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Keywords:

  • Epidemiology;
  • immunohistochemistry;
  • leprosy;
  • Mycobacterium leprae ;
  • oral mucosa;
  • polymerase chain reaction

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Transparency Declaration
  9. References

In leprosy, the nasal mucosa is considered as the principal route of transmission for the bacillus Mycobacterium leprae. The objective of this study was to identify M. leprae in the oral mucosa of 50 untreated leprosy patients, including 21 paucibacillary (PB) and 29 multibacillary (MB) patients, using immunohistochemistry (IHC), with antibodies against bacillus Calmette-Guérin (BCG) and phenolic glycolipid antigen-1 (PGL-1), and polymerase chain reaction (PCR), with MntH-specific primers for M. leprae, and to compare the results. The material was represented by 163 paraffin blocks containing biopsy samples obtained from clinically normal sites (including the tongue, buccal mucosa and soft palate) and visible lesions anywhere in the oral mucosa. All patients and 158 available samples were included for IHC study. Among the 161 available samples for PCR, 110 had viable DNA. There was viable DNA in at least one area of the oral mucosa for 47 patients. M. leprae was detected in 70% and 78% of patients using IHC and PCR, respectively, and in 94% of the patients by at least one of the two diagnostic methods. There were no differences in detection of M. leprae between MB and PB patients. Similar results were obtained using anti-BCG and anti-PGL-1 antibodies, and immunoreactivity occurred predominantly on free-living bacteria on the epithelial surface, with a predilection for the tongue. Conversely, there was no area of predilection according to the PCR results. Mleprae is present in the oral mucosa at a high frequency, implicating this site as a potential means of leprosy transmission.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Transparency Declaration
  9. References

There has been a notable decline in the global prevalence of leprosy, which has been primarily attributed to the use of multidrug therapy. However, 130 countries and territories submitted reports of leprosy to the World Health Organization (WHO) by the beginning of 2011, specifically from regions in Southeast Asia, North and South America, Africa and the Western Pacific. Worldwide, a total of 228 474 new cases were detected in 2010, and the global registered prevalence for the first quarter of 2011 was 192 246 cases [1]. Early diagnosis and the appropriate treatment of patients are the key elements for blocking disease transmission [2].

The mode of transmission of leprosy remains unclear; however, the upper respiratory tract, particularly the nasal mucosa, is considered as the primary route of entry and elimination of M. leprae [2]. There have been many studies showing the role of the nasal mucosa in the transmission of M. leprae; however, few studies have been conducted in the oral mucosa. Some reports indicated the participation of the oral mucosa in leprosy transmission [2, 3], particularly when there are leprosy-specific lesions [4].

Such lesions have been described in multibacillary (MB) patients in advanced stages of the disease [5-8]. Paucibacillary (PB) patients or those patients with incipient disease rarely present lesions in the oral mucosa [9].

Recent studies have described the rarity of oral lesions in leprosy [9-14], possibly due to the effectiveness of multidrug therapy. However, alcohol-acid-resistant bacilli (AARB) have been detected in the clinically normal oral mucosa of MB patients [7, 13, 15-17]. Recently, M. leprae DNA was amplified from oral mucosa samples [3, 18] in MB and PB patients using polymerase chain reaction (PCR), showing that sensitive techniques can detect the presence of bacilli even when they are undetectable by routine examination.

Immunohistochemistry (IHC) with antibodies that are directed against M. leprae antigens is another sensitive technique for detecting the bacillus, and this technique preserves tissue integrity. Among the various antibodies, anti-BCG [19] and anti-PGL-1 [20, 21] are the most widely used.

Our aim was to investigate the presence of M. leprae, using IHC and PCR, in the oral mucosa from leprosy patients and to compare the results from PB and MB patients, the preferred location of the bacillus, and the efficacy of the methods in detecting M. leprae antigens and DNA.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Transparency Declaration
  9. References

This was a cross-sectional retrospective study approved by the Research Ethics Committee of São Paulo Federal University (CEP 0609/04 and amended on 10 January 2010). The study analysed 163 oral mucosa biopsy samples included in paraffin blocks from 50 leprosy patients before starting treatment. The biopsy samples were systematically obtained from clinically normal mucosa, including the buccal mucosa, soft palate and tongue at pre-established points, totalling 150 samples, and 13 additional biopsy samples of clinically suspected leprosy-specific lesions that were noted in any part of the oral cavity. Histopathology revealed no specific impairment of the oral mucosa in any of the samples, excluding two normal mucosa samples from the same MB patient in which granulomas and AARB were evident [9, 13, 14].

The patient charts were reviewed, and their ages, gender and clinical forms of leprosy according to the WHO operational classification [22] were recorded. For inclusion in the study, the paraffin blocks for each patient must have contained sufficient material to generate the IHC sections and extract genomic DNA.

Immunohistochemistry

Immunohistochemistry was performed using the method of third-generation polymers labelled with immunoglobulins and peroxidase [23] with anti-BCG (rabbit anti-BCG, code B0124; Dako A/Sm, Glostrup, Denmark) and anti-PGL-1 (anti-M. leprae produced in rabbits, Institute of Tropical Medicine, College of Medicine, University of São Paulo, Brazil) polyclonal primary antibodies.

The microscopic analysis of immunoreactivity was performed in the cytoplasms of the epithelial cells, within macrophages and nerves in the corium and on free-living bacteria on the epithelial surface. The presence of a brown precipitate at the site indicated a reaction between the antigen and the primary antibody and was considered positive. Skin from an MB patient was used as a positive control, and the primary antibody was omitted for the negative control.

Polymerase chain reaction

For the PCR analysis, two 5.0-μm sections from each sample were deparaffinised with xylene, hydrated in decreasing concentrations of ethanol, and lysed using a buffer (1 M Tris, pH 8.0, 0.5 M EDTA, 10% SDS, 1 M NaCl, and sterile water) to which 500 μg/mL proteinase K was added every 24 h for 3 day. DNA was extracted using a 4-M ammonium acetate solution and precipitated with isopropanol. After quantification, 5.0 μL of DNA was used per reaction.

A previously designed pair of primers that was specific to a 336-bp internal sequence of the manganese ion transporter gene (MntH) was used to detect M. leprae bacilli, according to a standardized protocol for PCR [24].

The specificity of these primers was confirmed using cultured samples of Mycobacterium tuberculosis and Mycobacterium avium complex, in which no amplification was detected, thus excluding any cross-reactivity between the M. leprae MntH primers and sequences from other mycobacteria. In addition, 10 samples with positive PCR products for the M. leprae MntH gene were randomly selected, and PCR was performed using primers to amplify a 383-bp sequence for other Mycobacterium spp., which yielded a negative result for all of the tissue samples and positive amplification only for cultured M. tuberculosis [24].

For all of the oral mucosa samples that yielded negative PCR results for the M. leprae MntH gene, an additional PCR was performed with keratin-specific primers, which amplified a 343-bp fragment, to confirm that human genomic material was present [24].

To confirm the sequences of the PCR products that were amplified using the M. leprae MntH primers, six samples were sequenced, and they indicated identity and homogeneity to a M. leprae MntH gene sequence in GenBank (AL583924 and GI: 13093618).

Statistical analysis

The chi-squared test, Cochran-Mantel–Haenszel test and Fisher's exact test were used to evaluate the associations between variables, depending on the nature of the data. An analysis of variance was used to determine significant differences between the groups. The agreement between methods was assessed using a kappa analysis. The kappa values were interpreted as follows: <0, no agreement; 0–0.19, poor agreement; 0.20–0.39, fair agreement; 0.40–0.59, moderate agreement; 0.60–0.79, substantial agreement; 0.80–1.00, almost perfect agreement. The minimum level of significance considered was 5%.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Transparency Declaration
  9. References

The sample consisted of 50 leprosy patients, 26 (52%) women and 24 (48%) men, with a mean age of 53 years (range, 18–84). According to WHO operational classification [22], 29 (58%) were MB and 21 (42%) were PB. There was no association between gender (p 0.96) or age (p 0.67) and MB or PB patients.

Immunohistochemistry

Fifty patients were included in the IHC study, which resulted in 158/163 samples that contained sufficient material to generate the IHC sections (49 soft palate samples, 49 buccal mucosa samples, 48 tongue samples and 12 samples from other areas of the oral mucosa).

Overall, 70% (35/50) of the patients were positive in at least one sample, 68% (34/50) using anti-BCG antibodies and 66% (33/50) using anti-PGL-1 antibodies, demonstrating a strong agreement between the results obtained with the two antibodies (p <0.0001). However, there were no significant differences between the PB (80.95%, 17/21) and MB (62.07%, 18/29) patients and IHC positivity, including both anti-BCG and anti-PGL-1 antibodies (Table 1).

Table 1. Sensitivity of immunohistochemistry results for anti-BCG and anti-PGL-1 antibodies and PCR for the MntH gene in 50 leprosy patients according to clinical form
MethodPositive results
Total leprosy patients (n = 50) N (%)Multibacillary patients (n = 29) N (%)Paucibacillary patients (n = 21) N (%) p value
  1. N, number of patients.

Immunohistochemistry anti-PGL-1 antibody33 (66)18 (62.06)15 (71.43)0.69
Immunohistochemistry anti-BCG antibody34 (68)17 (58.62)17 (80.95)0.17
PCR - MntH gene39 (78)25 (86.20)14 (66.66)0.19
Immunohistochemistry (anti-BCG and/or anti-PGL-1 antibodies) and/or PCR-MntH gene47 (94)26 (89.65)21 (100)0.35

Using anti-BCG and anti-PGL-1 antibodies, regarding the areas of the oral mucosa, antigens were more frequently detected in the tongue (62.5%, 30/48) than in the soft palate (10.2%, 5/49; p <0.0001), the buccal mucosa (16.3%, 8/49; p <0.0001) and the other areas of the oral mucosa (25%, 3/12; p 0.04).

There was an irregular distribution of antigens in the tissue samples (Fig. 1). Including both antibodies, 50% (25/50) of the patients showed immunoreactivity on free-living bacteria on the epithelial surface compared with 14% (7/50) within macrophages in the corium (p 0.0003) and 22% (11/50) in the nerve (p 0.006). Notably, nerves were visible histologically in only 27 samples taken from 19 patients in previous studies performed on the same material [9, 13, 14]. No reactivity was observed within the cytoplasm of epithelial cells.

image

Figure 1. (a) and (c) Immunoreactivity of free bacteria (granular form) on the epithelial surface of the tongue. (b) and (d) Immunoreactivity in the cytoplasm of macrophages found in the corium of the soft palate (400×) (a, b, BCG marker; c, d, PGL-1 marker).

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Regarding tissue distribution among the areas of the oral mucosa, higher immunoreactivity was observed on free-living bacteria on the epithelial surface (50%, 25/50) of the tongue than on the soft palate (6%, 3/50) and the buccal mucosa samples (0/50; p <0.0001). There was no significant association between immunoreactivity within macrophages in the corium and areas of the oral mucosa (p 0.66). Immunoreactivity within macrophages in the corium was observed in 8% (4/50) of the soft palate samples, 6% (3/50) of the tongue samples and 2% (1/50) of the buccal mucosa samples.

Polymerase chain reaction

This analysis included 161/163 samples that contained sufficient material to extract genomic DNA; however, only 110 samples had viable DNA. There was viable DNA in at least one area of the oral mucosa for 47 patients. Among these patients, 82.98% (39/47) yielded positive PCR results for the MntH gene in at least one sample. Taking into account all patients, the positivity was 78% (39/50; Table 1). Representative PCR results are shown in Fig. 2. The samples from three patients who did not have viable DNA indicated IHC positivity.

image

Figure 2. Agarose gel electrophoresis of PCR products obtained with primers that amplified a MntH gene fragment (336 bp) to identify Mycobacterium leprae. 2–11: Oral mucosa biopsy samples of leprosy patients. Note the positive results and the specificity of samples 2, 5, 6, 7, 9, 10 and 11. 1: Negative control (no DNA added). 12: Mycobacterium avium complex. 13: Mycobacterium tuberculosis. 14: Positive control (plasmid that contained the cloned MntH gene of Mycobacterium leprae). 15: 100-bp marker.

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Regarding the clinical forms of the disease, there were no significant differences between the MB (86.20%, 25/29) and PB (66.66%, 14/21) patients and PCR for the MntH gene positivity (Table 1).

The distribution of the frequencies of positive PCR results in all areas of the oral mucosa was similar: 69.7% (23/33) in the soft palate samples, 60.61% (20/33) in the tongue samples, 69.45% (25/36) in the buccal mucosa samples and 62.5% (5/8) in the other areas.

IHC and PCR

Considering both the IHC and PCR methods, 94% (47/50) of the patients were positive in one or more areas of the oral mucosa using at least one method: 26% (13/50) were positive in one area, 32% (16/50) in two areas, 34% (17/50) in three areas, and 2% (1/50) in four areas.

To evaluate the agreement between the results for the methods, only the 110 samples that were validated for PCR were considered, and 40.91% (45/110) of the samples showed agreement between IHC and PCR methods. The kappa statistics for the results between PCR and anti-BCG antibodies and anti-PGL-1 antibodies resulted in <0 and no agreement, respectively. The kappa statistics for the results between anti-BCG and anti-PGL-1 in 110 samples indicated almost perfect agreement (κ = 0.969; 95% CI, 0.782 to >1.0). No significant differences were found between IHC and PCR methods in the PB or MB patients.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Transparency Declaration
  9. References

To the best of our knowledge, this is the first molecular and immunological study of M. leprae using oral mucosa biopsy material from leprosy patients. The results suggest that the oral mucosa is an important source of bacilli. This observation was reported in 2011 by Martinez et al. [3], who employed PCR with primers that amplified a 130-bp fragment in the RLEP3 genomic region of M. leprae and detected bacillus DNA in swabs of the buccal mucosa in 18.26% of the patients.

The higher rate of positivity found for PCR in this study in relation to IHC was expected because PCR is considered the reference standard method for detecting microorganisms [25]. However, an unexpected result was the high frequency of positive results obtained by both methods for PB patients, not significantly different from the frequency rates obtained for MB patients, a phenomenon also observed by Martinez et al. [3]. This finding is not consistent with reports in the literature, which uniformly suggest a greater involvement of the oral mucosa in MB patients [4-9, 13, 15]. It is likely that PCR and IHC, highly sensitive techniques, are able to demonstrate the presence of bacilli, even in minimal amounts, and this possibly occurs in the oral mucosa of PB patients.

In previous clinical studies, several authors have claimed that the soft palate [5, 6] and the hard palate [7, 8] are the preferred areas of M. leprae in the oral mucosa. In this study, bacillus DNA was amplified from all three areas at similar frequencies using PCR. This result may be explained by the possibility of cross-contamination, because the biopsies were performed using the same surgical instruments in each patient, because the material was originally obtained for previous histopathological studies [9, 13, 14]. Conversely, the IHC results using anti-PGL-1 and anti-BCG antibodies indicate a predilection for the tongue.

The presence of M. leprae on the epithelial surface of oral leprosy lesions has been reported in histopathological examinations [8, 16, 17]; however, its prevalence on free-living bacteria on the epithelial surface of clinically normal mucosa, as shown using IHC, was not reported previously. Possible explanations may include contamination by nasal secretions that contain the bacilli or penetration by the bacilli via the oral mucosa. Thus, the oral mucosa may act as an entry point for M. leprae, which has previously been proposed [3].

Therefore, despite the lower rate of positivity reported by Martinez et al. (18%), compared with this study, further epidemiological studies designed to investigate M. leprae in the oral mucosa should collect material with swabs, a technique that is less invasive and more easily performed than biopsies. However, swabs on the tongue (and not on the buccal mucosa as performed by the aforementioned researchers) may increase the likelihood of finding the bacillus. It would be interesting to conduct future research for PCR detection of Mleprae using swabs taken from the nasal mucosa and tongue, in order to compare the positivity rates between the two sites, because the nasal mucosa is considered the major port of entry and exit of bacilli.

There are some limitations regarding the present study: retrospective design, absence of healthy controls of the same population and, in particular, possible cross-contamination during the collection of surgical biopsy samples from different locations of the oral mucosa for all the PCR results. On the other hand, although anti-BCG antibodies react with Mycobacteria sp. antigens, results are validated by comparison and strong concordance with anti-PGL-1 antibodies, which react specifically with M. leprae antigens.

The high rate of positivity for M. leprae found in the studied patients demonstrates the epidemiological importance of the oral mucosa in the transmission of leprosy, suggesting that bacilli can be secreted in the saliva and transmitted to other individuals. Thus, investigating M. leprae in oral mucosa or saliva samples by PCR may represent an alternative for detecting infected individuals in populations at risk, particularly those who have had contact with leprosy patients. However, the presence of M. leprae in the oral mucosa without the occurrence of recognizable clinical signs or symptoms is not synonymous with leprosy infection and may simply represent a transient contamination process in the oral cavity.

In conclusion, we suggest that M. leprae is present in the oral mucosa of leprosy patients at high frequencies and that both MB and PB patients have M. leprae in their oral mucosa at similar frequencies. IHC indicates that the tongue is the site in the oral mucosa at which M. leprae is more likely to be found, particularly on free-living bacteria on epithelial surfaces, and both anti-BCG and anti-PGL-1 antibodies give similar results. PCR is more effective than IHC in detecting M. leprae in the oral mucosa.

Acknowledgement

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Transparency Declaration
  9. References

The authors would like to thank Professor Mario Tarumoto for performing the statistical analyses.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. Transparency Declaration
  9. References
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