disease Program,
Chicago, IL, USA and 'National Institute for Leprosy Research, Tokyo, Japan

(Received 14 October 1991, Accepted 14 November 1991)

An improved protocol for PCR analysis of Mycobacterium leprae-infected tissues, based on


enzymatic lysis, has been developed and used to demonstrate the feasibility of using PCR for
detecting M . leprae in routine skin biopsies taken from leprosy patients throughout the clinical
spectrum . Of 92 multibacillary patients tested, 99% were PCR-positive using gel electrophoresis or
DNA hybridization to detect the amplified product . Similar analysis of paucibacillary patients, in
which only one of 27 biopsies had demonstrable AFB microscopically, gave a positivity rate of 74% .
No PCR signals were demonstrated from skin biopsies from seven patients with non-leprosy
dermatoses and one AIDS patient with a disseminated atypical mycobacteriosis . Evaluation of
leprosy patients with antileprosy drug therapy prior to biopsy demonstrated that PCR signals were
either greatly diminished or absent after 2 months of continuous antibiotic therapy . PCR was also
able to detect the presence of M . leprae in tissues of patients receiving antibacterial therapy when
patients were suspected of harbouring drug-resistant M . leprae .

KEYWORDS : Mycobacterium leprae, polymerase chain reaction, drug therapy .

INTRODUCTION

Leprosy is a major world health problem affecting 10- methods of detecting Mycobacterium leprae in tissue
12 million people.' Clinical manifestations of leprosy samples could identify stages of infection preceding
continue to stigmatize affected individuals, and care overt disease and possibly elucidate preclinical mani-
for leprosy patients creates a tremendous economic festations of infection which lead to overt disease or
burden on endemic countries in the developing self-healing .
world . Early diagnosis and treatment constitute the The use of recombinant DNA methodologies, in
major public health strategy for controlling leprosy . particular, nucleic acid hybridization with species-
Routine diagnosis of leprosy requires clinical exper- specific DNA probes, has made possible the detection
tize and is effective only when the disease has of mycobacterial pathogens, such as, M. tuberculosis,
progressed to outward signs and symptoms . Prior to M . avium and M . leprae . ' Whereas these probes
the development of clinical symptoms, subclinical demonstrate excellent specificity, assays developed
leprosy can remain undetected for years . Improved with these probes lack the sensitivity necessary for

'Author to whom correspondence should be addressed .

0890-8508/92/050401 +10 $08.00/0 401 © 1992 Academic Press Limited


40 2 D . L . Williams et al.

detecting low numbers of mycobacteria directly from MATERIALS AND METHODS


clinical samples without prior culture amplification .
An alternative to direct detection of nucleic acids Patient data
using probes is the amplification of target sequences
using the polymerase chain reaction (PCR) .' The Skin biopsies were obtained from 124 leprosy
combined specificity and high degree of sensitivity of patients from various parts of the world including
PCR has been especially useful for the rapid, specific China, Vietnam, Thailand, Japan, Indonesia, Africa,
detection of slow-growing, fastidious or non-cultiv- Venezuela, Colombia, Brazil, Cuba, Puerto Rico, the
able micro-organisms, such as M . leprae, directly Philippines, Mexico, Nicaragua and the USA . Ninety-
from biological samples ." two patients were categorized for the basis of study
Recently, we have isolated and characterized a as multibacillary (MB) leprosy . This group contained
360 by DNA fragment of the M . leprae chromosome 72 patients with lepromatous (LL), 15 with borderline
encoding approximately 80% of the 18 kDa protein lepromatous (BL) and 5 with mid-borderline (BB)
of M . leprae ." Using this 360 by fragment as the leprosy (Table 1) . Twenty-seven patients were
'target' nucleic acid sequence, a rapid, specific and grouped as paucibacillary leprosy with 6 tuberculoid
sensitive method for detecting M . leprae DNA from a (TT) and 18 borderline tuberculoid (BT) patients . Three
variety of biological sources was developed ." The patients were diagnosed with indeterminate leprosy
assay employs PCR amplification and subsequent and were included in the paucibacillary group .
detection of amplification products by agarose gel Clinical histories available on MB leprosy patients
electrophoresis or hybridization with a radiolabelled indicated that 22 of the 92 MB patients had anti-
complementary DNA probe . leprosy drug therapy for varying periods of time prior
In this study we describe an improved protocol for to the date of skin biopsy . Drug sensitivity testing
PCR analysis of M . leprae-infected tissues and using the mouse footpad method 72 showed that the
demonstrate the feasibility of using PCR for the homogenates from five MB patients harboured dap-
detection of M . leprae DNA in routine skin biopsies sone-resistant M . leprae and one patient harboured
taken from leprosy patients throughout the clinical dapsone and rifampin-resistant M. leprae . In addition
spectrum of disease . In addition, we present prelimi- to the 22 MB patients described above, five patients
nary findings on the use of PCR for monitoring had inactive lepromatous leprosy for at least 11 years
antileprosy drug therapy . and were on continuous clofazimine therapy .

Table 1 . PCR of biopsy homogenate from multibacillary*, paucibacillaryt and inactive$ MB


leprosy patients and controls

PCR positive

Patient Number of Gel Slot-blot


samples electrophoresis hybridization§

Multibacillary 92 82/92 (89%) 91/92 (99%)


Untreated 70 70/70 (100%) 70/70 (100%)
Treated 22 10/22 (45%) 21/22 (95%)
Paucibacillary 27 1/27 (4%) 20/27 (74%)
Inactive lepromatous 5 ND¶ 0
Non-leprosy controls
Non-specific chronic 4 ND 0
dermatitis
Chronic granulomatous 2 ND 0
inflammation of skin
Sarcoidosis 1 ND 0
Systemic mycobacteriosis 1 ND 0
(AIDS patient)
Normal skin controls 5 ND 0

*Multibacillary; _>1 x 105 M . leprae ml - ' of biopsy homogenate.


t Paucibacillary ; < 1 x 10 5 M . leprae ml - ' of biopsy homogenate .
$ Inactive ; treated MB leprosy patients with no detectable AFB for 12 months or longer.
§ Slot-blot hybridization using "P-labelled 212 by DNA probe .
¶ ND; not done .

PCR in leprosy patients 403

Skin biopsy (24:1) . The DNA was precipitated with two volumes of

absolute ethanol at -20 °C for 2 h, then centrifuged


Skin biopsies (4 mm in diameter ti 40-100 mg) were 10,000 g for 15 min . Precipitates were resuspended in
taken from lesions of leprosy patients and held either 30 ul of sterile distilled water and 10 ul were added to
on ice (for no more than 48 h) or lyophilized and PCR reagents and template DNA was amplified as
shipped to the Gillis W . Long Hansen's Disease described below .
Center, Carville, Louisiana, USA. Lyophilized samples Human skin biopsies were obtained from healthy
were rehydrated in 100 ul Hank's balanced salt solu- volunteers showing no signs of infection with M .
tion (HBSS) for 30 min at 25 ° C . All biopsies were leprae and from patients who had a variety of other
minced with scissors, homogenized in 2 ml HBSS non-leprosy dermatologic disorders . Tissues were
using a Mickle homogenizer (Mickle Laboratory Engi- processed using the same procedure as described
neering, Surrey, UK) with approximately thirty, 3 mm above and extracts were used as negative controls for
diameter sterile glass beads (Sigma Chemical, St PCR experiments .
Louis, MO, USA), for 2 min at 25 ° C . Tissue debris was
allowed to settle at 1 g for 5 min, and the supernate
containing M . leprae was recovered . Enumeration of Sensitivity of PCR for detection of M . leprae
acid-fast bacilli (AFB) was assessed by microscopic
count, 13 which has a mean coefficient of variation of To assess the lower limit of detection of purified M .
29% . 14 Homogenates were stored at -70 ° C . Biopsy leprae DNA by agarose gel electrophoresis, serial
material was also fixed in neutral formalin, embedded two-fold dilutions, starting with 125 fg of M . leprae
in paraffin and evaluated for histopathology. All DNA, were amplified by PCR . To determine the lower
samples were coded and PCR was evaluated without limit of detection of M . leprae from homogenates of
knowledge of clinical or histopathologic data . skin biopsies, 1 x 10 5 M . leprae from an untreated
lepromatous leprosy patient were collected from the
tissue homogenate by centrifugation at 10,000g for
Extraction of M . leprae DNA for PCR 15 min . The pellet was resuspended in 100 ul of an
homogenate prepared from a normal human skin
Tissue homogenate samples (0 . 4 ml) were added to biopsy. Serial 10-fold dilutions were made using the
0. 1 ml of 5 x digestion buffer (150 M NaCl, 100 mm Tris control homogenate as diluent . Aliquots from each
HCI, 10 mm EDTA final concentrations) containing dilution of M . leprae were processed for PCR as
250 ug Proteinase K . Samples were incubated at 60 ° C described above. Each sample (10 ul) equivalent to
for 1 h, heat inactivated at 94° C for 8 min and cooled 1 x 10 4 to 1 bacillus was added to the PCR reagents
to 25 ° C . Lysozyme (1 mg) was added to each sample and amplified for 45 cycles . PCR products were
and samples were held at 37 ° C for 30 min . Proteinase analyzed by both agarose gel electrophoresis and
K was added again and samples were incubated for slot-blot hybridization as described previously" , "
1 h at 60 ° C . Each sample was extracted once with an using a 212 by probe which binds to an internal
equal volume of phenol/chloroform (1 :1) and once segment of the 360 by region of the M . leprae 18 kDa
with an equal volume of chloroform/isoamyl alcohol gene (Fig . 1) .

360 by

212 by

FI F2 R2 RI

18 k Do
Fig. 1 . Schematic representation of the 611 by Ode I-Dde I fragment of Mycobacterium leprae
DNA from the recombinant plasmid pML3 .10 The open reading frame for the 18 kDa protein
gene of M . leprae is shown as a rectangular open box . Forward (Fl, F2) and reverse (R1, R2)
primers are shown as smaller open boxes and direct the amplification of the 360 by and 212 by
PCR products from M. leprae DNA .
4 04 D . L . Williams et al.

PCR to monitor drug therapy the same solution at 65 ° C for 1 h . Hybridization was
detected by autoradiography as described pre-
Identical numbers of M. leprae from homogenates of viously . 16
either untreated or treated lepromatous leprosy
patients were processed for PCR as follows : prior to
enzymatic treatment, 1 x 105 M. leprae were washed DNA probe preparation
twice by centrifugation at 10,000 g for 15 min in 1 ml
of sterile, deionized water and the pellet was resus- The DNA probe used for the slot-blot hybridization
pended in 500 ul of 1 x digestion buffer containing analysis was a 212 by fragment of the 18 kDa protein
250 ug Proteinase K . The samples were further pro- gene ." This fragment is an internal fragment of the
cessed as described above. 360 by PCR target and was produced by amplifica-
tion of M . leprae DNA by 45 cycles of the PCR using
forward (F2) and reverse (R2) primers flanking this
Detection of PCR product by agarose gel region of the chromosome (Fig . 1) . The 212 by PCR
electrophoresis product was purified by agarose gel electrophoresis
followed by electroelution and concentration using a
The amplified product of the PCR was .analyzed by Centricon-30 microconcentrator (Amicon, Beverly,
agarose gel electrophoresis by adding 25 ul of the MA, USA) . The 212 by fragment was radiolabelled
PCR product to a 2% NuSieve :SeaKem (1 :1) agarose with 32 P-deoxycytidine triphosphate (New England
gel (FMC Corp ., Rockland, ME, USA) . The samples Nuclear Corp ., Boston, MA, USA) using a Nick transla-
were separated by electrophoresis for 1 h at 60 V in tion kit (Gibco BRL), separated from free nucleotides
tris-acetate-EDTA buffer (TAE) followed by ethidium by gel filtration using a Nick column (Pharmacia,
bromide staining (0 . 5 ug ml - ') and visualization by Piscataway, NJ, USA) and denatured into single
u .v . transillumination . The size of the PCR product strands by the addition of NaOH to a final concentra-
was determined by comparison to DNA size stan- tion of 0 . 2 mol 1 - ' .
dards using the 123 by DNA ladder (Gibco BRL,
Gaithersburg, MD, USA). Samples containing a band
at 360 by were considered M. leprae-positive . Those RESULTS
samples negative on ethidium bromide agarose gels
were analyzed by slot blot hybridization using the Quantitative aspects of PCR
radiolabelled 212 by probe as described below .
The lower limit of detection of the PCR products from
purified M . leprae DNA was between 31 . 25 and
Slot-blot hybridization 62 . 5 fg or approximately 8-16 genome equivalents
using 4 fg equivalent to 1 M . leprae genome (Fig. 2a) .
Slot-blots were performed on PCR products using a Analysis of the PCR products from M. leprae DNA
modification of a method previously described ." obtained from a biopsy of an untreated lepromatous
Briefly, 85 ul of the PCR product was added to 415 ul patient demonstrated that 100 organisms could be
of 0 . 4 NaOH and 0. 001 M EDTA and incubated at detected in two out of four extractions, while 10 3 M .
25 ° C for 10 min . Samples were added to individual leprae were required for 100% PCR positivity by gel
wells of a Biodot SF blotting apparatus (Bio Rad, electrophoresis (Fig . 2b) . When analyzing the total
Richmond, CA, USA) fitted with an Immobilon-P PCR product from these samples by slot-blot DNA
membrane which had been placed briefly in metha- hybridization analysis with 32 P-labelled 212 by probe,
nol, rinsed in distilled H 2 O, and then held in 0. 2 mol the PCR product from 10 organisms tested positive
I - ' NaOH for 1 min . PCR samples were immobilized (Fig . 2c) .
onto the membrane by vacuum filtration . The mem-
brane was removed from the apparatus, air-dried,
and then heated in a vacuum oven at 80 ° C for General characteristics of biopsy specimens
30 min . Next, the membrane was prehybridized for
1 h in 15 ml of hybridization solution (Sigma Chem- Biopsies from 92 MB patients contained from 1 x 105-
ical Co .) containing 50% formamide at 65°C and then 7. 1 x 107 AFB ml -1 of homogenate and individual
hybridized 2 h at 65 ° C in 4 ml of the same hybridiza- biopsies exhibited morphological indices (MI) ranging
tion solution containing 32 P-labelled 212 by DNA from 0-10% with bacterial indices (BI) from 2+-6+ .
probe at 5 x 106 cpm ml -1 . The blot was rinsed briefly Homogenates of 23 biopsies from tuberculoid and
in 200 ml 2 x SSC and 0. 1 % SDS, then in 800 ml of borderline tuberculoid patients were negative for AFB

PCR in leprosy patients 405

1 2 3 4 5 6 7 8 9

(a)
360 by

(b)
360 by

I I I 1 123 by
10 3 10 2 10'

(c )

10 4 103 10 2 10 1 10& 0

Fig. 2. Analysis of polymerase chain reaction (PCR) products from 45 cycles of PCR using
primers for 360 by product . Twenty-five microlitres of PCR products were separated by gel
electrophoresis through a 2% NuSieve :Seakem (1 :1) agarose gel in TAE buffer for 1 h at 60 V and
stained with ethidium bromide (0 . 5 ug ml -1 ) . (a) Lanes 1 and 2, PCR products from 125 fg M.
leprae DNA ; lanes 3 and 4, PCR products from 62 . 5 fg M . leprae DNA ; lanes 5 and 6, PCR
products from 31 . 25 fg M. leprae DNA ; lanes 7 and 8, PCR products from 15 . 6 fg M . leprae DNA;
and lane 9, 123 by DNA ladder . (b) Serial 10-fold dilutions of M . leprae from a skin biopsy
homogenate of a leprosy patient and diluted into homogenate from a normal human skin
biopsy . (c) Slot-blot containing 85 ul of denatured PCR product from the amplification of 10^ to
1 M . leprae diluted into homogenate from a normal human skin biopsy . Slot-blot was hybridized
with 32 P-labelled 212 by DNA probe .

by light microscopy, while one biopsy contained been shipped to our laboratory in the lyophilized
X
1 104 AFB ml - ' . Homogenates from biopsies of state, we compared the PCR signals obtained from
three patients with indeterminate leprosy were lyophilized biopsies with that obtained from fresh
uniformly negative for AFB . Inactive lepromatous biopsies from untreated patients . No differences were
leprosy patients on continuous clofazimine therapy observed between the PCR signals of 3 X 10" M .
had no acid-fast bacilli in biopsy homogenates nor leprae processed from either fresh or lyophilized
were any bacilli observed in homogenates from skin tissue .
biopsies of patients with skin disorders other than The M . leprae-specific, 360 by PCR product was
leprosy, except for the biopsy from an AIDS patient observed on agarose gels from 82 of 92 (89%)
with a disseminated non-cultivable acid-fast bacilli homogenates originating from multibacillary patients
infection . (Table 1) . Slot-blot hybridization of PCR products of
the entire MB group, including the 10 gel electrophor-
esis-negative samples, brought the overall percent
PCR of human biopsies positive to 99 with only one homogenate (MB patient
number 36) out of 92 testing negative (Table 1) .
Since some of the biopsies used in this study had Biopsies from all (n=70) untreated MB patients
406 D . L. Williams et al .

tested positive by gel electrophoresis and demon- nates from normal controls, were also negative by
strated a high degree of reproducibility with respect PCR (Table 1) .
to 360 by product made after 45 cycles of amplifica-
tion using 3 X 10 4 M . leprae (Table 1 and Fig . 3a) . In
contrast, of the 22 MB patients presenting with a Drug-treated patients and PCR
history of prior antileprosy drug therapy, only 10 or
45% tested positive when the PCR products from Having established the reproducibility of the PCR for
3 x 104 M . leprae were analyzed on agarose gels M. leprae derived from untreated MB patients, we
(Table 1) . Of the remaining 10 treated MB patients tested biopsies from previously treated patients to
testing negatively on gel electrophoresis, all were determine the potential effect of treatment on PCR .
positive by slot-blot, except MB patient number 36 . Effects on PCR were evident when PCR products from
Only one of 27 (4%) paucibacillary leprosy cases 3 x 104 M . leprae were analyzed from each patient on
produced a sufficient quantity of the 360 by product agarose gels . For example, patients on documented,
to be visible on agarose gels . This particular patient sustained antibiotic therapy for more than 2 months
had 1 x 104 AFB ml - ' of tissue homogenate, while all before the test biopsy was taken, regardless of the
other paucibacillary patients were negative for AFB antibiotic(s) used, gave significantly reduced or no
by light microscopy . Slot-blot hybridization of the 360 by product (Fig . 3b, lanes 1-4, 6, 7 and 10-13,
remaining 26 samples brought the percent positive to and Table 2) as compared to equivalent numbers of
74 (Table 1) with 2 of 3 indeterminate, 13 of 18 BT and M . leprae from untreated patients (Fig . 3a) . In con-
5 of 6 TT cases testing positive . trast, PCR products from lepromatous leprosy
Biopsy homogenates prepared from long-term patients with prior therapy for less than 2 months (Fig .
(,> 11 years) inactive leprosy patients were analyzed 3b, lane 5, and Table 2) or who received intermittent
by slot-blot hybridization and found negative . Homo- therapy prior to biopsy testing (Fig . 3b, lane 8, and
genates from patients who were diagnosed with Table 2) or those patients whose lesions contained
other non-leprosy skin disorders, as well as homoge- drug-resistant M . leprae (Fig. 3b, lane 9, and Table 2)

2 3 4 5 6 7 8 9 10 11 12 13

(a)
360 by

2 3 4 5 6 7 8 9 10 11 12 13 14 15

(b) 60 by

Fig. 3. Analysis of PCR products from 3 X 104 M . leprae obtained from skin biopsy
homogenates from treated and untreated lepromatous leprosy patients using 45 cycles of PCR
and primers for 360 by product. (a) Lanes 1-10, untreated leprosy patients ; lane 11, negative
biopsy homogenate control; lane 12, 3 x 104 M . leprae from nude mouse foot pad ; and lane 13,
123 by DNA ladder. (b) Leprosy patients with prior antileprosy drug therapy, lanes 1-13, MB
patient numbers 47, 46, 44, 39, 45, 42, 43, 41, 31a, 50, 40, 37 and 36, respectively (see Table 2) ;
lane 14, negative biopsy control ; lane 15, 123 by DNA ladder .

PCR in leprosy patients 407

Table 2 . PCR from multibacillary leprosy patients with prior antileprosy drug therapy

Patient Treatment Bacterial Morphological Growth$ Drug PCR¶


number Time` Drugt index index (%) resistance§

MB 45 1 w D+R 4+ 1 ND +
MB 48 2 w C 6+ 1 ND ND +
MB 49 2 w D+R 5+ 0 ND +
MB 50 2 m D+R+C 6+ 1 ND -
MB 40 2 m D+R 4+ 0 ND
MB 36 3 m D+R 4+ 0 ND
MB 37 4 m D+R 6+ 1 ND
MB 47 6 m D+R+C 5+ 0 ND wkII
MB 46 9 m D 3+ 0 ND
MB 44 10 m D+R 3+ 1 ND
MB 39 1 y D+R 5+ 0 ND
MB 41 2 y D (1) 5+ 2 S..
MB 42 2 y D (1) 5+ 0 ND
MB 43 3 y D+R 4+ 0 ND -
MB 38 6 y D+R 5+ 2 D wk
MB 31a 7 y D 6+ 5 D +
MB 31b 4 m R ND ND ND ND wk
MB 35 14 y D 5+ 0 D +
MB 30 16 y D+R ND ND D+R +
MB 34 17 y D ND ND D +
MB 33 20 y D+R 6+ 2 D +
MB 29 25 y D 2 + 0 ND -
MB 32 30 y D (I) ND ND ND ND +

Length of treatment before biopsy taken : w = weeks ; m = months; y = years .


j Drug: D = dapsone; R = rifampin ; C = clofazimine; I = intermittent .
f Growth of AFB from biopsy homogenate in the foot pads of BALB/c mice .
§ M. leprae resistant to optimal drug concentrations in mouse foot pad assay .
¶ Polymerase chain reaction product from 3 x 10° M. leprae and analyzed by agarose gel electrophoresis.
11 Weakly positive PCR result by agarose gel electrophoresis .
-Sensitive to dapsone, rifampin and clofazimine .

gave PCR results comparable to that of untreated early stages of infection with M . leprae . Accomplish-
lepromatous patients (Fig . 3a) . ing this goal will require the development of tests
In treated patients, where data was available, no capable of detecting infection prior to the observa-
correlation was observed between patients' BI and tion of clinically apparent symptoms . DNA amplifica-
PCR positively (Table 2) . For example, in the group of tin methodologies, such as PCR, may provide the
PCR-negative leprosy patients who received therapy level of sensitivity necessary to detect low levels of
from 2-12 months prior to biopsy date, BI's ranged infection with M . leprae . This is particularly important
from 3+-6+ (Table 2). in a disease like leprosy where the preclinical period
The morphological index (MI) and growth of M . has been estimated to be 2-5 years and the etiologic
leprae in foot pads of mice are currently used to agent is non-cultivable .
assess the viability of M . leprae . In the present study, We, as well as others, have shown that small
these assays were compared to the PCR analysis for numbers (10-100) of M . leprae could be detected
detection of the presence of viable M . leprae in tissue using PCR ."' In addition, we have shown that homo-
homogenates of lepromatous leprosy patients on genates of human skin biopsies from individuals with
antileprosy drug therapy (Table 2) . No correlation was lepromatous leprosy could be used in the PCR assay
found between the MI and PCR results . However, PCR to detect the presence of M . leprae DNA." In the
results and growth in the mouse foot pad showed present study, we have extended our earlier findings
similar results (16 of 17), in patients receiving 2 by improving our tissue extraction procedure for PCR
months or more of antileprosy drug therapy . amplification of M . leprae DNA and by testing the
broader applicability of PCR, coupled with DNA
hybridization analysis, to detect M . leprae DNA in
DISCUSSION skin biopsy homogenates of both treated and
untreated lepromatous and tuberculoid leprosy
A major goal of leprosy research is to understand the patients .
408 D . L. Williams et al .

In order to test the large number of samples for this PCR-based assay for detecting M . leprae from skin
study, it was necessary to develop an efficient sample biopsies is significantly improved over conventional
preparation method which would provide release of detection by light microscopy with the added dimen-
M . leprae DNA from tissues in a manner which would sion of specificity . Specificity was extended to include
reduce the possibility of cross-contaminating sam- analysis of samples from patients with non-leprosy
ples . Our original sample preparation for PCR relied skin disorders and an AIDS patient with a dissemi-
on sonication to release M. leprae DNA in tissues, nated, non-cultivable mycobacterial infection .
however, this method proved to be labour-intensive The ultrasensitive detection of M . leprae by PCR
and had the potential for cross-contaminating sam- provides a previously unavailable tool capable of
ples through aerosols and by using a common soni- authenticating the presence of M . leprae in tissue
cation probe . In this study we report a modification of samples from individuals suspected of having leprosy .
the previously-reported protocol of Woods and Cole' Of particular interest is the reasonably high rate of
for the release of M . leprae DNA from patient biopsies detection of M . leprae in indeterminate (66 . 7%) and
using enzymatic lysis . Using this protocol M . leprae tuberculoid (75%) leprosy, where bacilli are often
DNA was released from skin biopsy homogenates difficult to demonstrate in the biopsy . Since we tested
efficiently and resultant M . leprae DNA provided only 3 indeterminate and 24 tuberculoid cases, a
suitable template DNA for subsequent amplification more in-depth analysis of paucibacillary disease is
by PCR using previously defined synthetic oligonuc- needed to determine a more accurate level of positi-
leotide primers ." This method gave a lower limit of vity in these groups . De Wit et al. 20 has shown a rate
detection of 10 M . leprae by slot-blot hybridization of approximately 50% positivity from patients with
analysis, which was a 10-fold increase in sensitivity similar disease classifications using paraffin embed-
over sonication as a method of sample preparation . ded samples in the PCR . Even though these data are
This protocol was effective whether samples were encouraging in the area of M . leprae detection in PB
obtained fresh or lyophilized and stored at room leprosy, they in no way support the implementation
temperature prior to processing . of PCR in routine screening for leprosy . Rather, at this
Skin biopsy homogenates of leprosy patients with point in time PCR holds promise as a research tool to
clinically diagnosed disease, throughout the entire study early stages of infection with M . leprae and,
disease spectrum, were analyzed using PCR . In 99% where appropriate, to confirm cases difficult to diag-
of clinically diagnosed MB leprosy patients, where nose when clinical and histopathologic information
AFB were observed in the biopsy homogenates by is equivocal .
light microscopy, a positive PCR result was obtained . Since the samples analyzed in this study were
By comparison positive PCR results were obtained obtained from leprosy patients from many different
from 74% of the PB biopsies, where no AFB were geographical regions of the world and from patients
observed in all but one of the tissue homogenates . harbouring drug-resistant M . leprae strains (DDS and
The inability to detect M. leprae in the remaining rifampin) and from leprosy patients with different
26% of PB biopsies may be due to one or more of the clinical forms of leprosy, it was necessary to establish
following reasons : first, the patient may not have had the conservation of the 360 by PCR target region in
leprosy; second, biopsy site selection, the area of the these isolates . Data obtained from previous geno-
lesion chosen for sampling, may have been devoid of typic studies using restriction fragment polymor-
M . leprae due to an uneven distribution of M . leprae phism (RFLP) analysis suggested that sequences
in lesions ; third, random sampling of only a portion of within the M . leprae chromosome are highly con-
an homogenate containing very few bacilli could lead served among isolates .", " In particular, RFLP analysis
to sampling error resulting in bacilli-free test samples . using a DNA probe encoding 80% of the 18 kDa
To minimize this type of sampling error, bacilli- protein of M . leprae and 6 geographically distinct
negative homogenates testing negative by PCR were isolates of M . leprae supported the conserved nature
retested at least once . Fourth, tissue homogenates of sequences in this region of the chromosome ." , "
have been shown to contain inhibitors of enzymatic PCR data reported in this study using 124 patients
amplification of DNA, however, all PCR-negative bi- confirms the conserved nature of this gene fragment
opsy homogenates in our study tested positive when in M . leprae and strongly suggests that this 'target'
seeded with 1 pg of M . leprae DNA . Further improve- region should provide a suitable template for PCR
ments in PCR sample preparation (for example, meth- amplification of M . leprae DNA from any M . leprae
ods for concentrating bacilli) may enhance the cap- isolate .
ability of detecting even lower numbers of M . leprae, With the establishment of the specificity and utility
the rule in TT and indeterminate leprosy . of the PCR for detecting M. leprae in M. leprae-
This study clearly shows that the sensitivity of the infected skin biopsies, we were able to explore the

PCR in leprosy patients 409

effect of antibiotic therapy for leprosy on the ability multiple drug-resistant M. leprae would be unlikely .
to detect M. leprae by PCR. In our retrospective study Therefore, PCR may provide a useful way of detecting
we were unable to compare PCR results from pre- relapse, with the additional potential of differentiat-
treatment and post-treatment biopsies from the ing relapse from 'reactions' often a problem seen in
same patient, however, PCR results from 70 paucibacillary disease .
untreated patients clearly showed reproducible, In summary, a PCR-based assay has been de-
strongly positive PCR signals from all biopsies tested' veloped and characterized for detecting M . leprae
when analyzing 3 X 10" M . leprae . In contrast, PCR of from fresh and lyophilized skin biopsies of leprosy
M . leprae DNA from patients receiving continuous patients . The assay is sensitive and specific for M .
antileprosy therapy for periods of 2 months or longer leprae and it is anticipated that this assay may have a
showed greatly diminished or no 360 by product variety of clinical and research applications where
after 45 cycles of amplification . These data suggest specific detection of low numbers of M . leprae is
that effective antibiotic treatment regimens using critical . For instance, PCR could be used to comple-
dapsone, dapsone plus rifampin, clofazimine or a ment clinical and histopathological diagnosis of
combination of all three drugs resulted in physiologic patients where leprosy is suspected, but M. leprae
changes in M . leprae which were reflected in the cannot be detected by conventional means . This is
reduced ability to amplify the 360 by fragment of the not an infrequent problem in the differential diagnosis
18 kDa protein gene. One interpretation of this find- of tuberculoid leprosy from other granulomatous
ing is that as the bacteria die, autolytic or host- dermatoses, especially sarcoidosis, where the clinical
derived enzymatic degradation of M. leprae genomic picture is equivocal and skin biopsies demonstrate
DNA occurs, thereby reducing the efficiency of PCR granulomas without specific features of tuberculoid
amplification due to loss of target DNA . A previous leprosy . Also, PCR could be used to detect M . leprae
study has reported losses in PCR signals attributed to in clinical samples other than skin biopsies, for ex-
loss of viability of M . leprae due to drug treatment . 20 ample, skin slits, although due to its current cost and
Our study suggests that the degradation of M. leprae highly technical nature, it is unlikely that PCR would
DNA in lesions of lepromatous leprosy patients can ever supplant this form of simplified, appropriate
take considerable time l2 months as indicated by a technology for routine screening in the field .
comparison of length of antileprosy treatment, viabi-
lity in the mouse foot pad assay, and PCR results . For
ACKNOWLEDGEMENTS
example, treatment for as little as 2 weeks can result
in a loss of viability as indicated by the mouse foot We thank Ms Penne Cason for her excellent secretarial
pad assay ; however, a decrease in PCR signal was not assistance, and Mr Vernon Richard and Dr Suganthi Ravin-
observed for at least 2 months of drug therapy . dran for their excellent technical help . This research was
Some patients receiving various drug regimens supported in part by the World Health Organization, Grant
from 2 to 30 years, which were documented as #890096 .
irregular or intermittent, tested positive by PCR . In
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