Archives of Dermatological Research

, Volume 300, Issue 6, pp 269–290

Leprosy: diagnostic and control challenges for a worldwide disease

Authors

    • Leprosy National Reference Center, Clinics’ Hospital, School of MedicineFederal University of Uberlândia (UFU)
    • Departamento de Clínica Médica, Faculdade de MedicinaFederal University of Uberlândia (UFU)
  • Luiz Ricardo Goulart
    • Leprosy National Reference Center, Clinics’ Hospital, School of MedicineFederal University of Uberlândia (UFU)
    • Institute of Genetics and BiochemistryFederal University of Uberlândia (UFU)
Review Article

DOI: 10.1007/s00403-008-0857-y

Cite this article as:
Goulart, I.M.B. & Goulart, L.R. Arch Dermatol Res (2008) 300: 269. doi:10.1007/s00403-008-0857-y

Abstract

Leprosy is a curable disease with well-defined etiology, but lacks better diagnostic tools, preventive and therapeutic strategies. The continued application of the Ridley–Jopling clinical classification that recognizes the natural diversity of the immune response has provided the basis for understanding leprosy, and this review proposes its implementation in all Reference Centers in order to standardize the diagnostic resources, aiming at the improvement of the disease control. Due to the broad bioepidemiological aspects of infection its eradication is difficult, and proper diagnosis of the disease and the correct clinical classification are required to ensure proper treatment. Tools and markers for diagnosis and prognosis, and the novel use of nanotechnology, as well as strategies for disease control and monitoring populations at higher risk are still continuous challenges, which will be specifically reviewed with additional insights. The use of the current diagnostic tools, such as ELISA and PCR has a very limited approach for leprosy that has been considered as a marginal disease; therefore, the current diagnostic tools must be applied extensively in the routine to accumulate clinical experience in order to improve their precise application, like what has been done in many other infectious diseases. Since a vaccine for leprosy presents an unpredictable future, the proposed chemoprophylaxis of contacts (healthy carriers and/or with subclinical infection) must also be employed in referral centers of endemic countries not only to evaluate its efficacy, but also because of the favorable cost–benefit ratio, given that there is no other available approach, besides the multi-drug therapy of patients. This strategy should readily be applied as a public health policy, and may lead to a substantial breakage of the transmission chain aiming a world without leprosy.

Keywords

LeprosyMolecular epidemiologyDiagnostic toolsMonitoring strategiesClinical classificationSerological assaysPCR tests

Introduction

Leprosy, a chronic infectious disease of humans caused by Mycobacterium leprae, is still a major health problem in Asia, Latin America, and Africa [204]. It presents a variable incubation period ranging from 6 months to more than 20 years, with an average period of 2–4 years, due to its very slow growth [151]. Another critical issue is that bacterial culture is not possible, and infection of humans is mandatory for bacterial transmission.

Global efforts to control leprosy by intensive chemotherapy have led to a significant decrease in the number of registered patients, but the detection rate of new cases has been kept constant, with a very small reduction, meaning that control strategies have not accomplished the aimed efficacy.

Leprosy has no primary prevention, which means there is no specific vaccine against M. leprae, and diagnostics and prognostic tests are not feasible or not well established in clinical routine [173].

The classification of leprosy was well established by Ridley and Jopling in 1966 [165], and surprisingly, without any molecular tool, they came up with a very important description of classification forms, which was the most important contribution for the understanding of the disease in the twentieth century [174].

The disease presents a broad clinical and histopathological spectrum that is correlated with the immunological response of the patient [165]. At one end of the spectrum, in the tuberculoid form, a specific cell-mediated immune response to M. leprae is observed, with lesions characterized by epitheliod granulomas, participation of lymphocytes mainly of Th1 type, and few alcohol–acid-resistant bacilli [214]. In contrast, in the most severe, or lepromatous form, the specific cell immune against M. leprae is absent, with diffuse dermal lesions characterized by poorly differentiated young macrophages with a heavy load of bacilli and a small number of T cells predominantly of the Th2 type [214]. In the spectrum of borderline leprosy there are varying degrees of cell-mediated immune response characterized patients with a low response to the bacillus [164]. However, the disease manifestations and complications are determined by the immune response of the host. Therefore, proper classification of leprosy is one of the fundamental issues for treatment and prognosis.

Many patients experience nerve damage before, during or after treatment [151]. The purpose of controlling leprosy is to reduce the rate and severity of disabilities. Therefore, the main objectives in leprosy management are the early diagnosis and treatment, followed by an early recognition of nerve damage and effective intervention.

Due to the high complexity of leprosy, the development of a vaccine and the use of a unique marker for diagnosis are questioned. A thorough review on leprosy has been performed elsewhere [173], but novel tools, markers for diagnosis and prognosis as well as strategies for disease control are still a continuous challenge, which will be specifically reviewed with additional insights.

Conventional diagnostic tools

Leprosy is insidious; initially affecting the peripheral nervous system [113], with patients exhibiting contrasting clinical, immunological and pathological manifestations [71], despite minimal genetic variation among M. leprae isolates worldwide [137]. Because the infection presents bioepidemiological aspects that do not contribute to its eradication [118], a diagnosis to confirm the disease is required to ensure that proper treatment is applied. However, it is extremely difficult to detect M. leprae in an individual, and various clinical and laboratorial criteria are used due to the absence of an exam defined as a gold standard [11].

For treatment purposes, the World Health Organization recommends an operational classification (OC) whereby patients are classified under paucibacillar (PB), when they present five or fewer cutaneous lesions, or multibacillar (MB) when they have more than five lesions [202]. However, in places where bacilloscopic examination is available, patients whose skin-smear exam tested positive are MB regardless of the number of lesions. For a better operational classification, some studies have used the M. leprae serum lateral flow test (ML-Flow), which correlates the concentration of anti-PGL1 (specific antibody against M. leprae) in the patient’s peripheral blood with the bacillary load [22]. Serum-positive patients are classified as MB and serum-negative ones as PB [21]. The simplification of the operational classification may mask the true relationships of the immunological response, and other intrinsic genetic factors, limiting the information and preventing further molecular findings that could support epidemiological data collection, treatment, and control strategies [174].

The basic criteria in Ridley and Jopling’s [165] classification are the bacillary load measured by bacilloscopic exams (cutaneous biopsy and skin smear) and the cell-mediated immune response time, which is evaluated from the result of Mitsuda’s intradermal test. Based on these immunopathological criteria, patients are divided into six clinical categories: indeterminate (I), tuberculoid (TT), borderline-tuberculoid (BT), mid-borderline (BB), borderline-lepromatous (BL), and lepromatous (LL).

Although this classification is important to better understand the disease, it is often not standardized in health services [198], where the majority has assumed the simplified classification of the WHO [203]. However, the Mitsuda test has been used for research purposes to evaluate the patients’ response in many different countries, such as Brazil [57, 132], China, Vietnam [160] and India [32].

Moreover, due to the neural damage and consequent disabilities and the stigma of leprosy for humans, the correct histopathological diagnosis is mandatory to guide the doctor about the spectral form of the patient’s disease and its prognosis, favoring a therapeutic conduct in his follow-up [23].

Although the prevalence of leprosy has declined worldwide, the number of new cases diagnosed annually has only slowly declined and is stable in some regions. This paradox raises new, important, and interesting questions that will require application of the best scientific methods available to answer it [174].

The confirmation of the leprosy diagnosis for the determination of the load of the disease in a population is an important motive for carrying out the histopathological exam [164]. The pathologist is expected to give a definitive diagnosis; however, this exam has some limitations, since samples do not always indicate the presence of the bacillus in patients with the characteristic symptomatology, leading to controversies about the efficacy of microscopy for the identification of the bacillus in smears and biopsies [79].

Biopsies extracted from opposite edges of the same skin lesion, or even from different lesions, do not present significant morphological discrepancies, as the individual’s bacillary load and his immunological reactivity are determined systemically [40]. However, there are frequent reports of inter-observer variations, proving the need for studies to evaluate them and to put forward suggestions to minimize them [59].

Other researchers in various countries have shown concordance between the clinical diagnosis of leprosy and the histopathological classification based on Ridley and Jopling’s criteria [165], which vary from 29.7 to 89.0% as shown in Table 1 [11, 55, 96, 98, 103, 113, 129, 138, 175, 180, 189].
Table 1

Concordance between clinical diagnosis and histopathological examination for classification of leprosy clinical forms

Clinical form

Investigations from 1997 to 2007

Sehgal et al. [176]

Dubey et al. [56]

Jerath and Desai [97]

McDougall et al. [130]

Bathia et al. [11]

Kumar et al. [114]

Singh et al. [181]

Kalla et al. [104]

Moorthy et al. [139]

Vargas-Ocampo [199]

Teixeira et al. [190]

I

88.8

0.0

36.0

77.8

60.0

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

100.0

64.7

68.4

77.0

57.7

66.7

44.2

66.5

77.2

BB

66.7

71.7

53.8

16.7

26.0

30.8

37.0

50.0

68.6

BL

42.9

100.0

28.5

37.5

26.0

30.8

43.7

70.0

58.8

LL

66.7

93.6

61.5

100.0

91.0

90.0

75.6

80.0

63.9

92.5

TT + BT

49.4

96.6

55.8

80.0

60.0

83.0

93.5

BT + BB + BL

59.4

100.0

59.5

80.0

75.4

83.0

52.4

89.0

BL + LL

59.4

100.0

50.0

93.0

65.4

91.2

General

29.7

89.0

68.5

40.4

69.0

51.7

58.6

64.6

62.6

42.9

67.6

When analyzed comparatively (Table 1), the examination and their classifications differ in relation to their efficacy, which implies the need for a critical analysis taking as reference the objectives of the control programs and the reality of the different endemic areas and of standardization of classification adopted in the international literature.

The Immunopathology Committee of the tenth International Leprosy Congress, held in Bergen (1973), recommended the use of Ridley and Jopling’s classification [165], both to establish a general nomenclature to render the diagnostic criteria uniform and to standardize scientific research in several countries [27]. It is also reported that the generalized use of this classification requires human and infrastructural resources that do not always exist in developing countries, but that the establishment of reference laboratories that can meet the needs of different regions should be an important goal in the study and control of this disease [189].

It should be kept in mind that the basis for understanding leprosy is the recognition that—clinically, histologically and immunologically—the LL form differs from the BL form, and the BT from the TT [164]. This classification system recognizes the natural diversity of the immune response in leprosy which has challenged immunology for almost half a century.

Therefore, the present review demonstrates that the clinical and laboratorial discrepancies in the diagnosis of leprosy should be minimized, providing data to underpin the construction of public health policies, standardizing the diagnostic resources, and aiming at the improvement of Reference Centers for the control of this disease [189].

To accomplish this main objective, the routine use of the Ridley–Jopling classification is necessary [165]. The borderline leprosy group is classified within the spectrum between the tuberculoid and lepromatous poles. It is the most important part of the spectrum in terms of number of patients and severity of nerve damage. It causes most of the disability and deformity seen in leprosy [151]. While mid-borderline disease may be rare, the ratio of BT to BL patients shows an interesting geographical pattern. BT predominates in Africans while BL predominates in Asians and Europeans. This difference presumably reflects a genetic difference in the ability to express cell-mediated immunity to M. leprae, since differences in bacilli strains do not seem to be correlated with clinical forms that are more related to the transmission dynamics of M. leprae from different geographical regions [217]. Besides, it has been demonstrated that all existing cases of leprosy are attributable to a single clone whose dissemination worldwide 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 damage, and the mechanism of how it happens is still uncertain [206]. Neuropathy may partially occur by bacterial invasion of the Schwann cells from the outside in, first aggregating in epineurial lymphatics and blood vessels and then entering the endoneurial compartment through its blood supply, as suggested in experimentally infected armadillos [172]. Once these cells are not professional phagocytes, they cannot destroy the mycobacteria. The fact of being inside phagocytes also confers other advantages to the M. leprae, once the mycobacteria is located in a site protected from defense mechanisms of the host. It is acceptable that the long permanence of the M. leprae in the peripheral nervous system may affect the neural function even before stimulation of the immune response [159].

The pure neural leprosy (PNL) is characterized by signs and neural symptoms marked with sensitive alterations, like paresthesia, or sensorial deficit equivalent to the area of the nerve enlargement, associated or not to the motor or trophic deficits, or autonomic, without skin lesions [142]. This form of manifestation of the disease is a well-recognized clinical entity, accounting for 4–16% of patients with leprosy in India [68]. A recent study of PNL shows an approximate 9.0% incidence in the southeastern Brazil [170]. The most commonly affected nerves in the PNL are the ulnar and common fibular nerves [68, 123, 162, 170].

In literature, there are only a few limited to the pure neural leprosy [123, 140, 187]. Among the many reasons for this, it is considered that: (1) patients tend to ignore early symptoms of nerve damage in developing countries, (2) health professionals do not understand leprosy as a primary neurologic condition, (3) the PNL diagnosis is underestimated, and finally, (4) when the PNL is clinically suspected, a nerve biopsy is not usually easy to perform [14].

Pure neural leprosy (PNL) is also difficult to diagnose because skin lesions and acid-fast bacilli (AFB) in slit smears are absent. For patients who exhibit only neurological involvement, even when subjected to a careful investigation for differential diagnosis of various neuropathies [14, 154], the gold standard for PNL diagnosis is the histopathological examination of peripheral nerve biopsies. Even so, detection of bacteria is difficult and histological findings may be nonspecific. Furthermore, nerve biopsy is an invasive procedure that is only possible in specialized centers [93].

In addition to the complexity in achieving the PNL diagnosis, another problem with this kind of leprosy manifestation is its classification, since the treatment depends on it. Conventionally, these patients are considered as belonging to the tuberculoid pole (TT and BT) of the disease spectrum, since many of them are Mitsuda positive. As these patients also have negative smears, they are classified as belonging to the paucibacillary (PB) group. However, several studies have shown that some of these patients present the standard lepromatous profile in nerves, with high bacillary load [35, 56, 106, 107, 113, 148, 170]. Therefore, the wrong classification and the incorrect treatment of these patients may end up in resistance to the medications and disease relapse, factors that hinder the leprosy control [113].

Many reports on PNL diagnosis have used clinical, electrophysiological and histopathological aspects, emphasizing the nerve injury in the classical forms of the leprosy spectrum [16, 33, 49, 101, 124, 176]. In recent years, several publications have emerged highlighting specific information for the PNL diagnosis [14, 45, 6163, 169, 181].

The electroneuromyography (ENMG) is indispensable to the studies of peripheral neuropathies [16]. Approximately 98% of patients in whom leprosy is confirmed by the traditional methods present electroneuromyographic alterations [124]. The most common and early finding is the reduction of the extent of the motor and sensitive responses to variable degrees in different nerves tested, which explains its character of “neuropathy in mosaic,” or asymmetric multiple mononeuropathy [124, 187]. Studies have shown its usefulness in the diagnosis of the disease at any stage or clinical form, particularly in the initial stages. So the ENMG has been shown not only as an effective method in the early diagnosis of leprosy, but also as a useful tool in evaluating the effectiveness of therapy [49]. Moreover, it has become a tool of great value in the indication of nerve biopsy, once it is proved that this is the only one that can assure the PNL diagnosis [14, 16, 49, 63].

Therefore, there is a need for additional diagnostic methods that may help to confirm the clinical diagnosis of PNL and this includes the electroneuromyography and nerve biopsies for investigation of M. leprae by either the conventional technique of Ziehl–Neelsen or the polymerase chain reaction (PCR), with a further confirmation by reference centers, avoiding false positive results.

New diagnostic tools

Molecular and immunological tests have been developed for leprosy diagnostics and prognostics, and among these tools, the PCR and its variations, ELISA (enzyme-linked immunosorbent assay) and other serological tests such as the lateral flow (ML-Flow) are the main technologies employed with different markers and strategies.

Imaging techniques, such as ultrasonography (US) and magnetic resonance imaging (MRI) have also been recently used to evaluate the pure neural leprosy, and will be presented. We will also discuss the use of these techniques in association with specific markers and their utility in diagnosis or prognosis.

Nucleic acid detection—diagnosis and challenges

Due to the difficulty of finding bacilli alcohol–acid resistant (BAAR), through histopathological methods in the early stages of the disease, the PCR technique has been used successfully to detect small quantities of bacilli in tissues [52].

The major advantages of PCR on other diagnostic methods are based on its fast, specific and sensitive identification of organisms, which can be done by analyzing crude biological 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 relation to M. leprae detection in different samples (blood, skin, swabs, nerves and nasal inferior turbinate) of leprosy patients and their contacts by PCR [14, 47, 48, 52, 58, 99, 133, 145, 155, 190, 205, 213]. The PCR technology has no doubt brought a great advance in the M. leprae detection, and its sensitivity may be limited from one [155] to five bacilli in the collected sample [156].

The PCR technique makes possible the detection, quantification and determination of the bacillus viability at specific sites, through DNA or RNA detection, giving valuable information about the infection and transmission of M. leprae, and the verification of the effectiveness of the MDT, as it also detects transcriptional activity of the bacillus (RNA) [30, 114]. In addition, the PCR tests are statistically superior in comparison to microscopic tests of biopsies [179, 186].

Variations in PCR positivity have been observed in literature, mainly due to the different primers, amplified fragment sizes, and amplification techniques [52, 73, 76, 104, 105, 112, 114, 126, 133, 145, 147, 152, 153, 197, 211, 215] (Table 2).
Table 2

Comparison of molecular technologies for nucleic acid detection of Mycobacterium leprae as leprosy diagnostic tools

Marker (access no. M. leprae genomic region)

No. of patients

Molecular technique

Fragment size (bp)

Sensitivity (minimum no. of molecules or DNA concentration detected)

Total positivity (%)

Positivity by clinical form (%)

Positivity (%)

Positivity (%)

Literature

TT

BT

BB

BL

LL

BI (−)

BI (+)

PB

MB

RLEP3 (X 17153)

102

PCR

372

NA

 

NA

NA

NA

NA

NA

73.3

96.6

NA

NA

Yoon et al. [216]

110

PCR

372

NA

52.7

13.3

33.3

64.7

83.3

95.2

NA

NA

28.3

82

Goulart et al. [74]

110

PCR

130

40 Bacilli

73.6

40.0

55.5

100

100

100

58.2

97.7

51.7

100

67

PCR

129

NA

80.0

0

0

NA

85.7

100

67.7

100

NA

NA

Kang et al. [106]

6

Nested PCR

99

1,000-Fold more sensitive than 36 kDa

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

Donoghue et al. [53]

85-A-C intergenic region (X 60934)

69

PCR

250

100 pg

82.6

NA

NA

NA

NA

NA

NA

NA

62.5

100

Martinez et al. [127]

85-B antigen (X 60934)

69

PCR

263

10 pg

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

69

Real time

80

25 fg

91.3

       

79.2

100

16S rRNA (X 58888)

49

RT-PCR

171

10 Bacilli

82.0

NA

NA

NA

NA

NA

NA

NA

NA

NA

Kurabachew et al. [115]

60

RT-PCR nested

171

1 Bacillus

78.3

NA

NA

NA

NA

NA

NA

NA

54

94

Phetsuksiri et al. [153]

58

RT-PCR + colorimetry

171

10 Bacilli

91.3

NA

NA

NA

NA

NA

NA

NA

80

100

Haile and Ryon [78]

141

NASBA

NA

NA

65.2

NA

NA

NA

NA

NA

NA

NA

90.3

16.7

Van der Vliet et al. [198]

14

PCR

405

20 Bacilli

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

Pattyn et al. [148]

Ml0049

(NP_301162)

7

iMDA + real time

NA

100 fg

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

Groathouse et al. [77]

36 kDa (praline-rich antigen) (X 65546)

122

PCR

531

3.125 fg (0.5 bacillus)

73.0

20

52.8

NA

96

94.3

44.2

95.6

44.2

95.6

Kampirapap et al. [105]

6

Nested PCR

530

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

Donoghue et al. [53]

22

PCR

530

NA

63.3

50

40

NA

100

100

61

92

NA

NA

de Wit et al. [48]

39

Real-time PCR

76

194 M. leprae cells

95.0

NA

NA

NA

NA

NA

33.3

88.9

33.3

88.9

Kramme et al. [113]

55

PCR

531

3.125 fg

63.6

0

52.9

100

100

85

NA

NA

36.4

87.1

Wichtwechkarn et al. [206]

20

In situ PCR

530

1–100 Bacilli

60.0

NA

50

44.5

100

NA

NA

NA

NA

NA

Dayal et al. [47]

60

PCR

531

10 Bacilli

38.3

NA

NA

NA

NA

NA

10

100

0

46

Torres et al. [191]

18 kDa (M 22587)

6

Nested PCR

110

100-Fold more sensitive than 36 kDa

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

Donoghue et al. [53]

53

PCR + probe

360/212

NA

60.3

38.8

NA

NA

NA

92.6

NA

78.9

NA

NA

Williams et al. [212]

67

PCR

360

NA

56.7

0

0

NA

64.3

67.6

0

66.7

NA

NA

Kang et al. [106]

LSR/A15 (15 kDa) (NP 301294)

46

PCR

321

100 Bacilli

34.8

0

14.3

NA

50

75

18.9

100

8.3

63.6

Misra et al. [134]

PCR conventional PCR, RTPCR reverse transcription PCR, real time real-time PCR, iMDA isothermal multiple displacement amplification, NASBA nucleic acid-based amplification (isothermal multienzymatic amplification), NA not available

Among other amplification techniques that have been employed for M. leprae detection, the real-time PCR [126] has reached the highest sensitivity (91.3%) detecting as low as five molecules (25 fg) of M. leprae using primers targeting the antigen 85-B coding gene, which was 17.7% superior to the conventional PCR presented in this investigation; however, those authors have used only BT patients as paucibacillary form, excluding TT patients. It is important to emphasize that some BT cases may be classified as multibacillary patients, once they present from 0 to 2+ acid-fast bacilli in the granuloma [165], and this could have led to misclassification of patients. A different result has been published with a real-time PCR approach [112], which has demonstrated that sensitivity was not different from the conventional PCR and positive detection reached 88.9% in MB cases and 33.3% in PB cases; however, with a different set of primers (proline-rich antigen, LEP, 36 kDa), which were inferior to results presented elsewhere [73] with a set of primers that amplify a 130-bp fragment of the RLEP3 region of the M. leprae (Table 2).

Primers that amplify short amplicons of the M. leprae genome have been successfully used even in damaged or in low concentrations of DNA, especially in the paucibacillary forms, demonstrating that the amplicon size may be a strong limitation for the M. leprae DNA detection [52, 73]. One of the explanations for the high sensitivity of the RLEP3 sequence is presented elsewhere; [52] compared the sensitivity of primers targeting the 18-, 36-kDa and RLEP M. leprae genomic regions, have concluded that the RLEP primer set was 10 and 1,000 times more sensitive than the 18- and 36-kDa sets, respectively, a sequence that is repeated 28 times in the M. leprae genome.

The most sensitive technique published in the literature was a nested PCR protocol [153] that presented a detection limit of 3 fg, and although performed in a short time, it may be considered with caution, since contamination in this methodology is quite common [86]. Other technologies, such as the whole genomic amplification [76], have been used with great success for molecular typing of M. leprae with detection limit as low as 100 fg; however, it has not been used for leprosy diagnosis.

The use of the polymerase chain reaction (PCR) in diagnosing PNL has been recently investigated [14], and 50% of paucibacillary patients presented positive results. PCR analysis proved to be a useful method to investigate PNL, enabling confirmation of the diagnosis in more than a third of the cases that were negative for AFB by nerve biopsy. Another study corroborates those results through a semiquantitative PCR [170] that has also shown a good correlation among the bacillary load in nerves with the Mitsuda test response and ML-Flow assay.

Despite its major importance, references in the literature for qPCR are scarce. Only a few investigations reassure its value as a method for diagnosis and for therapeutic follow-up in leprosy [77, 152, 170]. Therefore, the development of methods for detection and quantification of M. leprae are necessary for studies involving the epidemiology, pathogenesis and evaluation of the efficacy of chemotherapy in leprosy as proposed elsewhere [72].

Considering all the above results, it is clear that there is an urgent need to standardize the PCR technique, especially taking into account the primer target, the amplicon size, and the technique, seeking a confirmatory diagnosis that may have important implications in the epidemiology and control of the disease, as well as an ethical and social impact [189].

In brief, the PCR has become the gold standard for amplifying DNA and RNA from many microorganisms in diagnostic tests. The conventional PCR consists of amplification followed by electrophoretic separation, ethidium bromide staining and documentation. The greatest advantage of PCR (sensitivity and specificity) may easily become its biggest disadvantage since the reaction must present very stringent and specific conditions. The conventional technique has been used for diagnosis of many diseases, besides its high sensitivity, for two major reasons: feasibility and low cost of equipments and reagents. However, there are infectious diseases that require technical variations, such as semi-quantitative/quantitative approaches or nested reactions, which may be difficult to optimize or to perform without contamination. The development of the real-time PCR has overcome all technical problems, and as it becomes wide spread and disseminated, equipments and reagents will become cheaper, allowing any reference laboratory to perform such a test.

We believe that the real-time PCR will soon surpass the conventional technique by becoming the gold standard laboratory test for leprosy diagnosis, and among all nucleic acid markers in the literature (Table 2), three of them present significant results (RLEP3, 85-B and 16S rRNA). The 16S rRNA may become one of the most important markers due to its high abundance (each cell contains 1,000–10,000 copies) the RNA reflects organisms’ viability, its use may indicate the efficacy of chemotherapy distinguishing relapse from late reaction, and also may be used for epidemiological studies, as reported elsewhere [77, 114, 152]. The only disadvantage of this marker is that samples must be frozen immediately in −70°C or must be collected, transported and stored in appropriate media to maintain RNA stability, which is not available everywhere, and is still expensive.

Therefore, it is possible that a DNA-based marker must be used for leprosy diagnostics. However, one should use a repetitive sequence, such as the RLEP3, with great care, which may increase the sensitivity of the test; but many homologous repetitive sequences may be present in other Mycobacterium species that have not been thoroughly investigated, generating false positive results, as reported for 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 of real-time PCR and serology with specific markers, supported by the health systems of endemic countries, may provide enough evidence of markers that present false positive and false negative results, as it has been shown for the Mycobacterium Tuberculosis Direct Test that cross-reacted with M. leprae infection in USA [34].

Mycobacterium leprae resistance to drugs

Current recommended control measures for treating leprosy with MDT are designed to prevent the spread of drug-resistant M. leprae. However, drug resistance has been reported since 1964 for dapsone [150], 1976 for rifampin [89], and 1996 for ofloxacin [97].

This in vivo method requires at least 6 months and relatively large numbers of bacteria. Recently, there have been advances in the elucidation of molecular events responsible for drug resistance in mycobacteria [92, 140, 158].

Rifampin resistance is associated with mutations in the rpoB gene that encodes the β subunit of RNA polymerase. All the mutations associated with resistance in mycobacteria are localized in the 500–540 domains, a numbering system used for Escherichia coli RpoB [140].

Resistance to ofloxacin is known to be associated with mutation 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 isolates investigated for rifampin and fluoroquinolone susceptibility by both genetic analysis and standard mouse footpad method is rather small [26, 83, 208].

Dapsone resistance has been associated with three mutations in the folP1 gene of dapsone-resistant M. leprae isolates at positions 157, 158 and 164, altering the amino acid positions 53 and 55. They corresponded to threonine → alanine, threonine → isoleucine, and proline → leucine in dihydropteroate synthase (DHPS), respectively [102, 121, 209].

Generally, discontinuation of treatments and monotherapy play a major role in production of multidrug-resistant (MDR) bacilli. To prevent the emergence and transmission of MDR leprosy and to identify and treat existing cases of MDR leprosy, it is necessary to establish rapid methods for detection of drug resistance in M. leprae. However, M. leprae has not been cultivated on artificial media; therefore, to identify drug susceptibility patterns, bacteria must be tested using Shepard’s mouse footpad assay, MFP [178]. Besides the long time taken by MFP method, multibacillary cases are becoming fewer. Therefore, the molecular methods will be of special interest for detection of drug resistance in paucibacillary cases.

The molecular methods for detection of drug resistance in 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 an informal consultation on rifampicin resistance in leprosy [141] that is valid for the other antibiotic resistances: (a) drug resistance surveillance should be established in reference centers; and (b) PCR-based sequence analysis is the chosen methodology for simultaneous analyses of the rpoB, folP1 and gyrA genes (rifampicin, dapsone and ofloxacin, respectively). The chosen methodology may be easily established, but in a few centers of endemic countries; therefore, it is possible that other screening methodologies that require only PCR and electrophoresis, such as PCR-SSCP (PCR-single strand conformation polymorphisms) or melting temperature (Tm) curves by real-time PCR should be used prior to sequencing in order to detect existent mutations and novel ones, which is cheaper and easier to perform. If a Tm can be standardized for each gene mutation, a pharmacogenetic surveillance and monitoring may be quickly established.

Immunological tests

The identification of specific informative diagnostic antigens is one of the most difficult aspects in developing new diagnostic tools, and this is particularly true with leprosy, because there is a paucity of information involving the roles of many of the expressed proteins or the metabolic state of the organism throughout infection and disease progression [75].

Many studies have exploited genomic and proteomic sequences for the identification of M. leprae-specific proteins or peptides that may be suitable for serodiagnosis of different disease states of leprosy. While many of these studies described novel antigens that show marked humoral and cellular immunogenicity, none have reached useful accuracy (sensitivity and specificity). Investigations on antigens that trigger cellular and humoral immunity will be presented.

Cellular immunity

Tests that measure cellular rather than humoral immunity, such as skin tests, have also been developed in various forms since Mitsuda [17].

It is well known that the late lepromin reaction is a measure of the individual’s ability to generate a cell-mediated immune response to an immunizing dose of M. leprae, and also a measure of granulomatous hypersensitivity. Although the molecular mechanisms involved in the Mitsuda reactivity and resistance are not yet fully established, it has been clearly demonstrated that the long-lasting late lepromin negativity in leprosy endemic areas is associated with an increased risk of developing lepromatous leprosy [78].

Tests that measure cellular immunity to mycobacteria historically have relied on the use of mycobacterial extracts, or purified complex mixtures of mycobacterial components. In leprosy, purified M. leprae was initially used in the lepromin skin test [134], followed later by the use of soluble extracts of the bacillus, designated leprosin. Two specific 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 a more potent antigen than the cytosol fraction, probably due to the dominance of the 65-kDa GroEL antigens [201]. In tuberculosis, purified protein derivative of boiled M. tuberculosis has been used since the beginning of the last century in the classical Tuberculin Skin Test (TST). However, the diagnostic value of almost all of these tests is compromised by the presence of conserved, immunologically cross-reactive components that are shared with other mycobacteria, which results in low test specificity. For leprosy, such cross reactivity is particularly problematic in countries with high incidence rates of tuberculosis, routine BCG vaccination practice, and high levels of exposure to non-pathogenic environmental mycobacteria [64].

Although specific tests are needed to distinguish previous infection with M. tuberculosis or M. leprae from each other as well as from exposure to other mycobacteria, including BCG [66], it has been demonstrated that the specific cellular response raised by the Mitsuda test (reaction > 7 mm) may be an indicator of acquired protective immunity (odds ratio = 0.16, CI95% = 0.05–0.46) rather than an expression of hypersensitivity in household contacts, and it has also been proposed that the application of this test in endemic countries may be an important epidemiological approach for monitoring household contacts of leprosy patients [74].

The search for M. leprae antigens aiming at improved leprosy diagnosis still remains a challenge; although there are many potential targets (Table 3) most of them have only preliminary results in cell culture stimulation assays and lack either specificity or sensitivity for the detection of asymptomatic infections and disease progression [75].
Table 3

Mycobacterium leprae antigens and their potential use in diagnostics

M. leprae antigens

Cellular/humoral response

Candidate tissue (test)

Observations

Reports

Mitsudin (Mycobacterium leprae whole cell extracts)

Cellular

Skin test

The measure of local intradermal reaction on the injection site in leprosy patients is an important indicator of efficient cell immunity and good prognostics

A negative result (<8 mm) presents a risk six times higher for the disease incidence

Negative Mitsuda associated with no BCG scar and positive PGL-1 indicate a risk 25 times higher for the disease occurrence

Mitsuda [135]

Brenan [17]

Goulart et al. [75]

GroES

Cellular

Blood (ELISA)

High titer of IgG1 in all disease spectrum

Hussain et al. [85]

ML 35 kDa

Humoral

Blood (ELISA)

A monoclonal antibody inhibition enzyme-linked immunosorbent assay based on the Mycobacterium leprae 35-kDa protein. Specificity = 97.5%; sensitivity = 90%. For multibacillary forms

Triccas et al. [192]

MLSA-LAM (cytosol fraction)

Cellular

Skin test

Associated with proteins of 70, 65, 45, 35, 28, 18 and 10 kDa

Weir et al. [202]

MLCwA (fraction of cell wall)

Cellular

Skin test and blood (ELISA)

Associated with proteins of 70, 65, 45, 35, 31, 30, 28 and 18 kDa

ML0576; ML1989; ML1990; ML2283; ML2567

Cellular

Blood (ELISA)

Significant IFN-γ expression.

All five detected 94% of negative PGL-1 patients

Geluk et al. [67]

ML0308; ML2498

Cellular/humoral

Blood (ELISA)

Strong IFN-γ expression

M. leprae antigens with orthologues in other organisms

Aráoz et al. [6]

CFP-10 (ML0050)

Cellular/humoral

Blood (ELISA)

Culture filtrate protein 10 (10 kDa)—early gamma interferon response

Cross-reactive with M. tuberculosis

Synthetic peptides presented differential response

Dockrell et al. [52]

Geluk et al. [66]

Mehra et al. [132]

Spencer et al. [185]

ESAT-6 (ML0049)

Cellular/humoral

Blood (ELISA)

Cross-reactive with M. tuberculosis

Arend et al. [9]

Dockrell et al. [52]

Skjøt et al. [182]

Spencer et al. [184]

MMP-II (major membrane protein II)

Cellular

Cell culture

T cell activating candidate

It can be recognized as an immunomodulating protein in terms of activation of antigen-presenting cells and innate immunity

Maeda et al. [123]

Mycobacterium specific polyclonal versus mAb for 12, 35 and 65 kDa

Cellular

Skin scrapings

The polyclonal antibody presented the best sensitivity: 100% of MB, 76% of multiple-lesion PB, and 62% of single-lesion PB patients

Suitable for field diagnosis—dot ELISA format

Chaturvedi et al. [31]

ML0008, ML0126, ML1057, ML2567

Cellular

PBMC

Showed interferon responses only in paucibacillary patients

Spencer et al. [186]

ML0678, ML0757, ML2177, ML2244, ML2498

Humoral

Blood (ELISA)

Strongly recognized by circulating antibodies in MB leprosy patients

Aráoz et al. [7]

ML0308, ML0410, ML1553 ML1829

Humoral

Blood (ELISA)

Induced responses that displayed statistically significant differences in blood from PB patients with respect to the MB, PC and TB groups

PGL-1

ND-O-BSA (disaccharide form)

NT-P-BSA (trisaccharide form)

Humoral

Blood (ELISA)

Detects 74–100% of MB and 13.6% of PB patients

Used for operational classification

Cho et al. [3638]

Gonzalez-Abreu et al. [70]

Cellona et al. [29]

Cunanan et al. [44]

Buhrer et al. [20]

Bakker et al. [10]

Buhrer-Sekula et al. [21]

Goulart et al. [75]

Mimotopes of PGL-1 (two heptapeptides)

Humoral

Blood (phage-ELISA)

Mimotopes were obtained through phage display and were highly reactive against sera of MB and PB patients, reaching a seropositivity of 76% overall (data not shown)

Capparelli et al. [28]

ML0405, ML2331

Humoral

Blood (ELISA)

Specifically identify LL/borderline lepromatous (BL) patients on the basis of immunoglobulin G (IgG) reactivity

Reece et al. [162]

Ep1-0308 (synthetic peptide base don ML0308)

Cellular/humoral

Blood (ELISA)

Propose a simple and robust whole blood test for the early diagnosis of leprosy

Aráoz et al. [7]

These studies of the human T cell response in leprosy patients have identified a number of antigens that induce T cell responses, measured by lymphocyte proliferation or gamma interferon (IFN-γ) secretion in patients with tuberculoid 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 homology among members of the heat shock family between M. leprae and M. tuberculosis, it is not possible to use such antigens as diagnostic reagents [111]. Other antigens, such as the M. leprae 35-kDa antigen, have also been shown to have homology to M. intracellulare and M. avium, containing both specific and conserved T cell epitopes [207].

Synthetic peptide antigens (15 amino acids), representing potentially M. leprae-specific epitopes have been used to evaluate responses in leprosy patients from the tuberculoid pole, contacts and healthy individuals from non-endemic leprosy areas. Although initial findings were promising, immunological responses to these peptides were of poor specificity and sensitivity [51].

The development of improved skin test antigens has used two specific approaches. First, armadillo-derived M. leprae cells have been fractionated, and the proteins associated with the membrane, cell wall and cytoplasm have been purified, and immunologically characterized [125]. This first generation of antigens, which comprised the most abundant M. leprae proteins [i.e., major membrane protein I (MMP-I), MMP-II, antigen 85-B (Ag85B), elongation factor Tu (EF-Tu), and GroES], encountered serious problems of cross-reactivity with their counterparts in pathogenic as well as environmental mycobacteria [15, 38, 122, 212]. Second, recent studies employing genomics, bioinformatics and experimental approaches to evaluate individual M. leprae proteins or small sets of proteins as potential serodiagnostic or T cell antigens have also been performed [6, 66, 67, 161, 185] and generated a series of potential antigens (Table 3).

Candidate M. tuberculosis proteins that were found to be lacking from the M. bovis BCG genome were shown to have considerable value as potential diagnostic reagents for tuberculosis in human or cattle [4, 9]. The most characterized antigens are ESAT-6 and CFP-10. Recently, several other candidate molecules were reported, which in combination with ESAT-6 and CFP-10 provided enhanced specificity and sensitivity [3, 19, 117].

Scrutiny of the M. leprae genome revealed the presence of two candidate genes, ML0049 and ML0050 that encode the M. leprae homologs of ESAT-6 and CFP-10, respectively [64, 65, 131, 183, 184]. It was reported that recombinant M. leprae ESAT-6 and CFP-10 proteins were efficiently recognized by T cells from the majority of M. leprae-responsive leprosy patients [50, 64]. Despite the limited sequence identity with their M. tuberculosis homologues Rv3875 and Rv3874 (36 and 40%, respectively), significant immunologic cross reactivity (i.e., recognition by T cells from TB patients) was detected. This clearly limits the diagnostic potential of ML0049 and ML0050 encoded proteins in leprosy-endemic areas with a high prevalence of tuberculosis.

It is suggested elsewhere [7, 67] that the combination of peptides should increase specificity and sensitivity, and in conjunction with anti-PGL-1 serology; such a test would give satisfactory coverage of most forms of leprosy.

Humoral immunity

Tests that measure humoral immunity have relied mainly on detection of circulating antibodies against the M. leprae phenolic glycolipid-1 (PGL-1) antigen [10, 20, 21, 29, 3638, 44, 69, 115] (Table 3).

Infection can be detected by the presence of elevated titers of IgM antibodies against PGL-1, and may be a reflection of total bacterial load in the body than the bacilloscopic index of a local skin smear [21]; however, these antibodies are generally low or absent in paucibacillary patients. The seroprevalence among contacts has varied from 1.7 to 8.7% [10], and may reach up to 11.1% [74]. Follow-up studies have shown that seropositive contacts run an increased risk of developing leprosy [44], presenting a relative risk almost six times higher for the appearance of the disease [74].

Therefore, a sensitive and specific method to identify subclinical 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 provide novel classes of antigens with potential use in diagnosis.

The recognition of peptides by human serum samples has been generally weaker overall, but this is expected once assays have detected the antibody response to a single peptide or antigen among the many antigens produced by the mycobacteria. Nevertheless, some individuals may have a distinct response according to peptide preferences [185]. These results imply that multiple targets must be used at the same time in order to improve leprosy diagnosis.

In brief, new investigations on M. leprae antigens are still necessary and comprehensive field work must be performed with potential antigens for improvement of immunodiagnostics. However, the anti-PGL-1 and Mistuda assays are two simple, easy and cheap tools to be employed in endemic countries that may help identifying higher risk individuals for developing leprosy from the population [74].

Nanotechnology and biosensors

Although PCR has become the gold standard diagnostic tool for many diseases, other recent technologies are arising and may revolutionize the diagnostic field, such as the nanotechnologies.

Nanomolecular diagnostics is the use of nanobiotechnology in molecular diagnostics and can be termed nanodiagnostics [90]. Nanotechnology is the creation and utilization of materials, devices, and systems through the control of matter on the nanometer (one billionth of a meter)-length scale. Various nanotechnologies and their applications in life sciences are described in detail elsewhere under the term nanobiotechnology [91].

Biosensors are defined as analytical devices incorporating a biological material, a biologically derived material, or biomimic, intimately associated with or integrated within a physicochemical transducer or transducing microsystem [70]. Biosensors should be distinguished from a bioassay or a bioanalytical system, which require additional processing steps such as reagent addition [144], where the assay design is permanently fixed in the construction of the device.

Biosensors are usually classified into various basic groups, according to the signal transduction and biorecognition principles. On the basis of the transducing element, biosensors can be categorized as electrochemical, optical, piezoelectric, and thermal sensors [168]. The electrochemical biosensors, and among them the amperometric and the potentiometric ones, are the best described in the literature; those based on optical principles are the next most commonly used transducers. In fact, most catalytic biosensors are based on electrochemical methods, whereas affinity biosensors have generally proved more amenable to optical detection methods [192]. The various types of optical transducers exploit properties such as simple light absorption, fluorescence/phosphorescence, bio/chemiluminescence, reflectance, Raman scattering, and refractive index [41]. Surface plasmon resonance (SPR) is another common transduction mechanism whose main advantage over most optical biosensors is that the analyte presence can be determined directly, without the use of labeled molecules. Finally, cantilever biosensors are an emerging group of biosensors, which are based on the bending of silicon cantilevers caused by the adsorption of target molecules onto the cantilever surface, where receptor molecules are immobilized.

Sensing occurs when there is an interaction between the target molecule and a biological macromolecule (e.g., enzyme, antibody, receptor or DNA strand). Therefore, according to the biorecognition principle, biosensors are classified into immunochemical, enzymatic, nonenzymatic receptor, whole-cell, and DNA biosensors. Immunosensors present the advantages of sensitivity and selectivity inherent to the use of immunochemical interactions [168], although cross-reactivity may be observed.

Recently, our group has reported experiments showing the coupling of electrochemical biosensors with PCR amplicons to detect the M. leprae DNA for diagnostic purposes [2]. Briefly, after the functionalization of polymers with aminophenols [18], the surface of the electrode was conjugated with a thermally denaturated 78-pb DNA fragment, which was PCR amplified from the RLEP3 repetitive sequence of the M. leprae genomic DNA. Detection of the target complementary DNA was performed using ferrocenecarboxyaldehyde as indicator of the hybridization. The M. leprae DNA detection without any amplification was performed in 3-min hybridization, indicating that a field portable electrochemical device may become a reality for bacilli DNA detection, which could also be used for detection of antibodies, consequently affecting diagnosis, epidemiological, pharmacogenetic, and monitoring programs of leprosy.

Diagnostic imaging

In leprosy, bone lesions due to direct invasion of bacilli are low in incidence and these lesions exhibit radiologic findings of acute and chronic osteomyelitis, similar to those of other granulomatous infectious agents. The more common bone lesions are those due to injurious effects of trauma and infection imposed upon denervated tissues [149]. Radiographically various degrees of reabsorption of the extremities are seen involving hands and feet with the loss of digits and disorganizing arthropathies in small joints. The radiologic appearance is similar to other conditions in which there is sensory impairment like scleroderma, syringomielia and diabetes mellitus. The ultrasonography (US) and magnetic resonance imaging (MRI) can be helpful in evaluation of the involvement of the peripheral nerves helping in the diagnosis of the neuritis, abscess and differential diagnosis in compressive syndromes [149]. The commitment of peripheral nerves in leprosy occurs by direct invasion of the bacillus in the reactional states, especially in the reverse reaction (RR), where the inflammatory process can result in intense irreversible damage [163].

Although the high-resolution US is an effective form of image to show morphological alterations of peripheral nerves, the value of the US for the diagnosis of diseases of the peripheral nervous system is poorly understood [82]. Furthermore, descriptions of MRI features of peripheral nerve involvement in leprosy are also sparse in literature [80]. In leprosy, these methods are also little studied to aid in the diagnosis of neuritis.

The refinement of high-frequency broadband linear-array transducers, and sensitive color and power Doppler technology, have improved the ability of US to detect fine textural abnormalities of tender tissues as well as to identify a variety of pathological conditions. In nerve imaging, US can support clinical and electrophysiological testing for detection of compressing lesions caused by nerve entrapment in a variety of osteofibrous tunnels of the limbs and extremities [128].

The improved soft-tissue definition afforded by MRI may be useful in evaluating neural involvement [80]. MRI may show diffuse edema and swelling of the involved nerve due to neuritis. However, these findings are quite non-specific and the differential diagnosis includes other hypertrophic neuropathies like Refsum’s disease, amyloid infiltration, chronic relapsing polyneuritis and Guillain–Barre syndrome [13]. Presence of nodules or nerve sheath granulomas is suggestive of leprosy [167]. Analyses of the peripheral nerves with ultrasonography and magnetic resonance imaging in leprosy have been performed elsewhere [127] which have classified leprosy nerves into three groups based on imaging appearance; group I consisted of normal appearing nerves, group II included enlarged nerves with fascicular abnormalities, and group III included nerves with absent fascicular structures. They found Doppler US and MRI to have sensitivity of 74 and 92%, respectively, in identifying active reversal reactions, based on detection of endoneural color flow signals, increased T2 signal and Gadolinium enhancement.

A recent study has showed that the US presented a well defined cord-like hypoechoic lesion along the left common peroneal nerve. On MRI the peroneal nerve was enlarged and was isointense to muscle on T1Wimage and had high signal on STIR sequence. It was concluded that MRI may exclude nerve abscess in cases of tender neuropathy. The differential diagnoses in the present case included ulnar nerve abscess, peripheral nerve tumor and reversal reaction. MRI appearance of well defined ovoid lesion with peripheral rim enhancement and central necrosis favors nerve abscess. Differentiation of ulnar nerve abscess from reversal reaction is important as reversal reaction can be managed conservatively with steroids whereas ulnar nerve abscess may need surgical decompression [95]. The MRI also helps in the differential diagnosis of osteomyelitis and neuropathic arthropathy, which is difficult with other techniques of image [149].

Nerves represent probably one of the best applications of musculoskeletal US due to the high-lesion detection rate and accuracy of US combined with its low cost, wide availability, and ease of use. A focused US examination can be performed more rapidly and efficiently than MR imaging [128]. It has also been suggested that the conduct of serial US examinations could be of value to monitor the reactive processes during treatment, particularly where it is clinically impossible to determine whether the patient is in remission. The US would be useful also in the selection of more affected nerves for which surgical decompression or even neurolysis would be indicated. Besides, the US may also be a useful tool in the PNL diagnosis [127].

Epidemiological aspects: transmission, infection, and monitoring strategies

Leprosy is a curable disease with well-defined etiology, but lacks better diagnostic tools and therapeutic strategies, which together with the socio-cultural prejudice becomes an important obstacle to overcome for early detection and protection of the susceptible population, especially for the household contacts of leprosy patients, who should be given priority in disease control programs in order to interrupt transmission and reduce physical and social disabilities [74]. Household contacts of leprosy patients are the highest risk group for the development of the disease, and although many risk or prevention factors have been identified, they have not been employed in leprosy-monitoring programs.

Leprosy prevalence is always low in statistical sensus. Even in high-endemic areas the prevalence rarely exceeds 5%. The explanation for these low prevalence indices is the unavailability of valid and reproducible tests for detection of sub-clinical infection [87].

Transmission of M. leprae infection is not significantly affected by current leprosy control measures. In addition to delayed or missed diagnosis of infectious leprosy patients, the lack of tests to measure asymptomatic M. leprae infection in contacts prevents assessment of transmission of M. leprae. Therefore, a key priority is the development of specific and sensitive diagnostic tools that detect M. leprae infection before clinical manifestations arise [87].

Possibly, there may be an undefined number of infected asymptomatic people, who may present an important active role on the disease transmission, preventing its effective control [119]. The evaluation of the exposure and the infection onset of the disease may be almost impossible, once bacterial culture is not possible, and the incubation period is long and variable, besides the existence of paucibacillary forms that are difficult to be detected by conventional optical microscopy [163].

The use of PCR in the field of molecular diagnostics has increased to the point where it is now accepted as the standard method for detecting nucleic acids from a number of sample and microbial types. PCR is the most commonly used nucleic acid amplification technique for the diagnosis of infectious diseases surpassing the probe and signal amplification methods. The PCR may be too sensitive for some applications including to detect a microbe that is present at non-pathogenic levels [120].

The nasal mucosa is the preferential site for the entry and exit of M. leprae as shown by the bacilli colonization of the nasal inferior turbinate [145]. Even in PNL patients in whom the disease is believed to be confined to the peripheral nerves there are widespread effects with specific changes of leprosy in the nasal mucosa seen in 51% of the patients, confirming that early leprosy involvement can be found in the nasal mucosa even before lesions become apparent in the skin or other parts of the body [187].

Untreated MB patients are probably the most important source of transmission of M. leprae. Household contacts of MB patients have been estimated to have a 5–10 times greater risk of developing leprosy than that of the general population [53, 54, 60, 195]. However, in many areas, the number of MB patients is very small and they may not represent the most important source of infection [87]. There is increasing evidence that subclinical transmission may occur [136], because even in highly endemic countries, no history of close contact with a leprosy patient can be established for many patients [60].

Nasal excretion of M. leprae by healthy carrier individuals could be responsible for transmission. M. leprae-specific DNA sequences have been detected by PCR on nasal swabs from many apparently healthy individuals residing in endemic areas [72, 81, 88, 110, 146, 156, 193] and large proportions of those who live in endemic areas show seropositivity against M. leprae specific antigens [88, 156, 195].

There is increasing evidence from nasal PCR studies of temporary carriage or even subclinical infection [39, 81, 100, 110, 194] that infected persons may go through a transient period of nasal excretion, indicating that the mycobacterium is highly infective [81]. Patients’ household contacts, neighbors, and social contacts have an increased risk of contracting the disease [196]. Nasal carriage of M. leprae in healthy people may have important implications for leprosy control, once it is difficult to visualize the widespread exposure without the existence of sources of transmission other than MB patients alone [110]. Other strong evidence on the involvement of contacts in the transmission chain is the presence of M. leprae DNA in the nasal mucosa biopsies (inferior turbinate) in 10% of household contacts [145] and in 4% in the nasal swab [100], which also confirms that the nose is major port of entry and exit of M. leprae. These findings also support results elsewhere [177] that have demonstrated the affinity of M. leprae for the nasal mucosa and head sinuses, which depend on the bacilli viability and mucosa integrity.

Although nasal carriage may not necessarily result in infection or excretion of bacilli, the finding of nasal carriage evidences the disseminated occurrence of M. leprae in contacts [100, 145] and leprosy-endemic populations [110] and its probable role as a reservoir for maintenance of bacteria [177].

In a preliminary investigation for the presence of M. leprae DNA in blood samples of 110 patients and 434 contacts, the general positivity was, respectively, 18.2 and 8.9% [8]. The presence of M. leprae DNA in the blood of healthy carriers provides additional epidemiological evidence that the route of M. leprae transmission is not only the upper airways, and may indicate possible transmission through the blood, which may affect blood bank routine tests in the future. This hypothesis may be corroborated by case reports in nonendemic areas [116, 135], in which leprosy was acquired after organ transplantation. However, these two case reports must be carefully investigated to demonstrate that recipient patients have not received contaminated blood during transfusion, since there is no scientific proof that indirect transmission, such as the chain armadillo–dog–man, is possible.

All evidences of M. leprae DNA detection in many tissues are important for epidemiological studies, but serological markers may also be a useful tool. Several studies have shown that antibody levels can be used as a substitute marker for the bacterial load in the sense that there is a positive correlation between antibody levels and the bacterial index [166]. Current evidence suggests that serological tests could be useful in defining high-risk contacts [12, 136]. But, it is difficult to predict which seropositive contact will develop leprosy, because the presence of circulating antibodies is not an indication of active disease and may only be an indication of a recent infection [44]. However, seropositivity contacts run an increased risk of developing leprosy, especially in MB leprosy [44, 74]. In addition, it has been demonstrated that seropositivity among contacts was most closely related to the serological status of the index patient, and thus, the serological status of the patient seems to be a better indicator for the transmission potential than the BI [10]. The fact that most of the MB patients produce antibodies against M. leprae suggests that the seropositivity in contacts may be a marker of incubation of the multibacillary infection, and not a marker of general infection [10]. This same study argues that the seroprevalence is higher among people living in close proximity to seropositive patients (≤75 m) and this may be important for a more accurate estimation of transmission potential to measure the serological status of all patients and contacts, with a concept of contact expanded [196], people living in neighboring houses, the stone-in-the-pond model as used in tuberculosis control [199].

Based on earlier findings it is possible to state that there is subclinical infection in leprosy and many times it corresponds to the incubation period of the multibacillary disease [54]. It has been found that a maximum duration of seropositivity prior to diagnosis of is 9 years, indicating the long incubation period prior to clinical diagnosis. This group of leprosy patients likely poses a serious threat to the control of the transmission of leprosy and should be given chemoprophylaxis aiming the prevention of new cases and opening the way for a rational program for eradication [54].

An epidemiological study [74] in a Brazilian endemic area was carried out over a 5-year period to measure the relative risks of leprosy occurrence and its clinical forms in household contacts. Three simple clinical procedures, the BCG vaccination, the Mitsuda test and the ML-Flow assay, have been evaluated to determine the specific risks for the development of leprosy or protective effects against this disease. Based on their results, it was suggested that an additional intradermal BCG booster dose be maintained in Leprosy Control Programs for household contacts, aiming for protection against leprosy, mainly against MB forms. The authors have shown that the BCG vaccination and the Mitsuda test showed a protective effect against leprosy of 0.27 (at least one scar) and 0.16 (>7 mm), respectively, and the positive ML-Flow test indicated a relative risk approximately sixfold higher for occurrence of the disease. All unfavorable combinations of two and three assays generated significant risk values that ranged from 5.76 to 24.47, with the highest risk given by the combination of no BCG scar, negative Mitsuda test, and positive ML-Flow test.

It was also suggested that the BCG vaccination may be given to stimulate Mitsuda test positivity, reducing the patient’s risk of developing multibacillary forms [74], which corroborates other reports [42, 43, 139] that were decisive in establishing the adoption of two intradermal BCG doses as a control measure for the household contacts of leprosy patients. Prior to the BCG vaccination, the Mitsuda specific cellular immunological assay and the ML-Flow test must also be used in contacts to identify individuals at a higher risk of developing leprosy.

Finally, elsewhere [74], the following approaches for reduction or control leprosy transmission have been suggested: (1) household contacts of leprosy patients must be monitored during the first year after diagnosis of the index case; (2) an additional intradermal BCG booster dose must be given in Leprosy Control Programs for household contacts, aiming for protection against leprosy, mainly against MB forms; and (3) the use of the combination of the three assays may discriminate individuals at a higher risk for developing leprosy from contacts with significant protection factors, which could lead to a closer monitoring program for those at risk, as well as a subsidized new and effective control strategy for leprosy. This proposal may justify the chemoprophylaxis of close contacts of leprosy patients who fit the highest risk categories defined in this study.

Chemoprophylaxis

The workshop on the use of chemoprophylaxis in the control of leprosy, held in Amsterdam, the Netherlands, in 2006 [143] is one of the most important reports to review current evidences and to discuss potential future courses of action with regard to the use of chemoprophylaxis to prevent leprosy, in this following, emphasis will be on the most important aspects reported.

Chemoprophylaxis is targeting those subjects with suspected sub-clinical leprosy infection. From an operational point of view, it would be highly desirable if chemoprophylaxis is administered in no more than a single dose. Consequently, the regimen should display powerful bactericidal activity against M. leprae by a single-dose of treatment.

Optimal chemoprophylaxis should provide maximum efficacy and minimal risks (adverse effects, resistance). Candidate antimicrobial agents for chemoprophylaxis should have the following: (1) fast oral absorption without gastro-intestinal interactions; (2) fast intracellular penetration into infected tissues; and (3) slow elimination (long half life) to allow prolonged effect and once-only regimens. Adverse reactions are generally a minor issue for (potential) chemoprophylactic agents.

There are four antimicrobial agents, i.e., rifampicin (RIF), rifapentine (RFP), moxifloxacin (MXF) and R207910 (a diarylquinoline), displaying similar but very powerful bactericidal activity, i.e., killing at least 90% of viable M. leprae, at a single dose. Therefore, the first choice of the chemoprophylactic regimen would be a single dose of 600 mg RIF, or 10 mg/kg body weight RIF for children. Although the combination of rifampicin–ofloxacin–minocycline (ROM) has been employed as monthly administered regimen for treatment of leprosy with promising results, a single dose of ROM is no more effective than a single dose of RIF alone; furthermore, addition of ofloxacin and minocycline to RIF will increase the cost and the risk of side-effects. Therefore, ROM should not be employed for prophylactic purpose. Finally, a person with sub-clinical leprosy infection is most likely skin-smear negative, and therefore, harbors no more than 106M. leprae, or 105 viable M. leprae, in the body; it is very unlikely that a single RIF-resistant mutant would be included in such bacterial population; therefore, the risk of emergence of RIF-resistance by a single dose of RIF monotherapy is probably negligible. On the other hand, if, for whatever reason, the bacterial population size is larger than expected, and even if it includes RIF-resistant mutants, the emergence of rifampicin resistance is still very unlikely, because a single dose RIF is insufficient to select the resistant mutants, as has been shown in MB patients who relapsed after a single dose of RIF’s treatment.

A number of requirements should be fulfilled, and it is advised that,
  1. 1.

    Contacts should be screened by a health worker for leprosy and TB prior to the provision of chemoprophylaxis.

     
  2. 2.

    Chemoprophylaxis should be provided under direct observation.

     
  3. 3.

    A system for recording and reporting of prophylaxis distribution should be available.

     
  4. 4.

    Health workers need to be informed of such a policy and those directly involved need to be trained in selection and distribution.

     
  5. 5.

    People receiving chemoprophylaxis should receive proper information about its effects so as to leave them with realistic expectations.

     
  6. 6.

    A system for antibiotic resistance monitoring should be in place.

     
  7. 7.

    There is a need for discussion and approval of any such program with the TB (and other infectious disease) authorities.

     

In our opinion, based on the various evidences, the chemoprophylaxis may be indicated for contacts of patients with subclinical infection (anti-PGL-1 positive serology and negative Mitsuda) in health services as an effective measure in preventing secondary leprosy cases, although it does not guarantee absolute and prolonged protective effect. For Reference Centers, it is also suggested and desired that contact healthy carriers that present positive M. leprae DNA in nasal swab, nasal mucosa and blood must receive prophylactic treatment and the epidemiological surveillance must be maintained for 5 years, the average period of incubation of the disease.

In conclusion, considering that a vaccine for leprosy is still a challenge, and with an unpredictable future, the proposed chemoprophylaxis of contacts as a routine practice must be employed by referral centers of endemic countries not only to evaluate its efficacy, but also because of the favorable cost–benefit ratio, since there is no other available approach that could lead to a substantial breakage of the transmission chain as a complementary action to the MDT (multi-drug therapy) treatment.

Summary and conclusions

The proper classification of leprosy, using the Ridley–Jopling system, is a fundamental tool to the basis of understanding the disease, allowing the selection of treatment modalities, prognosis evaluation, and the improvement of the disease control.

There is no specific vaccine against M. leprae, and it is still a great challenge for research development due to the possibility of eliciting a complex immunological response that could lead to neural damage in asymptomatic individuals, which may also be infected. It is well known that the protective immunity is the cellular response, which is responsible for the pathogenesis of the nerve injury, and the humoral response does not protect against the bacilli dissemination.

Therefore, the lack of an effective primary prevention measures has led scientists to search for tests that may detect at early stages the M. leprae in order to interrupt its transmission and to prevent nerve damage and deformity of leprosy patients.

The use of the current diagnostic tools, such as ELISA and PCR, has been used only for research purposes, and unfortunately has not been implemented in the leprosy clinical routine. However, this restricted clinical practice has been applied only in leprosy, which has been quite different from the other infectious disease management, such as AIDS, hepatitis B and C, among others that have thoroughly applied all the technological advances for the improvement of the clinical diagnosis and treatment. This seems to be a very limited approach for leprosy that has been considered as a marginal disease; therefore, the current diagnostic tools must be applied extensively in the routine to accumulate clinical experience in order to improve their precise application. The neglect of this approach may obstruct the progress in leprosy diagnosis and treatment strategies. Additionally, the confirmatory diagnosis of leprosy, beyond the obvious ethical issues, is very important for the correct clinical classification in order to allocate patients in the treatment schemes for PB or MB forms, with differentiated incapacity risks.

Continuous training programs are also required and may allow the immediate application of research innovations, including new diagnosis and treatment strategies. The great debt with leprosy patients is the broad attention, which goes from basic to the most complex assistance, and it is the only acceptable and decisive solution to eliminate the stigma of leprosy.

The search for new antigens, including short and specific recombinant peptides, using novel proteomic strategies, may provide novel approaches for diagnosis; immunogens for a putative vaccine development, although controversial, may avoid an immune reaction cascade similar to an autoimmune response that leads to nerve injury. These molecular strategies may also lead to the recognition of specific targets that are responsible for drug resistance and for the improvement of the understanding of scape mechanisms of the pathogen, which will give opportunities for the development of rational drug design and novel epidemiological tools. The identification of infectivity and pathogenecity markers may also alter the medical assistance modalities by contributing to the chemoprophylaxis of contacts and restricting the transmission chain by eliminating potential hosts, and leading to the development of new therapies, such as gene and immune therapies for the affected patients, especially those with neural damage.

The biotechnological revolution presents a brilliant perspective with a strong impact on the leprosy control. Recent advances in nanotechnology and nanomedicine may have a profound influence on medical actions, allowing the population of developing countries also access to the most important contributions of the science, and reducing the stigma of this disease, which is still considered as a minor one in most of the medical assistance centers.

Acknowledgments

The authors apologize for all the interesting studies by many other colleagues that were not mentioned in this review due to space and time limitations. The authors are grateful to all the staff of the National Reference Center of Leprosy of the Federal University of Uberlândia for fundamental support. This work was supported by grants from FAPEMIG, CNPq, CAPES, FINEP, and the Ministry of Health of the Brazilian Government.

Copyright information

© Springer-Verlag 2008