Detection of Leishmania in Unaffected Mucosal Tissues of Patients with Cutaneous Leishmaniasis Caused by Leishmania (Viannia) Species

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1 MAJOR ARTICLE Detection of Leishmania in Unaffected Mucosal Tissues of Patients with Cutaneous Leishmaniasis Caused by Leishmania (Viannia) Species Roger Adrian Figueroa, Leyder Elena Lozano, Ibeth Cristina Romero, Maria Teresa Cardona, Martin Prager, Robinson Pacheco, Yira Rosalba Diaz, Jair Alexander Tellez, and Nancy Gore Saravia Centro Internacional de Entrenamiento e Investigaciones Médicas, Cali, Colombia Background. Leishmania (Viannia) species are the principal cause of mucosal leishmaniasis. The natural history and pathogenesis of mucosal disease are enigmatic. Parasitological evaluation of mucosal tissues has been constrained by the invasiveness of conventional sampling methods. Methods. We evaluated the presence ofleishmania in the mucosa of 26 patients with cutaneous leishmaniasis and 2 patients with mucocutaneous leishmaniasis. Swab samples of the nasal mucosa, tonsils, and conjunctiva were analyzed using polymerase chain reaction with LV-B1 primers and Southern blot hybridization. Results. Two patients with mucocutaneous leishmaniasis and 21 (81%) of 26 patients with cutaneous leishmaniasis had Leishmania kinetoplast minicircle DNA (kdna) in mucosal tissues. kdna was amplified from swab samples of nasal mucosa from 14 (58%) of 24 patients, tonsils from 13 (46%) of 28 patients, and conjunctiva from 6 (25%) of 24 patients. kdna was detected in the mucosa of patients with cutaneous disease caused by Leishmania panamensis, Leishmania guyanensis, and Leishmania braziliensis. Conclusion. The asymptomatic presence of parasites in mucosal tissues may be common in patients with Leishmania (Viannia) infection. Although mucosal involvement has been occasionally described in human infection due to a broad range of Leishmania species [1 5], the risk of mucosal leishmaniasis is a constant concern in infection due to Leishmania of theviannia subgenus [6, 7]. Lymphatic [8 9] or hematogenous [10, 11] metastasis of parasites from the cutaneous site of inoculation to the nasopharyngeal mucosa is considered to be the underlying cause of mucosal disease [12]. However, the dissemination Received 13 November 2008; accepted 7 March 2009; electronically published 1 July Potential conflicts of interest: none reported. Financial support: Instituto Colombiano para el Desarrollo de la Ciencia y la Tecnología Francisco José de Caldas (Young Investigators Program awards to R.A.F. and M.T.C., grants and ; and grant to L.E.L. and J.A.T.); United States National Institutes of Health (NIH), National Institute of Allergy and Infectious Diseases ICDR (U19 AI to N.G.S.); Fogarty International Center (NIH) (grant D43 TW to N.G.S.). Reprints or correspondence: Nancy Gore Saravia, Centro Internacional de Entrenamiento e Investigaciones Médicas (CIDEIM), Carrera 125 #19-225, A.A. 5390, Cali, Colombia (saravian@cideim.org.co). The Journal of Infectious Diseases 2009; 200: by the Infectious Diseases Society of America. All rights reserved /2009/ $15.00 DOI: / of Leishmania to the nasopharyngeal mucosa and the pathogenesis of mucosal leishmaniasis are poorly understood. Systematic otorhinolaryngological evaluation can reveal early mucosal involvement. The frequency of early symptomatic mucosal disease has been described in 3% of cutaneous cases, which is similar to the prevalence of advanced mucosal leishmaniasis [13]. Nevertheless the early involvement of mucosa and indeed, the diagnosis of mucosal leishmaniasis, are constrained by the invasiveness and limited sensitivity of biopsy and histopathological methods, as well as by the fact that these methods require skilled medical specialists. The rationale for this exploratory study was twofold; we aimed to examine an alternative, less-invasive diagnostic approach for mucosal leishmaniasis and to establish proof-of-principle that swab samples combined with molecular amplification of kinetoplast minicircle DNA (kdna) could be an informative strategy that could aid in understanding the natural history of the dissemination of Leishmania (Viannia) infection to mucosal tissues in the human host. We have demon- 638 JID 2009:200 (15 August) Figueroa et al

2 strated the presence of Leishmania (Viannia) kdna in the unaffected mucosal sites of patients with active cutaneous or mucocutaneous disease using samples obtained with swabs and analyzed with polymerase chain reaction (PCR) of kdna and Southern blot hybridization. MATERIALS AND METHODS Study Population Patients consulting at Centro Internacional de Entrenamiento e Investigaciones Médicas (CIDEIM) clinics in Cali, Colombia, and in the level 1 health post La Independencia within the public health network of the municipality in Buenaventura Colombia, were invited to participate in this exploratory study. Inclusion criteria were all of the following: adult status (18 70 years of age), clinical and/or epidemiological history of exposure to transmission, 1 or more active lesions, parasitologic diagnosis, no receipt of prior antileishmanial drug treatment for current lesions, and voluntary participation after providing written informed consent. Pregnancy was an exclusion criterion because treatment is contraindicated for pregnant individuals. One patient (D1019) with mucosal disease who was 74 years old and had received antileishmanial treatment for 5 days was included with permission of the CIDEIM Institutional Review board for research involving human subjects. Twenty-eight patients with parasitologically confirmed active tegumentary leishmaniasis were included in the study after providing signed informed consent. The study protocol and sampling procedures were approved by the CIDEIM institutional review board for studies involving human subjects and conducted in compliance with national and international guidelines for the protection of human subjects from research risks. The study population included 26 patients who presented with cutaneous lesions only and 2 patients who presented with mucocutaneous leishmaniasis. Clinical Diagnosis All patients were evaluated by a physician with experience in the diagnosis and management of leishmaniasis. A complete medical history was obtained and a physical examination was performed by this physician before the inclusion of a patient in the study. During the review of systems, the patient was queried about signs and symptoms of mucosal involvement; these included nasal congestion, secretion, scab expulsion, nasal pruritus, epistaxis and mass sensation. Direct anterior rhinoscopy and mouth and palate examinations were performed for all patients to detect and evaluate mucosal involvement. Any patient reporting at least 1 symptom of mucosal involvement during the clinical history or with any evident abnormality in the mucosal evaluation was considered as a possible case of mucocutaneous leishmaniasis and was referred to an ear, nose, and throat specialist for evaluation. Mucosal biopsies were performed by this specialist. Laboratory Diagnosis Diagnosis of cutaneous lesions was performed by direct microscopic examination of tissue smears obtained from the lesion border and/or culture of tissue fluid aspirated with a tuberculin syringe from the lesion border [14]. Diagnosis of mucosal lesions was achieved by biopsy and histopathological examination [14]. Patients were enrolled in the study and swab samples obtained 1 4 weeks after the initial consultation and parasitological diagnosis. The mucosal areas that were evaluated by swabbing (except for lesions in patients with mucosal disease) were not manipulated or otherwise subjected to intervention at any time prior to obtaining swab samples. Patient demographic characteristics and the clinical characteristics of patients lesions are summarized in table 1. Parasite identification. Species were identified on the basis of the reactivity of the parasites with a panel of Viannia subgenus- and species-specific monoclonal antibodies and isoenzyme polymorphisms [15, 16]. Sampling. Swab samples of lesion tissue and the conjunctiva, nasal mucosa, and palatine tonsils were obtained from each patient using sterile swabs (BuccalAmp kit; Epicenter Biotechnologies) after parasitological diagnosis had been established. Biopsy samples of mucosal tissues were obtained only from the patients with mucosal lesions. To obtain nasal, tonsilar, and conjunctival swab samples, the nasal septum, tonsils, and the inner surface of the lower eyelid, respectively, were gently rubbed with the swab. Cutaneous lesion samples were obtained by swabbing the surface of an ulcerated lesion after thorough cleaning with 70% alcohol. All samples were stored in their original packaging at 20 C until processing. Sample processing. DNA extraction from lesion, conjunctival, and nasal swab samples was performed using the BuccalAmp kit and the protocol recommended by the manufacturer. Recovery of the parasite kdna was optimized by extraction of DNA with 350 ml of absolute ethanol and 40 ml of 3 mol/l sodium acetate and overnight refrigeration at 45 C. The extracted DNA was recovered by centrifugation at 16,100 g at 4 C for 15 min in an Eppendorf 5415C microcentrifuge, followed by resuspension in 500 ml of 70% ethanol and recentrifugation under the same conditions. The supernatant was discarded and the remaining liquid evaporated by drying in a thermal block at 60 C. Finally, DNA was resuspended in 50 ml of RNase- and DNase-free water. DNA was extracted from tonsil swab samples using the AllPrep DNA/ RNA Minikit (Qiagen) in accordance with the manufacturer s instructions. The following negative controls were included: extraction reagents; PCR reagents, and mucosal swabs from 2 Leishmania in Unaffected Mucosal Tissues JID 2009:200 (15 August) 639

3 Table 1. Summary of clinical and demographic characteristics and lesion characteristics of the patient with leishmaniasis included in the study. Patient code Age, years Lesions, no. Lesion type Lesion location Size, mm Lymphatic track Regional LAD Time of evolution, months Leishmania (Viannia) species recovered City and department of residence Primary infection D1002 D1003 D1005 D1006 D1007 D1008 D Ulcer Arm L. panamensis Cali, Papule Leg No No Ulcer Neck No No 1.5 L. panamensis Cali, Ulcer Arm 3 3 No No Ulcer Head 4 3 No No 3 L panamensis Roldanillo, Ulcer Head 3 x3 No No 2 Plaque Head ND No No Ulcer Neck L braziliensis Cali, Ulcer Head ND Granulomatous lesion Arm L. panamensis Jamundi, 21 1 Ulcer Head L. braziliensis Suarez, Cauca 22 4 Ulcer Leg No 4 L. guyanensis Guacari, Ulcer Leg No No 4 Ulcer Leg No 4 640

4 D1010 D1011 D1012 Ulcer Leg No No Ulcer Leg 10 6 No No 2 L. guyanensis Vijes, 23 3 Ulcer Arm 6 4 No No 2.5 L. panamensis Cali, Ulcer Leg 9 8 No No 2.5 Ulcer Leg No No Plaque Head No 1 L. panamensis Jamundi, Plaque Head 9 12 No 1 D1013 a 57 3 Ulcer Arm No No 12 NA Pradera, Plaque Thorax No No 12 Destructive lesion Septum NA NA NA 5 D Ulcer Leg No No 4 L. panamensis Trujillo, D1015 D1016 D1017 D1018 Ulcer Leg No No 4 Ulcer Leg No No Plaque Head No No 1.5 L. panamensis Vijes, 25 6 Ulcer Thorax 8 7 No No 1 L. panamensis Cali, Ulcer Thorax 7 9 No No 1 Ulcer Thorax 5 11 No No 1 Ulcer Thorax 8 6 No No 1 Ulcer Thorax 7 6 No No 1 Ulcer Arm 14 8 No No Ulcer Head L. braziliensis Timba, Cauca 20 2 Ulcer Arm 14 7 No No 2 L. panamensis Nubita, Choco 74 1 Filtrum and Right nostril lost D1020 D1021 Ulcer Arm 5 5 No No 1 Head NA NA NA 96 NA Caicedonia, 59 1 Ulcer Thorax No No 2 L. braziliensis Cali, 24 1 Ulcer Arm L. panamensis Santander de Quilichao, Cauca No 641

5 D1022 D1023 D1024 D1025 D1026 D1027 D1028 D1029 D Ulcer Head 6 10 No 0.6 L. panamensis Cali, 21 1 Plaque Head No No 3 NA Buenaventura, 34 1 Granulomatous lesion Arm NA Cali, 57 2 Ulcer Arm NA Buenaventura, Nodule Arm Ulcer Neck 8 5 No No 2 L. panamensis Cali, NA 23 5 Ulcer Leg L. panamensis El Charco, Nariño Ulcer Leg Nodule Leg Nodule Leg Other Leg Ulcer Arm No No 1.5 L. panamensis Chinchina, Caldas Ulcer Thorax 6 9 No No Ulcer Head No 1.5 NA Buenaventura, 19 1 Ulcer Head No No 1.5 L. panamensis Buenaventura, NOTE. LAD, lymphadenopathy; NA, not available; ND, not determined. a Patient with mucocutaneous leishmaniasis.

6 healthy donors who had not been exposed to Leishmania transmission. PCR PCR were performed using the cloned 700-bp sequence of kdna in a PCR 2.1 TOPO vector (Invitrogen) from L. panamensis strain MHOM/CO/86/1166; promastigotes of this strain were used as positive controls. Each PCR run included negative controls for PCR reagents and sample extraction. Primer pair LV-B1 were used to amplify Leishmania kdna using methods described elsewhere ( [17] and Ramirez et al, unpublished data) with the following modifications: the annealing temperature was increased to 66 C for 40 s, and a final extension at 72 C for 1 min was added. The final PCR volume was 25 ml, containing 28 ng total DNA from each sample, as determined by use of an ND-1000 spectrophotometer (Thermo Scientific NanoDrop). Electrophoresis and Hybridization The Leishmania (Viannia) specific amplification product was 700 bp. Southern blot analysis was performed with 10 ml of each PCR product separated in 1% agarose gels at 90 volts for 45 minutes; bands were visualized and recorded in a GelDoc 2000 (BioRad). The transfer was carried out by capillary action overnight onto a nylon membrane (Amersham Hybond-N+) using 10 sodium chloride sodium citrate (1.5 mol/l NaCl and 0.15 mol/l sodium citrate). DNA was fixed to the membrane with ultraviolet light in a Crosslinker (Spectronics) for 30 s in automatic mode. For hybridization and detection, the AlkPhos Direct Labeling and Detection System kit (Amersham Biosciences) was used in accordance with to the manufacturer s instructions. Hybridization was conducted at 60 C with a 700- bp kdna minicircle probe that had been purified and cloned into PCR 2.1 TOPO vector (Invitrogen). Radiographic films were processed automatically using a Computer Classic EOS developer (Agfa). Patient characteristics. Twenty-eight patients between 19 and 74 years of age were included in the study. Twenty-six presented with cutaneous lesions, and 2 presented with mucocutaneous disease. There were 25 men (89%) and 3 women (11%). The duration of lesions at the time of diagnosis ranged from!1 month to 8 years, and the majority of lesions were ulcerated. There were 14 patients (50%) with 11 active lesion (table 1). Leishmania species. Leishmania were isolated from 22 (79%) of the 28 patients at diagnosis. Sixteen isolates (73%) were identified as L. panamensis, 4 (18%) as L. braziliensis, and 2 (9%) as L. guyanensis. Leishmania kdna was detected in the apparently unaffected mucosal tissues of patients with cutaneous disease caused by L. panamensis, L. braziliensis, and L. guyanensis. PCR and Southern blot analysis. Overall, the mucosal tissues of 23 (82%) of 28 patients yielded Leishmania kdna (figure 1); only 2 of these patients had mucosal lesions. Nasal mucosa samples were the samples most frequently positive for Leishmania kdna (14 [58%] of 24), followed by tonsilar mucosa samples (13 [46%] of 28), and conjunctiva samples (6 [25%] of 24) (table 2). The location of cutaneous lesions was unrelated to the detection of Leishmania kdna in mucosal tissues; 11 of 14 patients with lesions on the face and/or head or neck and 12 of 14 patients with lesions only on their arms or legs had mucosal swab samples that were PCR positive. Of 24 cutaneous and mucocutaneous lesion swab samples analyzed, all but 2 (92%) were positive for Leishmania. Eighteen (63%) of 28 patients had samples that were positive by PCR alone; Southern blot hybridization revealed 6 additional patients with mucosal tissue samples that were positive for Leishmania and confirmed the specificity of PCR. DISCUSSION The availability of highly sensitive and specific molecular genetic technology and the development of less invasive, technically simple sampling methods have opened the way for early and efficient diagnosis and a greater understanding of the natural history of Leishmania infection in the human host. Indeed, RESULTS Figure 1. Amplified Leishmania (Viannia) kinetoplast minicircle DNA (kdna) product from swab samples of different sites using LV-B1 primers. A, Detection in 1% agarose gel. Lane 1, molecular ladder; lane 2, conjunctiva sample from a healthy donor; lane 3, tonsil sample from a healthy donor; lane 4, nasal mucosa sample from a healthy donor; lane 5, negative extraction control; lane 6, negative polymerase chain reaction (PCR) control; lane 7, positive PCR control; lane 8, conjunctiva sample from patient D1009; lane 9, tonsil sample from patient D1014; lane 10, tonsil sample from patient D1019; lane 11, nasal mucosa sample from patient D1009; lane 12, nasal mucosa sample from patient D1014; lane 13, mucosal lesion sample from patient D1019; lane 14, cutaneous lesion sample from patient D1020. B, Specificity confirmation and enhancement of detection by use of Southern blot analysis with a cloned 700-bp sequence of L. panamensis kdna. Leishmania in Unaffected Mucosal Tissues JID 2009:200 (15 August) 643

7 Table 2. Molecular analysis of swab samples from lesions and unaffected tissues of 28 patients with leishmaniasis. Patient code Swab sample result, by source and test type Tonsil Conjunctiva Nasal Lesion PCR SB PCR SB PCR SB PCR SB D NA NA NA NA NA NA D NA NA NA NA NA NA D1005 NA NA NA NA NA NA D1006 NA NA NA NA NA NA D D D D D D D1013 a D D D D D D1019 a D D D D D D D D D D D Total, proportion (%) 9/28 (32) 13/28 (46) 2/24 (8) 6/24 (25) 10/24 (42) 14/24 (58) 22/24 (92) 22/24 (92) NOTE. +, positive for Leishmania;, negative for Leishmania; NA, not available (used in the optimization process only and unavailable for analysis); PCR, polymerase chain reaction; SB, Southern blot. a Patient with mucocutaneous leishmaniasis. molecular diagnosis provides a new gold standard for the diagnosis of dermal leishmaniasis [18], especially for disease presentations that involve a low parasite burden, such as mucosal leishmaniasis [19]. The findings of this study show that the efficiency of recovery and detection of parasite kdna from swab samples of mucosal tissues analyzed with a combination of PCR using the LV-B1 primer pair and Southern blot hybridization provides noninvasive access to mucosal tissues and a promising diagnostic alternative to biopsy and histopathological evaluation. The presence of Leishmania in mucosal tissues in the apparent absence of pathology may be a common feature of the natural history of infection due to Leishmania species of the Viannia subgenus. The asymptomatic presence of Leishmania in mucosal tissues can now be readily assessed in infections due to different species by using the approach described in this study. Remarkably, in this patient series, the asymptomatic presence of Leishmania in mucosal tissue was the rule rather than the exception, being demonstrated in 21 (81%) of 26 patients who had cutaneous disease only. This finding suggests that metastasis to and presence of parasites in mucosal tissues per se does not explain the pathogenesis of mucosal leishmaniasis and supports the idea that the host response plays a role in the pathogenesis of mucosal disease [20, 21]. Case reports and series have demonstrated that a wide range of species of Leishmania from both the Old and New Worlds can invade and cause disease in mucosal tissues of the nasopharynx [22]. L. donovani and L. infantum have been identified in gastrointestinal mucosa [23], the genitourinary tract [24], and the eye and lacrimal excretory system [25, 26]. Although 644 JID 2009:200 (15 August) Figueroa et al

8 these clinical manifestations of infection affecting the gamut of mucosal surfaces involved viscerotropic species of Leishmania, they portray a pattern of dissemination to mucosal tissues that in the light of the findings of this study may be more frequent and common than previously suspected for other species of Leishmania. To our knowledge, this is the first demonstration of Leishmania in the apparently normal mucosal tissues of patients with cutaneous leishmaniasis. The high frequency of kdna in the nasal mucosa accords with the nasal septum being the most frequent location of mucosal lesions [6, 7]. Interestingly, cytologic evaluation of nasal samples from cutaneous and mucocutaneous patients revealed a correlation between the presence of inflammatory cells and mucosal pathology in L. braziliensis infection [27]. Leishmania have been cultured from the tonsils, nasal secretions, and urine of patients with visceral leishmaniasis [28, 29]. Nasal secretions in particular yielded propagable Leishmania in a high proportion of patients. The finding that nasal swab samples were the most common source of kdna in this study suggests that dissemination to nasal mucosa may also be a common feature in cutaneous leishmaniasis caused by Leishmania (Viannia) species. The presence of parasites in tonsilar tissue is consistent with lymphatic dissemination [30] to mucosal sites. Nevertheless hematologic dissemination also occurs in mucosal leishmaniasis, as shown by hemoculture [10, 31]; PCR and Southern blot analysis have provided evidence of parasitemia in at least 30% of blood samples from patients with cutaneous disease [6] and in asymptomatically infected individuals [26]. In a separate study, Leishmania (Viannia) kdna was amplified from blood monocytes from 19 (68%) of the 28 patients included in this study (I.C.R. et al, unpublished data). kdna was amplified from both mucosal tissues and blood for 16 (58%) of the 28 patients. Under the conditions of this study, the LV-B1 primer pair had a sensitivity of 0.2 parasites per reaction, and the 700-bp size of the amplification product distinguished Leishmania species of the Viannia subgenus from other trypanosomatids. Cross-hybridization with the amplification products of other trypanosomatids did not occur, and human DNA was not amplified (Vergel et al [11] and Ramirez et al, unpublished data). The validity of these findings is substantiated by the specificity of the amplification and hybridization, the use of negative extraction and PCR mix controls, and the absence of kdna amplification for the mucosal swab samples from 2 healthy donors. The asymptomatic presence of kdna in tonsilar and mucosal tissues, unaffected skin [17], and blood before and after successful treatment [11, 30] substantiates that Leishmania (Viannia) species commonly disseminate from the inoculation site, even in localized, cutaneous disease. 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