Acta Derm Venereol 2011 ; 91: 704-707

CLINICAL REPORT

Oral Lesion in Leprosy: Borderline Tuberculoid Diagnosis Basedon Detection of Mycobacterium leprae DNA by qPCRTalita DA SILVA MARTINEZ', Andr A. NAHASS Mrcia M.N.R FIGUEIRA\ Adeilson V. COSTA', Maria A. GONALVES', LuizR. GOULART'^ and Isabela M.B. GOULART' ^'National Reference Center in Sanitary Dermatology and Leprosy, Hospital das Clinicas, 'Graduate Program of Health Sciences, School of Medicine, Fe-deral University of Uberlndia, 'University Center of Triangulo-UNITRI, Brazil, and'Department of Medical Microbiology and Immunology, Universityof California. Davis, USA

Oral lesions are rarely reported in paucibacillary formsof leprosy. We report here a case with an erythematoushyposensitive lesion in the palate and no skin lesions.In addition to routine tests, biopsies of the lesion in thepalate and of clinically normal surrounding areas wereperformed and subjected to real-time PCR for detectionof Mycobacterium leprae DNA. The biopsy of the oral le-sion was positive for bacilli DNA, followed by positiveserum anti-PGL-1 and Mitsuda test, but with negativehistopathology. The patient was diagnosed with a bor-derline tuberculoid form. After multidrug therapy thelesion had significantly regressed and the bacilli DNAdetection in the former lesion was negative. The bacilliDNA detection in an oral lesion by real-time PCR notonly improved leprosy diagnosis, but also helped in theclassification of clinical form, and in the establishment ofthe appropriate therapeutic regime. Key words: leprosy;Mycobacterium leprae; diagnosis; PCR; mouth mucosa.

(Accepted April 4, 2011.)Acta Derm Venereol 2011; 91: 704-707.

Talita da Silva Martinez, National Reference Center inSanitary Dermatology and Leprosy, Av. Aspirante Mega,77, Bairro Jaragu, 38413-018 Uberlndia/MG, Brazil.E-mail: ttalittasm(^yahoo.com.br

Leprosy, an infectious disease caused by Mycobacteriumleprae, is a relevant public health issue in Brazil, whichhas the second highest prevalence worldwide, with37,610 new cases detected in 2009 (1).

An operational classification distinguishes multibacil-lary (MB) from paucibacillary (PB) forms of leprosy,according to the bacterial index (BI) and the number oflesions (2); for clinical form classification, Mitsuda'stest and PGL-1 antibody testing are reported in additionto the BI (3).

As a result of effective antimicrobial therapy, advan-ced lesions of the face, nasopharynx and oropharynx arenow rarely seen, but the presenting signs and symptomsof leprosy still arise occasionally in the nasal and oralmucosa (4). A case of oral lesion associated with leprosyhas been described elsewhere in a borderline tuberculoid

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(BT)-MB patient with papulo-nodular lesions in thecentre of the hard palate, by histopathological findings(5). However, oral mucosa lesions are not normallyfound in PB patients (5, 6), and when they are present,manifestations have been described exclusively in thetuberculoid and borderline forms in the reactional state,with few scientific studies and poorly characterizeddescriptions (7).

Oral lesions of leprosy occur more frequently in areasof the mouth with a lower surface temperature (8). Themain oral cavify sites of leprosy include the gingivalin the anterior portion of the maxilla, the hard and softpalate, the uvula and the tongue (9).

An earlier study detected M leprae DNA by PCRin oral mucosa samples of patients with MB forms ofleprosy (10), but literature is scarce regarding detectionof bacilli DNA in PB leprosy forms.

MATERIAL AND METHODSMitsuda's intradermal tests for evaluation of the cellular im-mune response to M. leprae, and the serum enzyme-linkedimmunoassay (ELISA) that evaluates the response of IgMantibodies against the PGL-1 bacillus antigen were performed,together with M leprae DNA detection in nasal swab, buccalswab, dermal swabs, peripheral blood, biopsies of the inferiornasal turbinate and of the sensory nerve, a research protocoladopted at CREDESH under the UFU Ethics Committee appro-val number 499/2008.

Real-time PCR (qPCR) was performed for M. leprae DNAdetection in all samples. The set of primers and probe (sense5'-TGACATGAATACGCAAGGGGCAGGA-3'; antisense5'-TCATCTCCTGCACCCCGA-3'; probe TaqMan 5'-CACA-TAGGGTCCAGTCGTGCCGC-3') were designed to amplifyand detect a 69-bp fragment of the ML0024 M leprae gene,a highly specific region. The genomic region was confirmedby dideoxy sequencing, which aligned only with the M. lepraegenome in a Basic Local Alignment Search Tool (BLAST)search (www.ncbi.nlm.nih.gov/BLAST). The sensitivity ofthe biomarker was close to 60%, detecting all BB, BL and LLclinical forms. Other Mycobacteria spp. and negative controlswere used, and confirmed 100% specificity. Interestingly, ba-cilli DNA were detected by the ML0024 primers in 25% of thepatients' samples with negative bacilloscopy.

Six-hundred nanograms of DNA (4 ni) from clinical sampleswere used in a 25-nl PCR reaction, which consisted of 2x Taq-Man Universal PCR Master Mix (Applied Biosystems), 200 nMeach primer, and 100 nM probe. The PCR reactions were carriedout at 50C for 2 min, 95C for 10 min, followed by 40 cycles

o 2011 The Authors, doi-. 10.2340/00015555-1175Journal Compilation O 2011 Acta Dermato-Venereologica. ISSN 0001-5555

of 95C for 15 s and 60C for 1 min. All tissues were weighedbefore DNA extraction to allow the absolute quantification permilligram of hiopsied tissues. The absolute quantification of eachDNA sample was calculated based on a standard curve construc-ted for the gene. All samples were run in triplicate.

ELISA followed the procedures described elsewhere (11).Briefly, microtitre plates (Maxisorp, NUNC, Rochester, NY,USA) were coated with 50 nl native PGL-I diluted in absoluteethyl alcohol at a concentration of 10 ng/ml. Serum sampleswere added in duplicate using a dilution of 1:100 (native PGL-I) in phosphate buffered saline/bovine serum albumin (PBS/BSA) 1%, and incuhated for 1 h at 37C, followed by washing.The anti-human IgM-peroxidase conjugate (Sigma ChemicalCo., St Louis, MO, USA) was added to the plates at a dilutionof 1:10,000 (PGL-I ELISA) in PBS/BSA 1%. The suhstrateo-phenylenediamine dihydrochloride (OPD, Sigma-Aldrich,Inc., St Louis, MO, USA) enzyme suhstrate was added to theplates and incuhated at room temperature for 10 min in the dark.The reaction was stopped hy the addition of 25 |jl H S^O^ 4N.The optical density (OD) was obtained in a microplate reader(Thermo Plate, TP-Reader, Rayto Life and Analytical SciencesC. Ltd, Germany) at 492 nm. Two positive and three negativecontrols were included in each plate. The cut-off level for posi-tivity was a standard deviation above the mean OD obtained forthe three negative controls. The antihody titres were expressedas the ELISA index (El) according to the following formula:^ ' " O s^ampie^ OD^^ ,^ ^ in whch the cut-off point was determinedhy the mean OD of the negative controls plus 3 standard devia-tions. Values of El > 1.1 were considered positive.

CASE REPORT

A 46-year-old Brazilian woman attended the NationalReference Center in Leprosy and Sanitary Dermatology(CREDESH), of the Hospital das Clnicas, at the Fede-ral University of Uberlndia, in January 2009, referredby her neurologist physician, presenting hypoesthesiaat the bottom of the right foot, high-stepping gait andan electroneuromyography demonstrating an axonalmononeuropathy with moderate to severe intensity ofthe right peroneal nerve, leading to suspicious leprosy.Although the referred patient had no known contactwith other patients, it is worth noting that this is an

Leprosy diagnosis by qPCR in oral lesion 705

endemic region for leprosy, and that the possible sourceof transmission could not be determined. Clinical eva-luation did not reveal any skin lesions in any part ofthe patient's body, but did present an important hypo-esthesia and pain at the bottom of the right foot, andparesis due to difficulty dorsiflexing the right foot, inthe region innervated by the right fibular.

For diagnosis, BI of dermal swabs was performed,collected at seven skin sites of the patient (two earlobes, two elbows, two knees and a suspected area atthe bottom of the right foot) as well as a biopsy of theintermediate cutaneous branch of the right superficialfibular nerve during surgical decompression of the rightcommon fibular, due to impairment of this nerve, withswelling and tenderness, associated with sensory lossand muscle paresis.

Other laboratory tests, such as liver and kidney func-tion, blood sugar, thyroid hormones, antinuclear anti-bodies (ANA), rheumatoid factor (RF) and urinalysis,were requested to rule out other causes of neuropathies,including autoimmune diseases and metabolic disorders.These tests were all negative.

During the intraoral examination, a hypoesthesicerythematous lesion was observed, without elevatedborders, in the region of the right hard palate, involvingthe medial line, of approximately 2.0 cm diameter (Fig.lA). Biopsies were collected from the lesion and fromthe clinically normal adjacent region (control), sparingthe central region of the lesion in order to avoid the areaof the greater palatine nerve.

The ELISA anti-PGL-1 serum test was positive, withan El of 2.37. The Mitsuda test was strongly positive,with a 10 mm-diameter nodule. BI and qPCR were nega-tive in all the dermal swabs samples, nerve and inferiornasal turbinate biopsies, buccal swab, nasal swab, andperipheral blood. The histopathological examinationof nerve biopsy for the cutaneous branch through theright superficial peroneal nerve showed no inflammatoryprocess of the neural tissue fragment and presented

Fig 1. Lesion before and after multidrug therapy. (A) Erythematous lesion of the hard palate involving the midline. (B) Regression of the hard palatelesion.

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706 T. da Silva Martinez et al.

negative acid-fast bacilli by Ziehl-Neelsen staining.The buccal lesion biopsy of the hard palate displayednegative bacilloscopy, without apparent pathologicalchanges (Fig. 2); however, the qPCR presented anamplification curve of the bacillus DNA and detected1,126 copies of the M leprae DNA per mg of biopsiedtissue. The clinically healthy mucosa of the hard palatehad negative results, both in the histopathological andthe qPCR tests.

The possibility of contamination of the sample fromthe buccal mucosa lesion was excluded with the undis-puted demonstration that qPCR for the detection of Mleprae DNA was negative in the clinically healthy areaadjacent to the lesion, in the buccal swab, the nasalswab, the blood, and the biopsy of the nasal turbinate,a fact that precludes any possibilify of contaminationby saliva, nasal secretion and/or blood at the site as aresult of the invasive biopsy procedure. Furthermore,the biopsy was split into three samples, which weresubmitted to normal extraction protocols with the othersampling replications prior to PCR reactions.

Considering the positive ELISA for serum PGL-1, thestrong positive Mitsuda test, and the positive PCR resultin the oral lesion, the patient was classified as borderlinetuberculoid, a PB leprosy form, and received multidrugtherapy (MDT) for 6 months, with a daily dose of 100mg dapsone, and a monthly dose of 600 mg rifampicin.Successful treatment was evidenced by the regressionof the hard palate lesion after the treatment (Fig. IB),by reversed results of qPCR with total absence of Mleprae DNA (negative), and by the reduction in El from2.37 to 1.16 (normal liii and eosin (H&Ex400) showed palatal fragment, showing the deep portion of the epithelial tissue lining and lamina propria of the mucosa. The lamina propria consists ofconnective tissue, not modelled, evidencing collagen bundles of various thicknesses and areas of neurovascular structures with common aspects of normality.At depth, there is fatty tissue in the sub-mucosa without apparent pathological changes. (B) and (C) Magnified view of the vascular-neural structures observedin the lamina propria and submucosal tissue (H&E x 400).

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Leprosy diagnosis by qPCR in oral lesion IQl

at the site as a result of the invasive biopsy procedure.Furthermore, the use of qPCR, with two primers andone intemal probe, avoids excessive manipulation ofthe sample, and supports a highly sensitive and specificdetection system.

Even though the histopathological examination inthis clinical case did not suggest or provide evidencefor the disease, the diagnosis of borderline tuberculoidleprosy was determined by detection of the bacilli DNAin the oral lesion, which was supported by other indirectevidence, such as: positive ELISA (meaning subclinicalinfection, once only 22% of patients with PB present po-sitive ELISA), positive Mitsuda (commonly detected inpaucibacillary patients that present a cellular response),and resolution of disease symptoms and oral lesion afierMDT, with absence of DNA post-treatment.

Our results demonstrated the great relevance of theqPCR detection system in oral samples of PB patients,which may be used not only for leprosy diagnosis, butalso for clinical classification, especially for the limitedsensitivity and specificity of the conventional diagnostictools (18).

ACKNOWLEDGEMENTSThe authors thank all the staff of the National Reference Centerin Sanitary Dermatology and Leprosy for fundamental support,FAPEMIG/CNPq/CAPES/FINEP and Ministry of Health forfinancial support, and the Integrated Laboratory of Biologyand Oral Pathology (LIBIPO) of the Federal University ofUberlndia for the histopathological analysis.

REFERENCES

1. WHO (World Health Organization). Global Leprosy Situa-tion, 2010. Weekly Epid Rec 2010; 85: 337-348.

2. WHO (World Health Organization). Expert committeeon leprosy sixth report. World Health Org Tech Rep Ser1988; 768.

3. Teixeira AC, Cmvinel DL, Roma FR, Luppino LF, ResendeLH, Sousa T, et al. Evaluation of the agreement betweenclinical and laboratorial exams in the diagnosis of leprosy.Rev Soc Bras Med Trop 2008; 41: 48-55.

4. Scollard DM, Skinsnes OK, 1999. Oropharyngeal leprosy inart, history, and medicine. Oral Surg Oral Med Oral PatholOral Radiol Endod 1999; 87: 463^70.

5. Ghosh S, Gadda Rohit-Bhailal, Vengal M, Pai KM, Ba-lachandran C, Rao R, et al. Oro-facial aspects of leprosy:report of two cases with literature review. Med Oral PatolOral Cir Bucal 2010; 15: 459^62.

6. De Abreu MA, Alchome MM, Michalany NS, Weckx LL,Pimentel DR, Hirata CH. The oral mucosa in paucibacillaryleprosy: a clinical and histopathological study. Oral Surg OralMed Oral Pathol Oral Radiol Endod 2007; 103: 48-52.

7. Opromolla DVA, Opromolla MA, Somei U. [Dimorphouslesions in oral cavity]. Hans Int 2003; 28: 151-155 (inPortuguese).

8. Rendall JR, McDougall AC, Willis LA. Intra-oral tempe-ratures in man with special reference to involvement ofthe central incisors and premaxillary alveolar process inlepromatous leprosy. Int J Lepr Other Mycobact Dis 1976;44: 462-468.

9. Bucci F Jr, Mesa M, Schwartz RA, McNeil G, LambertWC. Oral lesions in lepromatous leprosy. J Oral Med 1987-42: 4-6.

10. Santos GG, Marcucci G, Guimaraes Junior J, MargaridoLC, Lopes LHC. [Molecular detection of Mycobacteriumleprae by polymerase chain reaction in oral mucosal biopsyspecimens]. An Bras Dermatol 2007; 82: 245-249 (inPortuguese).

11. Lobato J, Silva DA, Mineo TW, Amaral JD, Segundo GR,Costa-Cruz JM, et al. Detection of immunoglobulin G anti-bodies to Neospora caninum in humans: high seropositivityrates in patients who are infected by human immunodefi-ciency virus or have neurological disorders. Clin VaccineImmunol 2006; 13: 84-89.

12. Goulart 1MB, Goulart LR. Leprosy: diagnostic and controlchallenges for a worldwide disease. Arch Dermatol Res2008; 300: 269-290.

13. Patrocinio LG, Goulart 1MB, Goulart LR, Patrocinio JA,Ferreira FR, Fleury RN. Detection of Mycobacterium lepraein nasal mucosa biopsies by the polymerase chain reaction.FEMS Immunol Med Mic 2005; 44: 311-316.

14. Naves MM, Patrocinio LG, Patrocinio JA, Mota FMN,Souza AD, Fleury RN, et al. Contribution of nasal biopsyto leprosy diagnosis. Am J Rhinol Allergy 2009- 23-177-180.

15. Scheepers A, Lemmer J, Lownie JF. Oral manifestations ofleprosy. Lep Rev 1993; 64: 37^3 .

16. Brand PW. Temperature variation and leprosy deformityInt J Lep 1959; 27: 1-7.

17. Girdhar BK, Desikan KV. A clinical study of the mouth inuntreated lepromatous patients. Lepr Rev 1979; 50: 25-35.

18. Goulart 1MB, Cardoso AM, Santos MS, Gonalves MA,Pereira JE, Goulart LR. Detection of Mycobacterium le-prae DNA in skin lesions of leprosy patients by PCR maybe affected by amplicon size. Arch Dermatol Res 2007'299:267-271.

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