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Arch Dermatol Res
DOI 10.1007/s00403-008-0857-yREVIEW ARTICLE
Leprosy: diagnostic and control challenges for a worldwide disease
Isabela Maria Bernardes Goulart · Luiz Ricardo Goulart
Received: 31 December 2007 / Revised: 5 April 2008 / Accepted: 10 April 2008© Springer-Verlag 2008
Abstract Leprosy is a curable disease with well-deWnedetiology, but lacks better diagnostic tools, preventive andtherapeutic strategies. The continued application of the Rid-ley–Jopling clinical classiWcation that recognizes the naturaldiversity of the immune response has provided the basis forunderstanding leprosy, and this review proposes its imple-mentation in all Reference Centers in order to standardize thediagnostic resources, aiming at the improvement of the dis-ease control. Due to the broad bioepidemiological aspects ofinfection its eradication is diYcult, and proper diagnosis ofthe disease and the correct clinical classiWcation are requiredto ensure proper treatment. Tools and markers for diagnosisand prognosis, and the novel use of nanotechnology, as wellas strategies for disease control and monitoring populationsat higher risk are still continuous challenges, which will bespeciWcally reviewed with additional insights. The use of thecurrent diagnostic tools, such as ELISA and PCR has a verylimited approach for leprosy that has been considered as amarginal disease; therefore, the current diagnostic tools mustbe applied extensively in the routine to accumulate clinicalexperience in order to improve their precise application, likewhat has been done in many other infectious diseases. Since
a vaccine for leprosy presents an unpredictable future, theproposed chemoprophylaxis of contacts (healthy carriersand/or with subclinical infection) must also be employed inreferral centers of endemic countries not only to evaluate itseYcacy, but also because of the favorable cost–beneWt ratio,given that there is no other available approach, besides themulti-drug therapy of patients. This strategy should readilybe applied as a public health policy, and may lead to a sub-stantial breakage of the transmission chain aiming a worldwithout leprosy.
Keywords Leprosy · Molecular epidemiology · Diagnostic tools · Monitoring strategies · Clinical classiWcation · Serological assays · PCR tests
Introduction
Leprosy, a chronic infectious disease of humans caused byMycobacterium leprae, is still a major health problem inAsia, Latin America, and Africa [204]. It presents a vari-able incubation period ranging from 6 months to more than20 years, with an average period of 2–4 years, due to itsvery slow growth [151]. Another critical issue is that bacte-rial culture is not possible, and infection of humans is man-datory for bacterial transmission.
Global eVorts to control leprosy by intensive chemother-apy have led to a signiWcant decrease in the number of regis-tered patients, but the detection rate of new cases has beenkept constant, with a very small reduction, meaning thatcontrol strategies have not accomplished the aimed eYcacy.
Leprosy has no primary prevention, which means thereis no speciWc vaccine against M. leprae, and diagnosticsand prognostic tests are not feasible or not well establishedin clinical routine [173].
I. M. B. Goulart · L. R. GoulartLeprosy National Reference Center, Clinics’ Hospital, School of Medicine, Federal University of Uberlândia (UFU), Uberlândia, MG, Brazil
L. R. GoulartInstitute of Genetics and Biochemistry, Federal University of Uberlândia (UFU), Uberlândia, MG, Brazil
I. M. B. Goulart (&)Departamento de Clínica Médica, Faculdade de Medicina, Federal University of Uberlândia (UFU), Av. Pará, 1720, Bloco 2H- Campus Umuarama, Uberlândia, MG 38400-902, Brazile-mail: [email protected]
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Arch Dermatol Res
The classiWcation of leprosy was well established byRidley and Jopling in 1966 [165], and surprisingly, withoutany molecular tool, they came up with a very importantdescription of classiWcation forms, which was the mostimportant contribution for the understanding of the diseasein the twentieth century [174].
The disease presents a broad clinical and histopatholo-gical spectrum that is correlated with the immunologicalresponse of the patient [165]. At one end of the spectrum, inthe tuberculoid form, a speciWc cell-mediated immuneresponse to M. leprae is observed, with lesions character-ized by epitheliod granulomas, participation of lympho-cytes mainly of Th1 type, and few alcohol–acid-resistantbacilli [214]. In contrast, in the most severe, or lepromatousform, the speciWc cell immune against M. leprae is absent,with diVuse dermal lesions characterized by poorly diVer-entiated young macrophages with a heavy load of bacilliand a small number of T cells predominantly of the Th2type [214]. In the spectrum of borderline leprosy there arevarying degrees of cell-mediated immune response charac-terized patients with a low response to the bacillus [164].However, the disease manifestations and complications aredetermined by the immune response of the host. Therefore,proper classiWcation of leprosy is one of the fundamentalissues for treatment and prognosis.
Many patients experience nerve damage before, duringor after treatment [151]. The purpose of controlling leprosyis to reduce the rate and severity of disabilities. Therefore,the main objectives in leprosy management are the earlydiagnosis and treatment, followed by an early recognitionof nerve damage and eVective intervention.
Due to the high complexity of leprosy, the developmentof a vaccine and the use of a unique marker for diagnosisare questioned. A thorough review on leprosy has been per-formed elsewhere [173], but novel tools, markers for diag-nosis and prognosis as well as strategies for disease controlare still a continuous challenge, which will be speciWcallyreviewed with additional insights.
Conventional diagnostic tools
Leprosy is insidious; initially aVecting the peripheral ner-vous system [113], with patients exhibiting contrasting clini-cal, immunological and pathological manifestations [71],despite minimal genetic variation among M. leprae isolatesworldwide [137]. Because the infection presents bioepide-miological aspects that do not contribute to its eradication[118], a diagnosis to conWrm the disease is required toensure that proper treatment is applied. However, it isextremely diYcult to detect M. leprae in an individual, andvarious clinical and laboratorial criteria are used due to theabsence of an exam deWned as a gold standard [11].
For treatment purposes, the World Health Organizationrecommends an operational classiWcation (OC) wherebypatients are classiWed under paucibacillar (PB), when theypresent Wve or fewer cutaneous lesions, or multibacillar(MB) when they have more than Wve lesions [202]. How-ever, in places where bacilloscopic examination is avail-able, patients whose skin-smear exam tested positive areMB regardless of the number of lesions. For a better opera-tional classiWcation, some studies have used the M. lepraeserum lateral Xow test (ML-Flow), which correlates theconcentration of anti-PGL1 (speciWc antibody against M.leprae) in the patient’s peripheral blood with the bacillaryload [22]. Serum-positive patients are classiWed as MB andserum-negative ones as PB [21]. The simpliWcation of theoperational classiWcation may mask the true relationshipsof the immunological response, and other intrinsic geneticfactors, limiting the information and preventing furthermolecular Wndings that could support epidemiological datacollection, treatment, and control strategies [174].
The basic criteria in Ridley and Jopling’s [165] classiW-cation are the bacillary load measured by bacilloscopicexams (cutaneous biopsy and skin smear) and the cell-med-iated immune response time, which is evaluated from theresult of Mitsuda’s intradermal test. Based on these immu-nopathological criteria, patients are divided into six clinicalcategories: indeterminate (I), tuberculoid (TT), borderline-tuberculoid (BT), mid-borderline (BB), borderline-lepro-matous (BL), and lepromatous (LL).
Although this classiWcation is important to better under-stand the disease, it is often not standardized in health ser-vices [198], where the majority has assumed the simpliWedclassiWcation of the WHO [203]. However, the Mitsuda testhas been used for research purposes to evaluate thepatients’ response in many diVerent countries, such as Bra-zil [57, 132], China, Vietnam [160] and India [32].
Moreover, due to the neural damage and consequent dis-abilities and the stigma of leprosy for humans, the correcthistopathological diagnosis is mandatory to guide the doctorabout the spectral form of the patient’s disease and its prog-nosis, favoring a therapeutic conduct in his follow-up [23].
Although the prevalence of leprosy has declined world-wide, the number of new cases diagnosed annually has onlyslowly declined and is stable in some regions. This paradoxraises new, important, and interesting questions that willrequire application of the best scientiWc methods availableto answer it [174].
The conWrmation of the leprosy diagnosis for the deter-mination of the load of the disease in a population is animportant motive for carrying out the histopathologicalexam [164]. The pathologist is expected to give a deWnitivediagnosis; however, this exam has some limitations, sincesamples do not always indicate the presence of the bacillusin patients with the characteristic symptomatology, leading
123
Arch Dermatol Res
to controversies about the eYcacy of microscopy for theidentiWcation of the bacillus in smears and biopsies [79].
Biopsies extracted from opposite edges of the same skinlesion, or even from diVerent lesions, do not present signiW-cant morphological discrepancies, as the individual’s bacillaryload and his immunological reactivity are determined system-ically [40]. However, there are frequent reports of inter-observer variations, proving the need for studies to evaluatethem and to put forward suggestions to minimize them [59].
Other researchers in various countries have shown con-cordance between the clinical diagnosis of leprosy and thehistopathological classiWcation based on Ridley andJopling’s criteria [165], which vary from 29.7 to 89.0% asshown in Table 1 [11, 55, 96, 98, 103, 113, 129, 138, 175,180, 189].
When analyzed comparatively (Table 1), the examina-tion and their classiWcations diVer in relation to theireYcacy, which implies the need for a critical analysis tak-ing as reference the objectives of the control programs andthe reality of the diVerent endemic areas and of standardiza-tion of classiWcation adopted in the international literature.
The Immunopathology Committee of the tenth Interna-tional Leprosy Congress, held in Bergen (1973), recom-mended the use of Ridley and Jopling’s classiWcation [165],both to establish a general nomenclature to render the diag-nostic criteria uniform and to standardize scientiWc researchin several countries [27]. It is also reported that the general-ized use of this classiWcation requires human and infrastruc-tural resources that do not always exist in developingcountries, but that the establishment of reference laborato-ries that can meet the needs of diVerent regions should be animportant goal in the study and control of this disease [189].
It should be kept in mind that the basis for understandingleprosy is the recognition that—clinically, histologicallyand immunologically—the LL form diVers from the BLform, and the BT from the TT [164]. This classiWcation sys-tem recognizes the natural diversity of the immuneresponse in leprosy which has challenged immunology foralmost half a century.
Therefore, the present review demonstrates that the clin-ical and laboratorial discrepancies in the diagnosis of lep-rosy should be minimized, providing data to underpin theconstruction of public health policies, standardizing thediagnostic resources, and aiming at the improvement ofReference Centers for the control of this disease [189].
To accomplish this main objective, the routine use of theRidley–Jopling classiWcation is necessary [165]. The bor-derline leprosy group is classiWed within the spectrumbetween the tuberculoid and lepromatous poles. It is themost important part of the spectrum in terms of number ofpatients and severity of nerve damage. It causes most of thedisability and deformity seen in leprosy [151]. While mid-borderline disease may be rare, the ratio of BT to BL T
able
1C
onco
rdan
ce b
etw
een
clin
ical
dia
gnos
is a
nd h
isto
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ical
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min
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n fo
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on o
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y cl
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al f
orm
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ions
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Sehg
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. [17
6]D
ubey
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56]
Jera
th
and
Des
ai [
97]
McD
ouga
ll
etal
. [13
0]B
athi
a et
al. [
11]
Kum
ar
etal
. [11
4]Si
ngh
etal
. [18
1]K
alla
et
al. [
104]
Moo
rthy
et
al. [
139]
Var
gas-
Oca
mpo
[199
]T
eixe
ira
etal
. [19
0]
I–
–88
.80.
036
.077
.860
.0–
20.0
19.9
33.3
TT
30.0
76.9
74.5
30.9
50.0
7.2
52.9
76.7
46.1
27.0
75.0
BT
26.3
100.
064
.768
.477
.057
.766
.744
.266
.5–
77.2
BB
66.7
71.7
53.8
16.7
26.0
–30
.837
.050
.0–
68.6
BL
42.9
100.
028
.537
.526
.0–
30.8
43.7
70.0
–58
.8
LL
66.7
93.6
61.5
100.
091
.0–
90.0
75.6
80.0
63.9
92.5
TT
+B
T49
.496
.6–
55.8
80.0
60.0
83.0
––
–93
.5
BT
+B
B+
BL
59.4
100.
0–
59.5
80.0
75.4
83.0
––
52.4
89.0
BL
+L
L59
.410
0.0
–50
.093
.0–
65.4
––
–91
.2
Gen
eral
29.7
89.0
68.5
40.4
69.0
51.7
58.6
64.6
62.6
42.9
67.6
123
Arch Dermatol Res
patients shows an interesting geographical pattern. BT pre-dominates in Africans while BL predominates in Asiansand Europeans. This diVerence presumably reXects agenetic diVerence in the ability to express cell-mediatedimmunity to M. leprae, since diVerences in bacilli strainsdo not seem to be correlated with clinical forms that aremore related to the transmission dynamics of M. lepraefrom diVerent geographical regions [217]. Besides, it hasbeen demonstrated that all existing cases of leprosy areattributable to a single clone whose dissemination world-wide can be retraced from analysis of very rare single-nucleotide polymorphisms [137].
Pure neural leprosy
Leprosy is the leading cause of peripheral neuropathy [94].There are no leprosy patients without peripheral nerve dam-age, and the mechanism of how it happens is still uncertain[206]. Neuropathy may partially occur by bacterial invasionof the Schwann cells from the outside in, Wrst aggregatingin epineurial lymphatics and blood vessels and then enter-ing the endoneurial compartment through its blood supply,as suggested in experimentally infected armadillos [172].Once these cells are not professional phagocytes, they can-not destroy the mycobacteria. The fact of being insidephagocytes also confers other advantages to the M. leprae,once the mycobacteria is located in a site protected fromdefense mechanisms of the host. It is acceptable that thelong permanence of the M. leprae in the peripheral nervoussystem may aVect the neural function even before stimula-tion of the immune response [159].
The pure neural leprosy (PNL) is characterized by signsand neural symptoms marked with sensitive alterations, likeparesthesia, or sensorial deWcit equivalent to the area of thenerve enlargement, associated or not to the motor or trophicdeWcits, or autonomic, without skin lesions [142]. Thisform of manifestation of the disease is a well-recognizedclinical entity, accounting for 4–16% of patients with lep-rosy in India [68]. A recent study of PNL shows an approx-imate 9.0% incidence in the southeastern Brazil [170]. Themost commonly aVected nerves in the PNL are the ulnarand common Wbular nerves [68, 123, 162, 170].
In literature, there are only a few limited to the pure neu-ral leprosy [123, 140, 187]. Among the many reasons forthis, it is considered that: (1) patients tend to ignore earlysymptoms of nerve damage in developing countries, (2)health professionals do not understand leprosy as a primaryneurologic condition, (3) the PNL diagnosis is underesti-mated, and Wnally, (4) when the PNL is clinically sus-pected, a nerve biopsy is not usually easy to perform [14].
Pure neural leprosy (PNL) is also diYcult to diagnosebecause skin lesions and acid-fast bacilli (AFB) in slitsmears are absent. For patients who exhibit only neurologi-
cal involvement, even when subjected to a careful investi-gation for diVerential diagnosis of various neuropathies[14, 154], the gold standard for PNL diagnosis is the histo-pathological examination of peripheral nerve biopsies.Even so, detection of bacteria is diYcult and histologicalWndings may be nonspeciWc. Furthermore, nerve biopsy isan invasive procedure that is only possible in specializedcenters [93].
In addition to the complexity in achieving the PNL diag-nosis, another problem with this kind of leprosy manifesta-tion is its classiWcation, since the treatment depends on it.Conventionally, these patients are considered as belongingto the tuberculoid pole (TT and BT) of the disease spec-trum, since many of them are Mitsuda positive. As thesepatients also have negative smears, they are classiWed asbelonging to the paucibacillary (PB) group. However, sev-eral studies have shown that some of these patients presentthe standard lepromatous proWle in nerves, with high bacil-lary load [35, 56, 106, 107, 113, 148, 170]. Therefore, thewrong classiWcation and the incorrect treatment of thesepatients may end up in resistance to the medications anddisease relapse, factors that hinder the leprosy control[113].
Many reports on PNL diagnosis have used clinical, elec-trophysiological and histopathological aspects, emphasiz-ing the nerve injury in the classical forms of the leprosyspectrum [16, 33, 49, 101, 124, 176]. In recent years, sev-eral publications have emerged highlighting speciWc infor-mation for the PNL diagnosis [14, 45, 61–63, 169, 181].
The electroneuromyography (ENMG) is indispensableto the studies of peripheral neuropathies [16]. Approxi-mately 98% of patients in whom leprosy is conWrmed bythe traditional methods present electroneuromyographicalterations [124]. The most common and early Wnding is thereduction of the extent of the motor and sensitive responsesto variable degrees in diVerent nerves tested, whichexplains its character of “neuropathy in mosaic,” or asym-metric multiple mononeuropathy [124, 187]. Studies haveshown its usefulness in the diagnosis of the disease at anystage or clinical form, particularly in the initial stages. Sothe ENMG has been shown not only as an eVective methodin the early diagnosis of leprosy, but also as a useful tool inevaluating the eVectiveness of therapy [49]. Moreover, ithas become a tool of great value in the indication of nervebiopsy, once it is proved that this is the only one that canassure the PNL diagnosis [14, 16, 49, 63].
Therefore, there is a need for additional diagnostic meth-ods that may help to conWrm the clinical diagnosis of PNLand this includes the electroneuromyography and nervebiopsies for investigation of M. leprae by either the con-ventional technique of Ziehl–Neelsen or the polymerasechain reaction (PCR), with a further conWrmation by refer-ence centers, avoiding false positive results.
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Arch Dermatol Res
New diagnostic tools
Molecular and immunological tests have been developedfor leprosy diagnostics and prognostics, and among thesetools, the PCR and its variations, ELISA (enzyme-linkedimmunosorbent assay) and other serological tests such asthe lateral Xow (ML-Flow) are the main technologiesemployed with diVerent markers and strategies.
Imaging techniques, such as ultrasonography (US) andmagnetic resonance imaging (MRI) have also been recentlyused to evaluate the pure neural leprosy, and will be pre-sented. We will also discuss the use of these techniques inassociation with speciWc markers and their utility in diagno-sis or prognosis.
Nucleic acid detection—diagnosis and challenges
Due to the diYculty of Wnding bacilli alcohol–acid resistant(BAAR), through histopathological methods in the earlystages of the disease, the PCR technique has been used suc-cessfully to detect small quantities of bacilli in tissues [52].
The major advantages of PCR on other diagnostic meth-ods are based on its fast, speciWc and sensitive identiWcationof organisms, which can be done by analyzing crude bio-logical samples without the need to culture the organism[213]. This is very important when it comes to M. leprae,whose culture is not possible [105].
Some reports have shown good perspectives in relationto M. leprae detection in diVerent samples (blood, skin,swabs, nerves and nasal inferior turbinate) of leprosypatients and their contacts by PCR [14, 47, 48, 52, 58, 99,133, 145, 155, 190, 205, 213]. The PCR technology has nodoubt brought a great advance in the M. leprae detection,and its sensitivity may be limited from one [155] to Wvebacilli in the collected sample [156].
The PCR technique makes possible the detection, quan-tiWcation and determination of the bacillus viability at spe-ciWc sites, through DNA or RNA detection, giving valuableinformation about the infection and transmission of M. lep-rae, and the veriWcation of the eVectiveness of the MDT, asit also detects transcriptional activity of the bacillus (RNA)[30, 114]. In addition, the PCR tests are statistically supe-rior in comparison to microscopic tests of biopsies [179,186].
Variations in PCR positivity have been observed in liter-ature, mainly due to the diVerent primers, ampliWed frag-ment sizes, and ampliWcation techniques [52, 73, 76, 104,105, 112, 114, 126, 133, 145, 147, 152, 153, 197, 211, 215](Table 2).
Among other ampliWcation techniques that have beenemployed for M. leprae detection, the real-time PCR [126]has reached the highest sensitivity (91.3%) detecting as lowas Wve molecules (25 fg) of M. leprae using primers target-
ing the antigen 85-B coding gene, which was 17.7% supe-rior to the conventional PCR presented in this investigation;however, those authors have used only BT patients as pau-cibacillary form, excluding TT patients. It is important toemphasize that some BT cases may be classiWed as multi-bacillary patients, once they present from 0 to 2+ acid-fastbacilli in the granuloma [165], and this could have led tomisclassiWcation of patients. A diVerent result has beenpublished with a real-time PCR approach [112], which hasdemonstrated that sensitivity was not diVerent from theconventional PCR and positive detection reached 88.9% inMB cases and 33.3% in PB cases; however, with a diVerentset of primers (proline-rich antigen, LEP, 36 kDa), whichwere inferior to results presented elsewhere [73] with a setof primers that amplify a 130-bp fragment of the RLEP3region of the M. leprae (Table 2).
Primers that amplify short amplicons of the M. lepraegenome have been successfully used even in damaged or inlow concentrations of DNA, especially in the paucibacillaryforms, demonstrating that the amplicon size may be astrong limitation for the M. leprae DNA detection [52, 73].One of the explanations for the high sensitivity of theRLEP3 sequence is presented elsewhere; [52] compared thesensitivity of primers targeting the 18-, 36-kDa and RLEPM. leprae genomic regions, have concluded that the RLEPprimer set was 10 and 1,000 times more sensitive than the18- and 36-kDa sets, respectively, a sequence that isrepeated 28 times in the M. leprae genome.
The most sensitive technique published in the literaturewas a nested PCR protocol [153] that presented a detectionlimit of 3 fg, and although performed in a short time, it maybe considered with caution, since contamination in thismethodology is quite common [86]. Other technologies,such as the whole genomic ampliWcation [76], have beenused with great success for molecular typing of M. lepraewith detection limit as low as 100 fg; however, it has notbeen used for leprosy diagnosis.
The use of the polymerase chain reaction (PCR) in diag-nosing PNL has been recently investigated [14], and 50%of paucibacillary patients presented positive results. PCRanalysis proved to be a useful method to investigate PNL,enabling conWrmation of the diagnosis in more than a thirdof the cases that were negative for AFB by nerve biopsy.Another study corroborates those results through a semi-quantitative PCR [170] that has also shown a good correla-tion among the bacillary load in nerves with the Mitsudatest response and ML-Flow assay.
Despite its major importance, references in the literaturefor qPCR are scarce. Only a few investigations reassure itsvalue as a method for diagnosis and for therapeutic follow-up in leprosy [77, 152, 170]. Therefore, the developmentof methods for detection and quantiWcation of M. lepraeare necessary for studies involving the epidemiology,
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Arch Dermatol Res
Tab
le2
Com
pari
son
of m
olec
ular
tech
nolo
gies
for
nuc
leic
aci
d de
tect
ion
of M
ycob
acte
rium
lepr
ae a
s le
pros
y di
agno
stic
tool
s
PC
R c
onve
ntio
nal P
CR
, RT
PC
R r
ever
se tr
ansc
ript
ion
PC
R, r
eal t
ime
real
-tim
e PC
R, i
MD
A is
othe
rmal
mul
tiple
dis
plac
emen
t am
pliW
catio
n, N
ASB
A n
ucle
ic a
cid-
base
d am
pliW
cati
on (
isot
herm
alm
ultie
nzym
atic
am
pliW
catio
n), N
A n
ot a
vaila
ble
Mar
ker
(acc
ess
no.
M. l
epra
e ge
nom
ic r
egio
n)
No.
of
patie
nts
Mol
ecul
ar
tech
niqu
eF
ragm
ent
size
(bp
)Se
nsiti
vity
(m
inim
um
no. o
f m
olec
ules
or
DN
A c
once
ntra
tion
dete
cted
)
Tot
al
posi
tivity
(%)
Posi
tivit
y by
clin
ical
fo
rm (
%)
Pos
itivi
ty
(%)
Posi
tivity
(%
)L
iter
atur
e
TT
BT
BB
BL
LL
BI
(¡)
BI
(+)
PBM
B
RL
EP3
(X
171
53)
102
PCR
372
NA
NA
NA
NA
NA
NA
73.3
96.6
NA
NA
Yoo
n et
al. [
216]
110
PCR
372
NA
52.7
13.3
33.3
64.7
83.3
95.2
NA
NA
28.3
82G
oula
rt e
tal.
[74]
110
PCR
130
40 B
acil
li73
.640
.055
.510
010
010
058
.297
.751
.710
0
67PC
R12
9N
A80
.00
0N
A85
.710
067
.710
0N
AN
AK
ang
etal
. [10
6]
6N
este
d P
CR
991,
000-
Fold
mor
e se
nsiti
ve th
an 3
6kD
aN
AN
AN
AN
AN
AN
AN
AN
AN
AN
AD
onog
hue
etal
. [53
]
85-A
-C in
terg
enic
re
gion
(X
609
34)
69PC
R25
010
0pg
82.6
NA
NA
NA
NA
NA
NA
NA
62.5
100
Mar
tinez
eta
l. [1
27]
85-B
ant
igen
(X
609
34)
69PC
R26
310
pgN
AN
AN
AN
AN
AN
AN
AN
AN
AN
A
69R
eal t
ime
8025
fg91
.379
.210
0
16S
rR
NA
(X
588
88)
49R
T-P
CR
171
10 B
acil
li82
.0N
AN
AN
AN
AN
AN
AN
AN
AN
AK
urab
ache
w e
tal.
[115
]
60R
T-P
CR
nes
ted
171
1 B
acill
us78
.3N
AN
AN
AN
AN
AN
AN
A54
94Ph
etsu
ksir
i eta
l. [1
53]
58R
T-P
CR
+co
lori
met
ry17
110
Bac
illi
91.3
NA
NA
NA
NA
NA
NA
NA
8010
0H
aile
and
Ryo
n [7
8]
141
NA
SB
AN
AN
A65
.2N
AN
AN
AN
AN
AN
AN
A90
.316
.7V
an d
er V
liet e
tal.
[198
]
14PC
R40
520
Bac
illi
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
Patty
n et
al. [
148]
Ml0
049
(NP_
3011
62)
7iM
DA
+re
al ti
me
NA
100
fgN
AN
AN
AN
AN
AN
AN
AN
AN
AN
AG
roat
hous
e et
al. [
77]
36kD
a (p
rali
ne-r
ich
antig
en)
(X 6
5546
)
122
PCR
531
3.12
5fg
(0.
5 ba
cill
us)
73.0
2052
.8N
A96
94.3
44.2
95.6
44.2
95.6
Kam
pira
pap
etal
. [10
5]
6N
este
d P
CR
530
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
Don
oghu
e et
al. [
53]
22PC
R53
0N
A63
.350
40N
A10
010
061
92N
AN
Ade
Wit
eta
l. [4
8]
39R
eal-
tim
e PC
R76
194
M. l
epra
e ce
lls95
.0N
AN
AN
AN
AN
A33
.388
.933
.388
.9K
ram
me
etal
. [11
3]
55PC
R53
13.
125
fg63
.60
52.9
100
100
85N
AN
A36
.487
.1W
icht
wec
hkar
n et
al. [
206]
20In
situ
PC
R53
01–
100
Bac
illi
60.0
NA
5044
.510
0N
AN
AN
AN
AN
AD
ayal
eta
l. [4
7]
60PC
R53
110
Bac
illi
38.3
NA
NA
NA
NA
NA
1010
00
46T
orre
s et
al. [
191]
18kD
a (M
225
87)
6N
este
d P
CR
110
100-
Fold
mor
e se
nsit
ive
than
36
kDa
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
Don
oghu
e et
al. [
53]
53PC
R+
prob
e36
0/21
2N
A60
.338
.8N
AN
AN
A92
.6N
A78
.9N
AN
AW
illia
ms
etal
. [21
2]
67PC
R36
0N
A56
.70
0N
A64
.367
.60
66.7
NA
NA
Kan
g et
al. [
106]
LSR
/A15
(15
kDa)
(N
P 30
1294
)46
PCR
321
100
Bac
illi
34.8
014
.3N
A50
7518
.910
08.
363
.6M
isra
eta
l. [1
34]
123
Arch Dermatol Res
pathogenesis and evaluation of the eYcacy of chemother-apy in leprosy as proposed elsewhere [72].
Considering all the above results, it is clear that there isan urgent need to standardize the PCR technique, especiallytaking into account the primer target, the amplicon size, andthe technique, seeking a conWrmatory diagnosis that mayhave important implications in the epidemiology and con-trol of the disease, as well as an ethical and social impact[189].
In brief, the PCR has become the gold standard foramplifying DNA and RNA from many microorganisms indiagnostic tests. The conventional PCR consists of ampliW-cation followed by electrophoretic separation, ethidiumbromide staining and documentation. The greatest advan-tage of PCR (sensitivity and speciWcity) may easily becomeits biggest disadvantage since the reaction must presentvery stringent and speciWc conditions. The conventionaltechnique has been used for diagnosis of many diseases,besides its high sensitivity, for two major reasons: feasibil-ity and low cost of equipments and reagents. However,there are infectious diseases that require technical varia-tions, such as semi-quantitative/quantitative approaches ornested reactions, which may be diYcult to optimize or toperform without contamination. The development of thereal-time PCR has overcome all technical problems, and asit becomes wide spread and disseminated, equipments andreagents will become cheaper, allowing any reference labo-ratory to perform such a test.
We believe that the real-time PCR will soon surpass theconventional technique by becoming the gold standard lab-oratory test for leprosy diagnosis, and among all nucleicacid markers in the literature (Table 2), three of them pres-ent signiWcant results (RLEP3, 85-B and 16S rRNA). The16S rRNA may become one of the most important markersdue to its high abundance (each cell contains 1,000–10,000copies) the RNA reXects organisms’ viability, its use mayindicate the eYcacy of chemotherapy distinguishingrelapse from late reaction, and also may be used for epide-miological studies, as reported elsewhere [77, 114, 152].The only disadvantage of this marker is that samples mustbe frozen immediately in ¡70°C or must be collected,transported and stored in appropriate media to maintainRNA stability, which is not available everywhere, and isstill expensive.
Therefore, it is possible that a DNA-based marker mustbe used for leprosy diagnostics. However, one should use arepetitive sequence, such as the RLEP3, with great care,which may increase the sensitivity of the test; but manyhomologous repetitive sequences may be present in otherMycobacterium species that have not been thoroughlyinvestigated, generating false positive results, as reportedfor the M. tuberculosis IS6110 marker elsewhere [108,130]. So, the use of a single gene, such as the 85-B antigen,
seems to be promising due to its high sensitivity in real-time PCR tests. We believe that only a large-scale use ofreal-time PCR and serology with speciWc markers, sup-ported by the health systems of endemic countries, mayprovide enough evidence of markers that present false posi-tive and false negative results, as it has been shown for theMycobacterium Tuberculosis Direct Test that cross-reactedwith M. leprae infection in USA [34].
Mycobacterium leprae resistance to drugs
Current recommended control measures for treating leprosywith MDT are designed to prevent the spread of drug-resis-tant M. leprae. However, drug resistance has been reportedsince 1964 for dapsone [150], 1976 for rifampin [89], and1996 for oXoxacin [97].
This in vivo method requires at least 6 months and rela-tively large numbers of bacteria. Recently, there have beenadvances in the elucidation of molecular events responsiblefor drug resistance in mycobacteria [92, 140, 158].
Rifampin resistance is associated with mutations in therpoB gene that encodes the � subunit of RNA polymerase.All the mutations associated with resistance in mycobacte-ria are localized in the 500–540 domains, a numbering sys-tem used for Escherichia coli RpoB [140].
Resistance to oXoxacin is known to be associated withmutation in gyrA, encoding the A subunit of DNA gyrase,of various mycobacteria [24, 25] including M. leprae [26].Nevertheless, until now, the number of M. leprae isolatesinvestigated for rifampin and Xuoroquinolone susceptibilityby both genetic analysis and standard mouse footpadmethod is rather small [26, 83, 208].
Dapsone resistance has been associated with three muta-tions in the folP1 gene of dapsone-resistant M. lepraeisolates at positions 157, 158 and 164, altering the aminoacid positions 53 and 55. They corresponded tothreonine ! alanine, threonine ! isoleucine, andproline ! leucine in dihydropteroate synthase (DHPS),respectively [102, 121, 209].
Generally, discontinuation of treatments and mono-therapy play a major role in production of multidrug-resistant (MDR) bacilli. To prevent the emergence andtransmission of MDR leprosy and to identify and treatexisting cases of MDR leprosy, it is necessary to establishrapid methods for detection of drug resistance in M. lep-rae. However, M. leprae has not been cultivated on artiW-cial media; therefore, to identify drug susceptibilitypatterns, bacteria must be tested using Shepard’s mousefootpad assay, MFP [178]. Besides the long time taken byMFP method, multibacillary cases are becoming fewer.Therefore, the molecular methods will be of special inter-est for detection of drug resistance in paucibacillarycases.
123
Arch Dermatol Res
The molecular methods for detection of drug resistancein leprosy are: PCR-SSCP [83], PCR and sequencing [157,218], DNA heteroduplex analysis [210], touchdown PCR-SSCP/sequencing [109, 216], reverse line probe assay[171] and DNA microarray [188].
Due to the urgent need to recognise drug resistance in M.leprae, there are two main recommendations made by aninformal consultation on rifampicin resistance in leprosy[141] that is valid for the other antibiotic resistances: (a)drug resistance surveillance should be established in refer-ence centers; and (b) PCR-based sequence analysis is thechosen methodology for simultaneous analyses of the rpoB,folP1 and gyrA genes (rifampicin, dapsone and oXoxacin,respectively). The chosen methodology may be easilyestablished, but in a few centers of endemic countries;therefore, it is possible that other screening methodologiesthat require only PCR and electrophoresis, such as PCR-SSCP (PCR-single strand conformation polymorphisms) ormelting temperature (Tm) curves by real-time PCR shouldbe used prior to sequencing in order to detect existent muta-tions and novel ones, which is cheaper and easier to per-form. If a Tm can be standardized for each gene mutation, apharmacogenetic surveillance and monitoring may bequickly established.
Immunological tests
The identiWcation of speciWc informative diagnostic anti-gens is one of the most diYcult aspects in developing newdiagnostic tools, and this is particularly true with leprosy,because there is a paucity of information involving the rolesof many of the expressed proteins or the metabolic state ofthe organism throughout infection and disease progression[75].
Many studies have exploited genomic and proteomicsequences for the identiWcation of M. leprae-speciWc pro-teins or peptides that may be suitable for serodiagnosis ofdiVerent disease states of leprosy. While many of thesestudies described novel antigens that show marked humoraland cellular immunogenicity, none have reached usefulaccuracy (sensitivity and speciWcity). Investigations onantigens that trigger cellular and humoral immunity will bepresented.
Cellular immunity
Tests that measure cellular rather than humoral immunity,such as skin tests, have also been developed in variousforms since Mitsuda [17].
It is well known that the late lepromin reaction is a mea-sure of the individual’s ability to generate a cell-mediatedimmune response to an immunizing dose of M. leprae, andalso a measure of granulomatous hypersensitivity.
Although the molecular mechanisms involved in the Mit-suda reactivity and resistance are not yet fully established,it has been clearly demonstrated that the long-lasting latelepromin negativity in leprosy endemic areas is associatedwith an increased risk of developing lepromatous leprosy[78].
Tests that measure cellular immunity to mycobacteriahistorically have relied on the use of mycobacterialextracts, or puriWed complex mixtures of mycobacterialcomponents. In leprosy, puriWed M. leprae was initiallyused in the lepromin skin test [134], followed later by theuse of soluble extracts of the bacillus, designated leprosin.Two speciWc fractions from M. leprae have been prepared,generating the MLSA-LAM (cytosol), and the MLCwA(cell wall) components, in which the latter appeared to be amore potent antigen than the cytosol fraction, probably dueto the dominance of the 65-kDa GroEL antigens [201]. Intuberculosis, puriWed protein derivative of boiled M. tuber-culosis has been used since the beginning of the last centuryin the classical Tuberculin Skin Test (TST). However, thediagnostic value of almost all of these tests is compromisedby the presence of conserved, immunologically cross-reac-tive components that are shared with other mycobacteria,which results in low test speciWcity. For leprosy, such crossreactivity is particularly problematic in countries with highincidence rates of tuberculosis, routine BCG vaccinationpractice, and high levels of exposure to non-pathogenicenvironmental mycobacteria [64].
Although speciWc tests are needed to distinguish previ-ous infection with M. tuberculosis or M. leprae from eachother as well as from exposure to other mycobacteria,including BCG [66], it has been demonstrated that the spe-ciWc cellular response raised by the Mitsuda test(reaction > 7 mm) may be an indicator of acquired protec-tive immunity (odds ratio = 0.16, CI95% = 0.05–0.46) ratherthan an expression of hypersensitivity in household con-tacts, and it has also been proposed that the application ofthis test in endemic countries may be an important epidemi-ological approach for monitoring household contacts ofleprosy patients [74].
The search for M. leprae antigens aiming at improvedleprosy diagnosis still remains a challenge; although thereare many potential targets (Table 3) most of them have onlypreliminary results in cell culture stimulation assays andlack either speciWcity or sensitivity for the detection ofasymptomatic infections and disease progression [75].
These studies of the human T cell response in leprosypatients have identiWed a number of antigens that induce Tcell responses, measured by lymphocyte proliferation orgamma interferon (IFN-�) secretion in patients with tuber-culoid leprosy. Such antigens include the M. leprae 70-, 65-,45-, 35-, 18-, and 10-kDa antigens [1, 5, 31, 38, 50, 84,200, 207]. However, due to the high conservation and
123
Arch Dermatol Res
Tab
le3
Myc
obac
teri
um le
prae
ant
igen
s an
d th
eir
pote
ntia
l use
in d
iagn
ostic
s
M. l
epra
e an
tige
nsC
ellu
lar/
hum
oral
res
pons
eC
andi
date
ti
ssue
(te
st)
Obs
erva
tions
Rep
orts
Mit
sudi
n (M
ycob
acte
rium
lepr
ae
who
le c
ell e
xtra
cts)
Cel
lula
rS
kin
test
The
mea
sure
of
loca
l int
rade
rmal
rea
ctio
n on
the
inje
ctio
n si
te
in le
pros
y pa
tient
s is
an
impo
rtan
t ind
icat
or o
f eY
cien
t cel
l im
mun
ity a
nd g
ood
prog
nost
ics
A n
egat
ive
resu
lt (<
8m
m)
pres
ents
a r
isk
six
times
hig
her
for
the
dise
ase
inci
denc
eN
egat
ive
Mit
suda
ass
ocia
ted
wit
h no
BC
G s
car
and
posi
tive
PG
L-1
indi
cate
a r
isk
25 ti
mes
hig
her
for
the
dise
ase
occu
rren
ce
Mits
uda
[135
]B
rena
n [1
7]G
oula
rt e
tal.
[75]
Gro
ES
Cel
lula
rB
lood
(E
LIS
A)
Hig
h ti
ter
of I
gG1
in a
ll d
isea
se s
pect
rum
Hus
sain
eta
l. [8
5]
ML
35
kDa
Hum
oral
Blo
od (
EL
ISA
)A
mon
oclo
nal a
ntib
ody
inhi
bitio
n en
zym
e-lin
ked
imm
unos
orbe
nt a
ssay
ba
sed
on th
e M
ycob
acte
rium
lepr
ae 3
5-kD
a pr
otei
n. S
peciW
city
=97
.5%
; se
nsiti
vity
=90
%. F
or m
ultib
acill
ary
form
s
Tri
ccas
eta
l. [1
92]
ML
SA
-LA
M (
cyto
sol f
ract
ion)
Cel
lula
rS
kin
test
Ass
ocia
ted
with
pro
tein
s of
70,
65,
45,
35,
28,
18
and
10kD
aW
eir
etal
. [20
2]
ML
Cw
A (
frac
tion
of
cell
wal
l)C
ellu
lar
Ski
n te
st a
nd b
lood
(E
LIS
A)
Ass
ocia
ted
with
pro
tein
s of
70,
65,
45,
35,
31,
30,
28
and
18kD
a
ML
0576
; ML
1989
; M
L19
90; M
L22
83; M
L25
67C
ellu
lar
Blo
od (
EL
ISA
)Si
gniW
cant
IFN
-� e
xpre
ssio
n.A
ll W
ve d
etec
ted
94%
of
nega
tive
PGL
-1 p
atie
nts
Gel
uk e
tal.
[67]
ML
0308
; ML
2498
Cel
lula
r/hu
mor
alB
lood
(E
LIS
A)
Stro
ng I
FN
-� e
xpre
ssio
nM
. lep
rae
antig
ens
with
ort
holo
gues
in o
ther
org
anis
ms
Ará
oz e
tal.
[6]
CF
P-10
(M
L00
50)
Cel
lula
r/hu
mor
alB
lood
(E
LIS
A)
Cul
ture
Wlt
rate
pro
tein
10
(10
kDa)
—ea
rly
gam
ma
inte
rfer
on r
espo
nse
Cro
ss-r
eact
ive
with
M. t
uber
culo
sis
Synt
hetic
pep
tides
pre
sent
ed d
iVer
enti
al r
espo
nse
Doc
krel
l eta
l. [5
2]G
eluk
eta
l. [6
6]M
ehra
eta
l. [1
32]
Spen
cer
etal
. [18
5]
ESA
T-6
(M
L00
49)
Cel
lula
r/hu
mor
alB
lood
(E
LIS
A)
Cro
ss-r
eact
ive
with
M. t
uber
culo
sis
Are
nd e
tal.
[9]
Doc
krel
l eta
l. [5
2]Sk
jøt e
tal.
[ 182
]Sp
ence
r et
al. [
184]
MM
P-I
I (m
ajor
mem
bran
e pr
otei
n II
)C
ellu
lar
Cel
l cul
ture
T c
ell a
ctiv
atin
g ca
ndid
ate
It c
an b
e re
cogn
ized
as
an im
mun
omod
ulat
ing
prot
ein
in te
rms
of a
ctiv
atio
n of
ant
igen
-pre
sent
ing
cell
s an
d in
nate
imm
unity
Mae
da e
tal.
[123
]
Myc
obac
teri
um s
peciW
c po
lycl
onal
ver
sus
mA
b fo
r 12
, 35
and
65kD
a
Cel
lula
rS
kin
scra
ping
sT
he p
olyc
lona
l ant
ibod
y pr
esen
ted
the
best
sen
sitiv
ity: 1
00%
of
MB
, 76%
of
mul
tiple
-les
ion
PB
, and
62%
of
sing
le-l
esio
n P
B p
atie
nts
Suita
ble
for W
eld
diag
nosi
s—do
t EL
ISA
for
mat
Cha
turv
edi e
tal.
[31]
ML
0008
, ML
0126
, ML
1057
, ML
2567
Cel
lula
rP
BM
CSh
owed
inte
rfer
on r
espo
nses
onl
y in
pau
ciba
cilla
ry p
atie
nts
Spen
cer
etal
. [18
6]
ML
0678
, ML
0757
, ML
2177
, M
L22
44, M
L24
98H
umor
alB
lood
(E
LIS
A)
Stro
ngly
rec
ogni
zed
by c
ircu
lati
ng a
ntib
odie
s in
MB
lepr
osy
patie
nts
Ará
oz e
tal.
[7]
ML
0308
, ML
0410
, ML
1553
ML
1829
Hum
oral
Blo
od (
EL
ISA
)In
duce
d re
spon
ses
that
dis
play
ed s
tatis
tical
ly s
igniW
cant
diV
eren
ces
in b
lood
fro
m P
B p
atie
nts
with
res
pect
to th
e M
B, P
C a
nd T
B g
roup
s
123
Arch Dermatol Res
homology among members of the heat shock familybetween M. leprae and M. tuberculosis, it is not possible touse such antigens as diagnostic reagents [111]. Other anti-gens, such as the M. leprae 35-kDa antigen, have also beenshown to have homology to M. intracellulare and M.avium, containing both speciWc and conserved T cell epi-topes [207].
Synthetic peptide antigens (15 amino acids), represent-ing potentially M. leprae-speciWc epitopes have been usedto evaluate responses in leprosy patients from the tubercu-loid pole, contacts and healthy individuals from non-endemic leprosy areas. Although initial Wndings werepromising, immunological responses to these peptides wereof poor speciWcity and sensitivity [51].
The development of improved skin test antigens hasused two speciWc approaches. First, armadillo-derived M.leprae cells have been fractionated, and the proteins associ-ated with the membrane, cell wall and cytoplasm have beenpuriWed, and immunologically characterized [125]. ThisWrst generation of antigens, which comprised the mostabundant M. leprae proteins [i.e., major membrane proteinI (MMP-I), MMP-II, antigen 85-B (Ag85B), elongationfactor Tu (EF-Tu), and GroES], encountered serious prob-lems of cross-reactivity with their counterparts in patho-genic as well as environmental mycobacteria [15, 38, 122,212]. Second, recent studies employing genomics, bioinfor-matics and experimental approaches to evaluate individualM. leprae proteins or small sets of proteins as potentialserodiagnostic or T cell antigens have also been performed[6, 66, 67, 161, 185] and generated a series of potentialantigens (Table 3).
Candidate M. tuberculosis proteins that were found to belacking from the M. bovis BCG genome were shown tohave considerable value as potential diagnostic reagents fortuberculosis in human or cattle [4, 9]. The most character-ized antigens are ESAT-6 and CFP-10. Recently, severalother candidate molecules were reported, which in combi-nation with ESAT-6 and CFP-10 provided enhanced speci-Wcity and sensitivity [3, 19, 117].
Scrutiny of the M. leprae genome revealed the presenceof two candidate genes, ML0049 and ML0050 that encodethe M. leprae homologs of ESAT-6 and CFP-10, respec-tively [64, 65, 131, 183, 184]. It was reported that recombi-nant M. leprae ESAT-6 and CFP-10 proteins wereeYciently recognized by T cells from the majority of M.leprae-responsive leprosy patients [50, 64]. Despite thelimited sequence identity with their M. tuberculosis homo-logues Rv3875 and Rv3874 (36 and 40%, respectively),signiWcant immunologic cross reactivity (i.e., recognitionby T cells from TB patients) was detected. This clearly lim-its the diagnostic potential of ML0049 and ML0050encoded proteins in leprosy-endemic areas with a highprevalence of tuberculosis.T
able
3co
ntin
ued
M. l
epra
e an
tige
nsC
ellu
lar/
hum
oral
res
pons
eC
andi
date
ti
ssue
(te
st)
Obs
erva
tion
sR
epor
ts
PG
L-1
ND
-O-B
SA
(di
sacc
hari
de f
orm
)N
T-P
-BSA
(tr
isac
char
ide
form
)
Hum
oral
Blo
od (
EL
ISA
)D
etec
ts 7
4–10
0% o
f M
B a
nd 1
3.6%
of
PB p
atie
nts
Use
d fo
r op
erat
iona
l cla
ssiW
catio
nC
ho e
tal.
[36–
38]
Gon
zale
z-A
breu
eta
l. [7
0]C
ello
na e
tal.
[29]
Cun
anan
eta
l. [4
4]B
uhre
r et
al. [
20]
Bak
ker
etal
. [10
]B
uhre
r-S
ekul
a et
al. [
21]
Gou
lart
eta
l. [7
5]
Mim
otop
es o
f P
GL
-1
(tw
o he
ptap
epti
des)
Hum
oral
Blo
od (
phag
e-E
LIS
A)
Mim
otop
es w
ere
obta
ined
thro
ugh
phag
e di
spla
y an
d w
ere
high
ly r
eact
ive
agai
nst s
era
of M
B a
nd P
B p
atie
nts,
re
achi
ng a
ser
opos
itiv
ity o
f 76
% o
vera
ll (d
ata
not s
how
n)
Cap
pare
lli e
tal.
[28]
ML
0405
, ML
2331
Hum
oral
Blo
od (
EL
ISA
)S
peciW
cally
iden
tify
LL
/bor
derl
ine
lepr
omat
ous
(BL
) pa
tien
ts o
n th
e ba
sis
of im
mun
oglo
bulin
G (
IgG
) re
activ
ityR
eece
eta
l. [1
62]
Ep1
-030
8 (s
ynth
etic
pep
tide
base
don
ML
0308
)C
ellu
lar/
hum
oral
Blo
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It is suggested elsewhere [7, 67] that the combination ofpeptides should increase speciWcity and sensitivity, and inconjunction with anti-PGL-1 serology; such a test wouldgive satisfactory coverage of most forms of leprosy.
Humoral immunity
Tests that measure humoral immunity have relied mainlyon detection of circulating antibodies against the M. lepraephenolic glycolipid-1 (PGL-1) antigen [10, 20, 21, 29, 36–38, 44, 69, 115] (Table 3).
Infection can be detected by the presence of elevatedtiters of IgM antibodies against PGL-1, and may be areXection of total bacterial load in the body than the bacillo-scopic index of a local skin smear [21]; however, theseantibodies are generally low or absent in paucibacillarypatients. The seroprevalence among contacts has variedfrom 1.7 to 8.7% [10], and may reach up to 11.1% [74].Follow-up studies have shown that seropositive contactsrun an increased risk of developing leprosy [44], presentinga relative risk almost six times higher for the appearance ofthe disease [74].
Therefore, a sensitive and speciWc method to identify sub-clinical infection is a priority, and yet to be developed.Recent technologies, such as protein-based microarrays [75]and phage display [28] have been employed, and may pro-vide novel classes of antigens with potential use in diagnosis.
The recognition of peptides by human serum sampleshas been generally weaker overall, but this is expected onceassays have detected the antibody response to a single pep-tide or antigen among the many antigens produced by themycobacteria. Nevertheless, some individuals may have adistinct response according to peptide preferences [185].These results imply that multiple targets must be used at thesame time in order to improve leprosy diagnosis.
In brief, new investigations on M. leprae antigens are stillnecessary and comprehensive Weld work must be performedwith potential antigens for improvement of immunodiagnos-tics. However, the anti-PGL-1 and Mistuda assays are twosimple, easy and cheap tools to be employed in endemiccountries that may help identifying higher risk individualsfor developing leprosy from the population [74].
Nanotechnology and biosensors
Although PCR has become the gold standard diagnostictool for many diseases, other recent technologies are arisingand may revolutionize the diagnostic Weld, such as thenanotechnologies.
Nanomolecular diagnostics is the use of nanobiotechnol-ogy in molecular diagnostics and can be termed nanodiag-nostics [90]. Nanotechnology is the creation and utilizationof materials, devices, and systems through the control of
matter on the nanometer (one billionth of a meter)-lengthscale. Various nanotechnologies and their applications inlife sciences are described in detail elsewhere under theterm nanobiotechnology [91].
Biosensors are deWned as analytical devices incorporat-ing a biological material, a biologically derived material, orbiomimic, intimately associated with or integrated within aphysicochemical transducer or transducing microsystem[70]. Biosensors should be distinguished from a bioassay ora bioanalytical system, which require additional processingsteps such as reagent addition [144], where the assay designis permanently Wxed in the construction of the device.
Biosensors are usually classiWed into various basicgroups, according to the signal transduction and biorecog-nition principles. On the basis of the transducing element,biosensors can be categorized as electrochemical, optical,piezoelectric, and thermal sensors [168]. The electrochemi-cal biosensors, and among them the amperometric and thepotentiometric ones, are the best described in the literature;those based on optical principles are the next most com-monly used transducers. In fact, most catalytic biosensorsare based on electrochemical methods, whereas aYnity bio-sensors have generally proved more amenable to opticaldetection methods [192]. The various types of optical trans-ducers exploit properties such as simple light absorption,Xuorescence/phosphorescence, bio/chemiluminescence,reXectance, Raman scattering, and refractive index [41].Surface plasmon resonance (SPR) is another commontransduction mechanism whose main advantage over mostoptical biosensors is that the analyte presence can be deter-mined directly, without the use of labeled molecules.Finally, cantilever biosensors are an emerging group of bio-sensors, which are based on the bending of silicon cantile-vers caused by the adsorption of target molecules onto thecantilever surface, where receptor molecules are immobi-lized.
Sensing occurs when there is an interaction between thetarget molecule and a biological macromolecule (e.g.,enzyme, antibody, receptor or DNA strand). Therefore,according to the biorecognition principle, biosensors areclassiWed into immunochemical, enzymatic, nonenzymaticreceptor, whole-cell, and DNA biosensors. Immunosensorspresent the advantages of sensitivity and selectivity inher-ent to the use of immunochemical interactions [168],although cross-reactivity may be observed.
Recently, our group has reported experiments showingthe coupling of electrochemical biosensors with PCRamplicons to detect the M. leprae DNA for diagnostic pur-poses [2]. BrieXy, after the functionalization of polymerswith aminophenols [18], the surface of the electrode wasconjugated with a thermally denaturated 78-pb DNA frag-ment, which was PCR ampliWed from the RLEP3 repeti-tive sequence of the M. leprae genomic DNA. Detection of
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the target complementary DNA was performed using ferro-cenecarboxyaldehyde as indicator of the hybridization. TheM. leprae DNA detection without any ampliWcation wasperformed in 3-min hybridization, indicating that a Weldportable electrochemical device may become a reality forbacilli DNA detection, which could also be used for detec-tion of antibodies, consequently aVecting diagnosis, epide-miological, pharmacogenetic, and monitoring programs ofleprosy.
Diagnostic imaging
In leprosy, bone lesions due to direct invasion of bacilli arelow in incidence and these lesions exhibit radiologic Wnd-ings of acute and chronic osteomyelitis, similar to those ofother granulomatous infectious agents. The more commonbone lesions are those due to injurious eVects of trauma andinfection imposed upon denervated tissues [149]. Radio-graphically various degrees of reabsorption of the extremi-ties are seen involving hands and feet with the loss of digitsand disorganizing arthropathies in small joints. The radio-logic appearance is similar to other conditions in whichthere is sensory impairment like scleroderma, syringomieliaand diabetes mellitus. The ultrasonography (US) and mag-netic resonance imaging (MRI) can be helpful in evaluationof the involvement of the peripheral nerves helping in thediagnosis of the neuritis, abscess and diVerential diagnosisin compressive syndromes [149]. The commitment ofperipheral nerves in leprosy occurs by direct invasion of thebacillus in the reactional states, especially in the reversereaction (RR), where the inXammatory process can result inintense irreversible damage [163].
Although the high-resolution US is an eVective form ofimage to show morphological alterations of peripheralnerves, the value of the US for the diagnosis of diseases ofthe peripheral nervous system is poorly understood [82].Furthermore, descriptions of MRI features of peripheralnerve involvement in leprosy are also sparse in literature[80]. In leprosy, these methods are also little studied to aidin the diagnosis of neuritis.
The reWnement of high-frequency broadband linear-array transducers, and sensitive color and power Dopplertechnology, have improved the ability of US to detect Wnetextural abnormalities of tender tissues as well as to identifya variety of pathological conditions. In nerve imaging, UScan support clinical and electrophysiological testing fordetection of compressing lesions caused by nerve entrap-ment in a variety of osteoWbrous tunnels of the limbs andextremities [128].
The improved soft-tissue deWnition aVorded by MRImay be useful in evaluating neural involvement [80]. MRImay show diVuse edema and swelling of the involved nervedue to neuritis. However, these Wndings are quite non-speciWc
and the diVerential diagnosis includes other hypertrophicneuropathies like Refsum’s disease, amyloid inWltration,chronic relapsing polyneuritis and Guillain–Barre syn-drome [13]. Presence of nodules or nerve sheath granulo-mas is suggestive of leprosy [167]. Analyses of theperipheral nerves with ultrasonography and magnetic reso-nance imaging in leprosy have been performed elsewhere[127] which have classiWed leprosy nerves into threegroups based on imaging appearance; group I consisted ofnormal appearing nerves, group II included enlarged nerveswith fascicular abnormalities, and group III included nerveswith absent fascicular structures. They found Doppler USand MRI to have sensitivity of 74 and 92%, respectively, inidentifying active reversal reactions, based on detection ofendoneural color Xow signals, increased T2 signal and Gad-olinium enhancement.
A recent study has showed that the US presented a welldeWned cord-like hypoechoic lesion along the left commonperoneal nerve. On MRI the peroneal nerve was enlargedand was isointense to muscle on T1Wimage and had highsignal on STIR sequence. It was concluded that MRI mayexclude nerve abscess in cases of tender neuropathy. ThediVerential diagnoses in the present case included ulnarnerve abscess, peripheral nerve tumor and reversal reaction.MRI appearance of well deWned ovoid lesion with periphe-ral rim enhancement and central necrosis favors nerveabscess. DiVerentiation of ulnar nerve abscess from rever-sal reaction is important as reversal reaction can be man-aged conservatively with steroids whereas ulnar nerveabscess may need surgical decompression [95]. The MRIalso helps in the diVerential diagnosis of osteomyelitis andneuropathic arthropathy, which is diYcult with other tech-niques of image [149].
Nerves represent probably one of the best applications ofmusculoskeletal US due to the high-lesion detection rateand accuracy of US combined with its low cost, wide avail-ability, and ease of use. A focused US examination can beperformed more rapidly and eYciently than MR imaging[128]. It has also been suggested that the conduct of serialUS examinations could be of value to monitor the reactiveprocesses during treatment, particularly where it is clini-cally impossible to determine whether the patient is inremission. The US would be useful also in the selection ofmore aVected nerves for which surgical decompression oreven neurolysis would be indicated. Besides, the US mayalso be a useful tool in the PNL diagnosis [127].
Epidemiological aspects: transmission, infection, and monitoring strategies
Leprosy is a curable disease with well-deWned etiology, butlacks better diagnostic tools and therapeutic strategies,
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which together with the socio-cultural prejudice becomesan important obstacle to overcome for early detection andprotection of the susceptible population, especially for thehousehold contacts of leprosy patients, who should begiven priority in disease control programs in order to inter-rupt transmission and reduce physical and social disabilities[74]. Household contacts of leprosy patients are the highestrisk group for the development of the disease, and althoughmany risk or prevention factors have been identiWed, theyhave not been employed in leprosy-monitoring programs.
Leprosy prevalence is always low in statistical sensus.Even in high-endemic areas the prevalence rarely exceeds5%. The explanation for these low prevalence indices is theunavailability of valid and reproducible tests for detectionof sub-clinical infection [87].
Transmission of M. leprae infection is not signiWcantlyaVected by current leprosy control measures. In addition todelayed or missed diagnosis of infectious leprosy patients,the lack of tests to measure asymptomatic M. leprae infec-tion in contacts prevents assessment of transmission of M.leprae. Therefore, a key priority is the development of spe-ciWc and sensitive diagnostic tools that detect M. lepraeinfection before clinical manifestations arise [87].
Possibly, there may be an undeWned number of infectedasymptomatic people, who may present an important activerole on the disease transmission, preventing its eVectivecontrol [119]. The evaluation of the exposure and the infec-tion onset of the disease may be almost impossible, oncebacterial culture is not possible, and the incubation periodis long and variable, besides the existence of paucibacillaryforms that are diYcult to be detected by conventional opti-cal microscopy [163].
The use of PCR in the Weld of molecular diagnostics hasincreased to the point where it is now accepted as the stan-dard method for detecting nucleic acids from a number ofsample and microbial types. PCR is the most commonlyused nucleic acid ampliWcation technique for the diagnosisof infectious diseases surpassing the probe and signalampliWcation methods. The PCR may be too sensitive forsome applications including to detect a microbe that is pres-ent at non-pathogenic levels [120].
The nasal mucosa is the preferential site for the entry andexit of M. leprae as shown by the bacilli colonization of thenasal inferior turbinate [145]. Even in PNL patients inwhom the disease is believed to be conWned to the periphe-ral nerves there are widespread eVects with speciWcchanges of leprosy in the nasal mucosa seen in 51% of thepatients, conWrming that early leprosy involvement can befound in the nasal mucosa even before lesions becomeapparent in the skin or other parts of the body [187].
Untreated MB patients are probably the most importantsource of transmission of M. leprae. Household contacts ofMB patients have been estimated to have a 5–10 times
greater risk of developing leprosy than that of the generalpopulation [53, 54, 60, 195]. However, in many areas, thenumber of MB patients is very small and they may not rep-resent the most important source of infection [87]. There isincreasing evidence that subclinical transmission mayoccur [136], because even in highly endemic countries, nohistory of close contact with a leprosy patient can be estab-lished for many patients [60].
Nasal excretion of M. leprae by healthy carrier individu-als could be responsible for transmission. M. leprae-spe-ciWc DNA sequences have been detected by PCR on nasalswabs from many apparently healthy individuals residing inendemic areas [72, 81, 88, 110, 146, 156, 193] and largeproportions of those who live in endemic areas show sero-positivity against M. leprae speciWc antigens [88, 156,195].
There is increasing evidence from nasal PCR studies oftemporary carriage or even subclinical infection [39, 81,100, 110, 194] that infected persons may go through a tran-sient period of nasal excretion, indicating that the mycobac-terium is highly infective [81]. Patients’ householdcontacts, neighbors, and social contacts have an increasedrisk of contracting the disease [196]. Nasal carriage of M.leprae in healthy people may have important implicationsfor leprosy control, once it is diYcult to visualize the wide-spread exposure without the existence of sources of trans-mission other than MB patients alone [110]. Other strongevidence on the involvement of contacts in the transmissionchain is the presence of M. leprae DNA in the nasal mucosabiopsies (inferior turbinate) in 10% of household contacts[145] and in 4% in the nasal swab [100], which also con-Wrms that the nose is major port of entry and exit of M. lep-rae. These Wndings also support results elsewhere [177]that have demonstrated the aYnity of M. leprae for thenasal mucosa and head sinuses, which depend on the bacilliviability and mucosa integrity.
Although nasal carriage may not necessarily result ininfection or excretion of bacilli, the Wnding of nasal car-riage evidences the disseminated occurrence of M. leprae incontacts [100, 145] and leprosy-endemic populations [110]and its probable role as a reservoir for maintenance of bac-teria [177].
In a preliminary investigation for the presence of M. lep-rae DNA in blood samples of 110 patients and 434 con-tacts, the general positivity was, respectively, 18.2 and8.9% [8]. The presence of M. leprae DNA in the blood ofhealthy carriers provides additional epidemiological evi-dence that the route of M. leprae transmission is not onlythe upper airways, and may indicate possible transmissionthrough the blood, which may aVect blood bank routinetests in the future. This hypothesis may be corroborated bycase reports in nonendemic areas [116, 135], in which lep-rosy was acquired after organ transplantation. However,
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these two case reports must be carefully investigated todemonstrate that recipient patients have not received con-taminated blood during transfusion, since there is no scien-tiWc proof that indirect transmission, such as the chainarmadillo–dog–man, is possible.
All evidences of M. leprae DNA detection in many tis-sues are important for epidemiological studies, but serolog-ical markers may also be a useful tool. Several studies haveshown that antibody levels can be used as a substitutemarker for the bacterial load in the sense that there is a pos-itive correlation between antibody levels and the bacterialindex [166]. Current evidence suggests that serologicaltests could be useful in deWning high-risk contacts [12,136]. But, it is diYcult to predict which seropositive con-tact will develop leprosy, because the presence of circulat-ing antibodies is not an indication of active disease and mayonly be an indication of a recent infection [44]. However,seropositivity contacts run an increased risk of developingleprosy, especially in MB leprosy [44, 74]. In addition, ithas been demonstrated that seropositivity among contactswas most closely related to the serological status of theindex patient, and thus, the serological status of the patientseems to be a better indicator for the transmission potentialthan the BI [10]. The fact that most of the MB patients pro-duce antibodies against M. leprae suggests that the seropos-itivity in contacts may be a marker of incubation of themultibacillary infection, and not a marker of general infec-tion [10]. This same study argues that the seroprevalence ishigher among people living in close proximity to seroposi-tive patients (·75 m) and this may be important for a moreaccurate estimation of transmission potential to measure theserological status of all patients and contacts, with a con-cept of contact expanded [196], people living in neighbor-ing houses, the stone-in-the-pond model as used intuberculosis control [199].
Based on earlier Wndings it is possible to state that thereis subclinical infection in leprosy and many times it corre-sponds to the incubation period of the multibacillary dis-ease [54]. It has been found that a maximum duration ofseropositivity prior to diagnosis of is 9 years, indicating thelong incubation period prior to clinical diagnosis. Thisgroup of leprosy patients likely poses a serious threat to thecontrol of the transmission of leprosy and should be givenchemoprophylaxis aiming the prevention of new cases andopening the way for a rational program for eradication [54].
An epidemiological study [74] in a Brazilian endemicarea was carried out over a 5-year period to measure the rel-ative risks of leprosy occurrence and its clinical forms inhousehold contacts. Three simple clinical procedures, theBCG vaccination, the Mitsuda test and the ML-Flow assay,have been evaluated to determine the speciWc risks for thedevelopment of leprosy or protective eVects against thisdisease. Based on their results, it was suggested that an
additional intradermal BCG booster dose be maintained inLeprosy Control Programs for household contacts, aimingfor protection against leprosy, mainly against MB forms.The authors have shown that the BCG vaccination and theMitsuda test showed a protective eVect against leprosy of0.27 (at least one scar) and 0.16 (>7 mm), respectively, andthe positive ML-Flow test indicated a relative risk approxi-mately sixfold higher for occurrence of the disease. Allunfavorable combinations of two and three assays gener-ated signiWcant risk values that ranged from 5.76 to 24.47,with the highest risk given by the combination of no BCGscar, negative Mitsuda test, and positive ML-Flow test.
It was also suggested that the BCG vaccination may begiven to stimulate Mitsuda test positivity, reducing thepatient’s risk of developing multibacillary forms [74],which corroborates other reports [42, 43, 139] that weredecisive in establishing the adoption of two intradermalBCG doses as a control measure for the household contactsof leprosy patients. Prior to the BCG vaccination, the Mit-suda speciWc cellular immunological assay and the ML-Flow test must also be used in contacts to identify individu-als at a higher risk of developing leprosy.
Finally, elsewhere [74], the following approaches forreduction or control leprosy transmission have been sug-gested: (1) household contacts of leprosy patients must bemonitored during the Wrst year after diagnosis of the indexcase; (2) an additional intradermal BCG booster dose mustbe given in Leprosy Control Programs for household con-tacts, aiming for protection against leprosy, mainly againstMB forms; and (3) the use of the combination of the threeassays may discriminate individuals at a higher risk fordeveloping leprosy from contacts with signiWcant protec-tion factors, which could lead to a closer monitoring pro-gram for those at risk, as well as a subsidized new andeVective control strategy for leprosy. This proposal mayjustify the chemoprophylaxis of close contacts of leprosypatients who Wt the highest risk categories deWned in thisstudy.
Chemoprophylaxis
The workshop on the use of chemoprophylaxis in the con-trol of leprosy, held in Amsterdam, the Netherlands, in2006 [143] is one of the most important reports to reviewcurrent evidences and to discuss potential future courses ofaction with regard to the use of chemoprophylaxis to pre-vent leprosy, in this following, emphasis will be on themost important aspects reported.
Chemoprophylaxis is targeting those subjects withsuspected sub-clinical leprosy infection. From an opera-tional point of view, it would be highly desirable if che-moprophylaxis is administered in no more than a singledose. Consequently, the regimen should display powerful
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bactericidal activity against M. leprae by a single-dose oftreatment.
Optimal chemoprophylaxis should provide maximumeYcacy and minimal risks (adverse eVects, resistance).Candidate antimicrobial agents for chemoprophylaxisshould have the following: (1) fast oral absorption withoutgastro-intestinal interactions; (2) fast intracellular penetra-tion into infected tissues; and (3) slow elimination (longhalf life) to allow prolonged eVect and once-only regimens.Adverse reactions are generally a minor issue for (poten-tial) chemoprophylactic agents.
There are four antimicrobial agents, i.e., rifampicin(RIF), rifapentine (RFP), moxiXoxacin (MXF) andR207910 (a diarylquinoline), displaying similar but verypowerful bactericidal activity, i.e., killing at least 90% ofviable M. leprae, at a single dose. Therefore, the Wrstchoice of the chemoprophylactic regimen would be a sin-gle dose of 600 mg RIF, or 10 mg/kg body weight RIFfor children. Although the combination of rifampicin–oXoxacin–minocycline (ROM) has been employed asmonthly administered regimen for treatment of leprosywith promising results, a single dose of ROM is no moreeVective than a single dose of RIF alone; furthermore,addition of oXoxacin and minocycline to RIF willincrease the cost and the risk of side-eVects. Therefore,ROM should not be employed for prophylactic purpose.Finally, a person with sub-clinical leprosy infection ismost likely skin-smear negative, and therefore, harborsno more than 106 M. leprae, or 105 viable M. leprae, inthe body; it is very unlikely that a single RIF-resistantmutant would be included in such bacterial population;therefore, the risk of emergence of RIF-resistance by asingle dose of RIF monotherapy is probably negligible.On the other hand, if, for whatever reason, the bacterialpopulation size is larger than expected, and even if itincludes RIF-resistant mutants, the emergence of rifampi-cin resistance is still very unlikely, because a single doseRIF is insuYcient to select the resistant mutants, as hasbeen shown in MB patients who relapsed after a singledose of RIF’s treatment.
A number of requirements should be fulWlled, and it isadvised that,
1. Contacts should be screened by a health worker for lep-rosy and TB prior to the provision of chemoprophy-laxis.
2. Chemoprophylaxis should be provided under directobservation.
3. A system for recording and reporting of prophylaxisdistribution should be available.
4. Health workers need to be informed of such a policyand those directly involved need to be trained in selec-tion and distribution.
5. People receiving chemoprophylaxis should receiveproper information about its eVects so as to leave themwith realistic expectations.
6. A system for antibiotic resistance monitoring should bein place.
7. There is a need for discussion and approval of any suchprogram with the TB (and other infectious disease)authorities.
In our opinion, based on the various evidences, the chemo-prophylaxis may be indicated for contacts of patients withsubclinical infection (anti-PGL-1 positive serology andnegative Mitsuda) in health services as an eVective measurein preventing secondary leprosy cases, although it does notguarantee absolute and prolonged protective eVect. ForReference Centers, it is also suggested and desired that con-tact healthy carriers that present positive M. leprae DNA innasal swab, nasal mucosa and blood must receive prophy-lactic treatment and the epidemiological surveillance mustbe maintained for 5 years, the average period of incubationof the disease.
In conclusion, considering that a vaccine for leprosy isstill a challenge, and with an unpredictable future, the pro-posed chemoprophylaxis of contacts as a routine practicemust be employed by referral centers of endemic countriesnot only to evaluate its eYcacy, but also because of thefavorable cost–beneWt ratio, since there is no other avail-able approach that could lead to a substantial breakage ofthe transmission chain as a complementary action to theMDT (multi-drug therapy) treatment.
Summary and conclusions
The proper classiWcation of leprosy, using the Ridley–Jopling system, is a fundamental tool to the basis of under-standing the disease, allowing the selection of treatmentmodalities, prognosis evaluation, and the improvement ofthe disease control.
There is no speciWc vaccine against M. leprae, and it isstill a great challenge for research development due to thepossibility of eliciting a complex immunological responsethat could lead to neural damage in asymptomatic individu-als, which may also be infected. It is well known that theprotective immunity is the cellular response, which isresponsible for the pathogenesis of the nerve injury, and thehumoral response does not protect against the bacilli dis-semination.
Therefore, the lack of an eVective primary preventionmeasures has led scientists to search for tests that maydetect at early stages the M. leprae in order to interrupt itstransmission and to prevent nerve damage and deformity ofleprosy patients.
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The use of the current diagnostic tools, such as ELISAand PCR, has been used only for research purposes, andunfortunately has not been implemented in the leprosy clin-ical routine. However, this restricted clinical practice hasbeen applied only in leprosy, which has been quite diVerentfrom the other infectious disease management, such asAIDS, hepatitis B and C, among others that have thor-oughly applied all the technological advances for theimprovement of the clinical diagnosis and treatment. Thisseems to be a very limited approach for leprosy that hasbeen considered as a marginal disease; therefore, the cur-rent diagnostic tools must be applied extensively in the rou-tine to accumulate clinical experience in order to improvetheir precise application. The neglect of this approach mayobstruct the progress in leprosy diagnosis and treatmentstrategies. Additionally, the conWrmatory diagnosis of lep-rosy, beyond the obvious ethical issues, is very importantfor the correct clinical classiWcation in order to allocatepatients in the treatment schemes for PB or MB forms, withdiVerentiated incapacity risks.
Continuous training programs are also required and mayallow the immediate application of research innovations,including new diagnosis and treatment strategies. The greatdebt with leprosy patients is the broad attention, which goesfrom basic to the most complex assistance, and it is the onlyacceptable and decisive solution to eliminate the stigma ofleprosy.
The search for new antigens, including short and speciWcrecombinant peptides, using novel proteomic strategies,may provide novel approaches for diagnosis; immunogensfor a putative vaccine development, although controversial,may avoid an immune reaction cascade similar to an auto-immune response that leads to nerve injury. These molecu-lar strategies may also lead to the recognition of speciWctargets that are responsible for drug resistance and for theimprovement of the understanding of scape mechanisms ofthe pathogen, which will give opportunities for the devel-opment of rational drug design and novel epidemiologicaltools. The identiWcation of infectivity and pathogenecitymarkers may also alter the medical assistance modalities bycontributing to the chemoprophylaxis of contacts andrestricting the transmission chain by eliminating potentialhosts, and leading to the development of new therapies,such as gene and immune therapies for the aVected patients,especially those with neural damage.
The biotechnological revolution presents a brilliant per-spective with a strong impact on the leprosy control. Recentadvances in nanotechnology and nanomedicine may have aprofound inXuence on medical actions, allowing the popu-lation of developing countries also access to the mostimportant contributions of the science, and reducing thestigma of this disease, which is still considered as a minorone in most of the medical assistance centers.
Acknowledgments The authors apologize for all the interestingstudies by many other colleagues that were not mentioned in this re-view due to space and time limitations. The authors are grateful to allthe staV of the National Reference Center of Leprosy of the FederalUniversity of Uberlândia for fundamental support. This work was sup-ported by grants from FAPEMIG, CNPq, CAPES, FINEP, and theMinistry of Health of the Brazilian Government.
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