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Volume 26, Number 10—October 2020
Research Letter

Mycobacterium leprae on Palatine Tonsils and Adenoids of Asymptomatic Patients, Brazil

Marilda Aparecida Milanez Morgado de Abreu1Comments to Author , Gisele Alborghetti Nai, Juliana D’Andrea Molina, Rafael Tomaz Gomes, and Ana Maria Roselino Natalia de Paula1
Author affiliations: Regional Hospital, Presidente Prudente, Brazil (M.A.M. Morgado de Abreu, R.T. Gomes); University of Oeste Paulista, Presidente Prudente (M.A.M. Morgado de Abreu, G.A. Nai, J.A. Molina); and University of São Paulo, Ribeirão Preto, Brazil (N. de Paula, A.M. Roselino).

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Abstract

We investigated palatine tonsil and adenoid specimens excised from otorhinolaryngological patients in a leprosy-endemic region of Brazil. Fite-Faraco staining identified Mycobacterium spp. in 9 of 397 specimen blocks. Immunohistochemistry and molecular analysis confirmed the presence of Mycobacterium leprae, indicating that these organs can house M. leprae in persons inhabiting a leprosy-endemic region.

Leprosy is a chronic infectious disease caused by Mycobacterium leprae that especially affects skin and peripheral nerves (1). In 2018, the registered global prevalence in the 6 World Health Organization regions was 184,238 cases (0.24/10,000 population), showing a decrease of 8,475 cases over the previous year (2). Although its incidence in Brazil has declined during 2009–2018, leprosy continues to be a major public health problem at the national level (1). Reports of M. leprae resistance against antimicrobial drugs used in multidrug therapy raise concern about the future of leprosy treatment (2). Therefore, not only does leprosy persist, but the emergence of multidrug-resistant M. leprae is a potential threat to global public health (3,4).

Although the exact mode of leprosy transmission is not known, it is thought that the upper respiratory tract, in particular the nasal mucosa, is the usual site of primary infection (3). Because we have previously identified M. leprae in oral mucosa of leprosy patients (5), we aimed to investigate other anatomic sites that could host this microorganism to clarify the epidemiology and transmission mechanisms of leprosy.

In this study, we hypothesized that M. leprae, after penetration through the airway mucosa, could infect the palatine tonsils and adenoids, because these organs represent the first immune defense line against inhaled or ingested antigens (6). We also theorized that if leprosy is a highly contagious disease (1), a considerable part of the population in endemic regions might be infected with M. leprae.

We conducted a cross-sectional study of 397 paraffin-embedded blocks of palatine tonsils and adenoids extracted from 144 patients due to otorhinolaryngological indication during 2011–2016 at the Regional Hospital, Presidente Prudente, Brazil. The local Research Ethics Committee approved the study (protocol #1.920.994).

Microscopic analysis using hematoxylin-eosin staining did not reveal granulomas. We analyzed 50 fields in the 100× objective (1,000× magnification) per slide stained with Fite-Faraco; of the positive cases (9 [2.3%] slides from 8 [5.6%] patients, 6 men and 2 women [mean age 11 + 5.5 years]), we observed only 1 acid-fast rod per slide. We studied all the blocks of these 8 patients, a total of 20 blocks (Table).

Immunohistochemistry with 1:20,000 anti–phenolic glycolipid-I (anti-PGL-I) antibody (Bei Resources, https://www.beiresources.org), specific for M. leprae, was conducted with the Mach 1 polymer-based biotin-free detection kit (Biocare Medical, https://biocare.net) (5). We used deparaffinized skin sections from multibacillary leprosy as positive control. For the negative control, we omitted the antibody. To confirm specificity, we used deparaffinized skin sections from paucibacillary leprosy and atopic dermatitis (excluding inflammatory cell recognition by the antibody), normal human scalp, and tuberculous lung section.

We extracted DNA from paraffin sections with isopropanol-ammonium acetate (1). The resulting DNA was used in conventional PCR with sense 5¢-ATTTCTGCCGCTGGTATCGGT-3¢ and antisense 5¢-TGCGCTAGAAGGTTGCCGTAT-3¢ primers (ThermoFisher Scientific, https://www.thermofisher.com) to amplify M. leprae microsatellite sequences, according to a previous report (7). We assessed amplicons of 148 bp on 2% agarose gel. The assays included negative (DNA omission) and positive (DNA from multibacillary leprosy skin sample) controls. We confirmed specificity with DNA extracted from M. tuberculosis culture. In addition, we conducted PCR with TB1/TB2 primers to detect Mycobacterium spp. and with T4/T5 primers to detect M. tuberculosis (5).

Immunohistochemistry was positive in 18/20 (90%) blocks. By PCR, 19 (95%) were positive with RLEP and 5 were simultaneously positive with TB1/TB2; all 19 positive by PCR were negative by T4/T5 primers. Samples that tested negative in IHC or PCR were also negative in Fite-Faraco staining (Table).

Little attention has been paid to the role that mucosa-associated lymphoid tissue (MALT) plays in the mechanisms used by mycobacteria during host invasion. In tuberculosis infection, bacilli cross mucous membranes and penetrate into palatine tonsils and adenoids, where they initiate an immune response. However, bacilli may develop immune-evasion strategies and disseminate into the organism or return to the mucosa surface and be eliminated to the environment (8). This process might also occur in leprosy, but to our knowledge there are no reports on this subject.

Our results corroborate the hypothesis that M. leprae bacilli infect palatine tonsils and adenoids. Prospective studies with a larger population group are necessary to clarify these findings. We could not infer from this retrospective study with paraffinized samples whether patients who had positive results for M. leprae identification had leprosy or were asymptomatic carriers. In both clinical scenarios, however, our findings indicate that palatine tonsils and adenoids may represent reservoirs for M. leprae bacilli in persons inhabiting a leprosy-endemic region.

Dr. Morgado de Abreu is a professor and coordinator at the program of specialization in dermatology, Regional Hospital and University of Oeste Paulista, Presidente Prudente. Her research interests include infectious diseases, with emphasis on the epidemiology and mechanisms of leprosy transmission.

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Acknowledgments

We thank Sandra Rodrigues and Aline Turatti for their technical laboratory assistance.

The Paulista Foundation against Leprosy provided partial research support.

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References

  1. Lastória  JC, Abreu  MA. Leprosy: review of the epidemiological, clinical, and etiopathogenic aspects - part 1. An Bras Dermatol. 2014;89:20518. DOIPubMed
  2. World Health Organization. Global leprosy update, 2018: moving towards a leprosy-free world. WER No. 35/36. Wkly Epidemiol Rec. 2019;94:389412.
  3. Levis  W, Rendini  T, Martiniuk  F. Increasing virulence in leprosy indicated by global Mycobacterium spp. Emerg Infect Dis. 2018;24:183a184. DOIPubMed
  4. Cambau  E, Saunderson  P, Matsuoka  M, Cole  ST, Kai  M, Suffys  P, et al.; WHO surveillance network of antimicrobial resistance in leprosy. Antimicrobial resistance in leprosy: results of the first prospective open survey conducted by a WHO surveillance network for the period 2009-15. Clin Microbiol Infect. 2018;24:130510. DOIPubMed
  5. Morgado de Abreu  MAM, Roselino  AM, Enokihara  M, Nonogaki  S, Prestes-Carneiro  LE, Weckx  LLM, et al. Mycobacterium leprae is identified in the oral mucosa from paucibacillary and multibacillary leprosy patients. Clin Microbiol Infect. 2014;20:5964. DOIPubMed
  6. Brandtzaeg  P. Immunology of tonsils and adenoids: everything the ENT surgeon needs to know. Int J Pediatr Otorhinolaryngol. 2003;67(Suppl 1):S6976. DOIPubMed
  7. Azevedo  MC, Ramuno  NM, Fachin  LR, Tassa  M, Rosa  PS, Belone  AF, et al. qPCR detection of Mycobacterium leprae in biopsies and slit skin smear of different leprosy clinical forms. Braz J Infect Dis. 2017;21:718. DOIPubMed
  8. Lugton  I. Mucosa-associated lymphoid tissues as sites for uptake, carriage and excretion of tubercle bacilli and other pathogenic mycobacteria. Immunol Cell Biol. 1999;77:36472. DOIPubMed

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Cite This Article

DOI: 10.3201/eid2610.191267

Original Publication Date: September 02, 2020

1These authors contributed equally to this article.

Table of Contents – Volume 26, Number 10—October 2020

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Marilda A.M. Morgado de Abreu, rua São Paulo, 1949, centro, CEP 17900-000, Dracena, SP, Brazil

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Page created: September 02, 2020
Page updated: September 17, 2020
Page reviewed: September 17, 2020
The conclusions, findings, and opinions expressed by authors contributing to this journal do not necessarily reflect the official position of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors' affiliated institutions. Use of trade names is for identification only and does not imply endorsement by any of the groups named above.
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