PCR detection of specific Leishmania-DNA in patients ... - Siapec - IAP

© Springer-Verlag 2002

Pathologica (2002) 94:28-31 A RT I C O L O O R I G I NA L E

G. Premoli-de-Percoco · N. Gonzalez · N. Añez · P. Guevara · J.L. Ramirez

PCR detection of specific Leishmania-DNA in patients with periodontal disease Amplificazione per mezzo della PCR della presenza del DNA di Leishmania in pazienti con malattie periodontali

Summary This study deals with the detection of Leishmania braziliensis DNA in gingival specimens from 10 individuals who all had suffered from cutaneous leishmaniasis 5-10 years prior to the examination and all had been treated with anti-leishmaniasis drugs. This preliminary study gives an interesting contribution to the oral microbiology of this disease, with the observation that inflamed periodontal tissues can serve as a factor affecting the dispersion of Leishmania parasites in individuals who had suffered from cutaneous leishmaniasis. These finding are corroborated by the results of polymerase chain reaction (PCR) which demonstrated the presence of Leishmania DNA in tissue samples of patients with periodontal diseases. Key words American tegumentary leishmaniasis • Polymerase chain reaction (PCR) • Periodontal disease • Inflammatory process • Leishmania braziliensis Parole chiave Leishmaniasis tegumentaria americana • Reazione a catena delle polimerasi (PCR) • Malattia periodontale • Processo infiammatorio • Leishmania braziliensis

G. Premoli-de-Percoco () Center for Odontological Research, Faculty of Dentistry, University of the Andes, calle 23 entre Av. 2 y 3, Edificio el Rectorado, Mérida 5101, Venezuela e-mail: [email protected] Tel.: +58-274-2402388 Fax: +58-274-2402418 N. Gonzalez • N. Añez Faculty of Science University of the Andes, Mérida, Venezuela P. Guevara • J.L. Ramirez Molecular Genetics Group Institute of Experimental Biology Central University of Venezuela, Caracas, Venezuela

Introduction American tegumentary leishmaniasis (ATL) is an infectious disease caused by species of the genus Leishmania. Among these, Leishmania (Viannia) braziliensis is an important etiologic agent in endemic areas of Latin America [1]. A characteristic of L. (V.) braziliensis infection is its tendency to produce metastasis to nasopharyngeal mucosae. Another characteristic of Leishmania species is their tendency to establish inapparent infections, or to persist after clinical resolution of the disease [2]. It has been considered that when an inflammatory process occurs, the individual suffers a temporary immunosuppressive stage because the macrophages invading the inflammation site are immature and thus unable to kill the parasites that they phagocyte [3]. Altes et al. [4] recognized leishmaniasis as an opportunistic infection in patients with human immunodeficiency virus (HIV), where the parasite disseminates to unusual places including the digestive tract and fluids such as urine, feces, sperm and pharyngeal secretion. In the oral cavity, inflammatory processes are common, periodontal diseases being one of them [5]. Different kinds of microorganisms colonizing the gingival crevice can cause periodontal disease, which is characterized by the presence of periodontal pocket and active bone resorption with inflammation. Oral mucosal leishmaniasis associated with chronic periodontitis has been previously described in the Old World [6, 7]. However, in the neotropical region there is not enough information about the association of Leishmania and chronic periodontal disease. This preliminary study gives an interesting contribution to the oral microbiology with the observation that the inflamed periodontal tissues (IPT) may serve as a factor affecting the dispersion of Leishmania parasites in individuals who had suffered from cutaneous leishmaniasis. These finding are corroborated by using polymerase chain

G. Premoli-de-Percoco et al.: PCR detection of specific Leishmania-DNA in patients with periodontal disease

reaction (PCR) technique demonstrating the presence of Leishmania DNA in tissue samples of patient with periodontal diseases.

Materials and methods Patient selection We studied 10 individuals (8 male and 2 female), 35-50 years of age coming from areas where leishmaniasis is endemic (located at 71000’-71058’W and 8020’-9000’N in Mérida, Venezuela). All the patients had suffered cutaneous leishmaniasis 5-10 years prior to examination at the outpatient clinic of the Faculty of Dentistry, Universidad de Los Andes. The clinical records indicated that all of them were cured after receiving anti-Leishmania treatment, based on injection of N-methyl-glumamine (glucantime). The 10 patients underwent oral surgery for chronic marginal periodontitis. During a preliminary screening, all patients met the following entry criteria: 1. A minimum of at least 2 sites with ≥5 mm of attachment loss in each quadrant (only one site was analyzed by PCR). 2. The absence of any systemic medical problem that might impact directly on the periodontal status (i.e. rheumatic fever requiring antibiotic prophylaxis, diabetes mellitus). 3. No current use of medications that could potentially affect the periodontal conditions (i.e. the regular use of prescription or non-steroidal anti-inflammatory drugs) and no history of antibiotic use drugs the previous 6 months. 4. No history of periodontal treatment in the past year. Periodontal reports (plaque index, color of the gingiva, swelling of the gingiva, periodontal pocket depth, attachment loss) were obtained from dental records. They were included in our protocol after receiving written consent. The biopsies from gingival tissue were divided into three groups: one was embedded in paraffin wax for histopathological study, another was analysed by PCR and the third was smeared on glass slides. The tissue sections were stained with the Giemsa-collophonium method, and the smears were stained with Giemsa stain in phosphate buffer (pH 7.2). Polymerase chain reaction DNA from all gingival tissue specimens was extracted as previously reported by Brown et al. [8]. Briefly, 50 µl of a 20% suspension of Chellex-100-iron‚ (Bio-Rad Laboratories, Hercules, CA) and 1 µl of a 20 mg/ml solution of proteinase K were added to 150 µl of the cell material and mixed vigorously. After 1 h incubation at 65°C, the samples were boiled in a water bath for 10 min. The Chellex resin was spun down in an Eppendorf microcentrifuge (Brinkman Instruments, Eppendorf, Germany) for 1 min. The super-

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natant was transferred to a new plastic tube, 2.5 µl of which was used for each PCR assay, adjusting the final volume to 50 µl. The primers used to drive the PCR reaction were derived from L. braziliensis non-transcribed ribosomal gene spacer DNA sequences (GenBank accession no. M75133) [9]. The forward primer sequence was 5’GCA GCA CAG GGA AAG 3’, and the reverse primer sequence was 5’ATG GAG AGA GGC ACT AGC 3’. The reaction mixture consisted of 2.5 µl supernatant liquid from gingival biopsy in a final volume of 50 µl, containing 50 mM KCl, 10 mM Tris-HCl pH 8.8, 1.5 mM MgCl2, 1% Triton X-100, 0.2 mM of each of the NTPs, 1 µM of each primer, and 1 unit of Taq polymerase (Promega Corporation, Madison, WI). The DNA amplifications were done in a thermal cycler (Perkin-Elmer Cetus, Norwalk, CT) programmed as follows: – First cycle, 5 min at 94°C; – Second cycle, 1 min at 60°C, 1 min at 72°C, and 1 min 94°C (repeated 10 times); – Third cycle, 1 min at 60°C, 1 min at 72°C, and 1 min at 93°C, (repeated 25 times); – Fourth cycle, 1 min at 60°C and 5 min at 72°C. To assess the suitability of the sample for PCR, we used a PCR assay targeted on human β-globin that amplifies a 268-bp product. To check carry-over contamination, we included a sample with the reaction mixture but no DNA. Tissue section of a 34-year-old male volunteer who has never lived in an endemic area (no leishmaniasis) and with periodontal disease was used as a negative control. As positive control, we used a blood sample from a patient who had leishmaniasis and whose primary cutaneous lesion evolved into secondary cutaneous lesions. All reactions were done simultaneously in one-day period with the same stock of reagents and the same equipment and controls. Precautions were taken to avoid contamination of the samples. The PCR products (5 µl) were electrophoresed in 3% agarose gels. DNA amplification bands were transferred to nylon filters and hybridized with a digoxigenin-labeled probe. To prepare the probe, we amplified 126-bp target sequence within plasmid pLbbrs4 using the same primers [9]. The 126-bp band was labelled with digoxigenin by multiprimer incorporation of dUTP-digoxigenin using a Genuis kit (Boehringer, Mannheim). The hybridized probe was detected by chemiluminescence with a Lumi-Phos 530 (Boehringer Mannheim); the exposure time ranged from 5 min to a maximum of 20 min (as recommended by the manufacturer).

Results The age, sex, and mean values of periodontal clinical parameters for the 10 patients are shown in Table 1. Presence of plaque, redness, swelling, and bleeding upon probing were

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Table 1 Mean values of the periodontal clinical parameters of the 10 patients included in the study Patient

1 2 3 4 5 6 7 8 9 10

Age (years)

35 38 40 47 43 35 50 36 38 40

Sex

F M F M M M M M M M

PII

0.54 1.96 1.67 1.58 1.04 1.46 1.71 0.46 0.37 1.46

R

0.87 1.00 0.79 1.00 0.87 1.00 0.96 0.71 0.75 0.71

SW

0.87 1.67 1.00 1.04 0.96 1.21 1.29 0.87 0.71 0.72

PD (mm)

2.15 3.79 2.72 3.41 2.27 2.67 3.09 2.27 2.42 2.50

PPBI

0.42 1.00 0.67 0.67 0.42 0.50 0.86 0.25 0.58 0.71

Sites with AL N.

Mean AL (mm)

PCR for Leshmania DNA

6 7 5 8 5 6 6 5 4 4

2.00 2.14 1.80 1.87 2.10 2.20 2.16 1.60 1.50 1.75

– + – + – + + + + +

PI, plaque index; R, color of the gingiva; Sw, swelling of the gingiva; PPBI, periodontal pocket bleeding index; PD, probing pocket depth; AL, attachment loss; – negative; + positive

generally observed. At the sampled sites, loss of attachment ranged from 2 to 4 mm, with probing pocket depths ranging from 2.14 to 3.41 mm. Radiographs of the maxillary and mandibular alveolar bones showed horizontal destruction of interdental bone and widening of the periodontal membrane. In severely affected areas, there were v-shaped defects between the roots and alveolar bone. Histopathological examination of biopsy specimens from the oral mucosae showed a chronic inflammatory cellular reaction with plasma cells usually predominant, with lymphocytes, macrophages and polymorphonuclear leukocytes also present. None of them showed Leishmania parasites in the sections. The PCR results are shown in Fig. 1. In positive samples, a clear 126-bp band was observed (lanes 10-16). Lanes 4-7 were negative samples. Lane 2 is the positive control, and lane 8 is the carry-over negative control. Leishmania DNA was found in 7 or 10 patients (Table 1). To confirm the positive samples, we transferred the gel to a nylon filter and hybridized it with the digoxigenin-labeled probe (data not shown). Human β-globin was amplified (268-bp band on gel electrophoresis) in all biopsies including controls.

Discussion The detection of Leishmania DNA in 7 of 10 patients with chronic periodontal disease, who had been cured of ATL, indicates the persistence of the parasite. Despite the possible low parasite number in the inflammation area, we obtained a robust amplification product, which supported previous finding [10].

Fig.1 L. braziliensis PCR assay. Ethidium bromide-stained, 3% agarose gel of one-fifth of the PCR products. Lanes 1 and 9, DNA used as molecular mass marker. Lane 2, positive control. Lanes 4-7, negative samples. Lanes 10-16, positive samples showing L. braziliensis from Viannia-subgenus-specific 126-bp amplification. Lane 8, negative control (no DNA)


Chronic periodontal disease is characterized by inflammatory and immunopathological reactions accompanied by migration and accumulation of both humoral and cell-mediated immunity. It is possible that peripheral blood monocytes harboring L. braziliensis might transport the parasite to the inflamed area [11, 12]. Supporting this view, we found (unpublished) in experimentally infected animals that inflammatory processes affect migration of Leishmania. The patients with advanced periodontal diseases studied here harbored Leishmania with no apparent ill effects. However, it is important to take extreme precautions should they be considered for steroid therapy or any other immunosuppressing drug. Leishmaniasis may arise subsequently from the downregulation of the immune system, as in HIV infection and other systemic diseases [2, 13]. A way to control the transmission of the disease in nonendemic areas is to include new clinical criteria in dental history, warning about the possibility of professional infection by inoculation of infected patients. Finally, the present results lead us to recommend that leishmaniasis be considered as an oral disease in differential diagnosis of oral and dental infections of patients coming from endemic areas for leishmaniasis. Acnowledgments Financial support was provided by CDCHT-ULA Grant O-47-96-07-C (GPP), O-46-96-07-A (GPP), O-40-95-A-07 (GPP), 0-41-95-C (GPP), C-743-95-07-AA (NA), CONICIT-S195000889 (NA) and CONICIT-S1-95000524 (JLR).

Riassunto Questo studio descrive la presenza di Leishmania braziliensis nei tessuti gengivali di 10 individui che avevano sofferto di leishmaniasi cutanea 5-10 anni prima dell’esame e tutti erano stati trattati con farmaci anti-leishmaniasi. Questo studio preliminare fornisce un interessante contributo alla microbiologia orale attraverso l’osservazione che i tessuti periodontali infiammati potrebbero servire come un fattore che favorisce la dispersione dei parassiti della Leishmania in individui che hanno sofferto di leishmaniasi cutanea. Questa osservazione è stata verificata utilizzando la reazione a catena della polimerasi (PCR) che dimostra la presenza del DNA della Leishmania nel tessuto dei pazienti con malattie periodontali.

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