Mixed mucosal leishmaniasis infection caused by Leishmania tropica and Leishmania

26 Mixed infection by different Leishmania species could explain differences in the clinical 27 course of these infections. On identification of Leishmania parasites from Iranian patients 28 with mucosal leishmaniasis (ML), a patient with both oral and nasal lesions was found to be 29 concomitantly infected with L. tropica and L. major. Mixed infection was identified by PCR 30 amplification of Leishmania kinetoplast DNA on scraping of cytological smears and 31 histopathological sections. Leishmania major and L. tropica were isolated from the nasal and 32 oral lesions, respectively. These species were also confirmed by immunohistochemistry. This 33 seems to be the first reported case of concurrent infection of ML with two Leishmania 34 species. It indicates that, at least in this patient, previous infection with a one of these 35 Leishmania species did not protect against another. This result has important implications in 36 development of vaccines against leishmaniases, and implies careful attention in treatment of 37 this infectious disease. 38 39 Case description and methods 40 A 34-year-old immunocompetent male subject presented with lesions on the mucous 41 membranes of nose and mouth. The patient was from Fars Province, southern Iran. He 42 presented with a 7 and 5 months history of intranasal and oral lesions, respectively. No scar 43 or other lesion was found in other parts of the body. On examination, there were multiple tiny 44 erythematous lesions, varying in size from 0.1 to 0.3 cm in diameter. The nasal pyramid was 45 edematous and bloody crusts were observed on the inferior conchae, septum, and floor of the 46 nasal fossa. The nasal lesions were located in intranasal portion in the mucous membrane 47 over the turbinates far from the cutaneous lesions. Clinically, diffuse yellowish white 48 erosions with grayish fibrinous membranes were seen on a reddish edematous background on 49 the involved oral mucosa (Fig. 1). 50 on A uust 0, 2017 by gest ht://jcm .sm .rg/ D ow nladed fom His blood biochemistry and complete blood count were within reference range. His 51 hemoglobin was 13.6 g/dl, total leukocyte count 6600/mm, serum creatinine 0.8 mg/dl, and 52 blood urea nitrogen was 18 mg/dl. Serological studies for human immunodeficiency virus 53 and hepatitis B and C virus were negative. 54 Tissue samples from the lesions were fixed in 10% neutral buffered formalin, embedded in 55 paraffin, sectioned at 5-μm in thickness, and stained with haematoxylin and eosin. 56 The cytologic smears were prepared by scraping from the oral lesions with a scalpel. In 57 addition, exfoliative cytology from the nasal lesions was performed by washing the nasal 58 cavity as previously noted (7). Multiple smears were made on slides and were both air dried 59 and alcohol-fixed and then stained by Wright method. Review of the cytologic smears and 60 histologic sections were conducted blindly by three pathologists. Microscopic examination 61 showed the amastigote forms of Leishmania (Fig. 2 A, B). 62 The following antibodies that were kindly provided by Special Programme for Research and 63 Training in Tropical Disease (TDR), WHO: IS2-2B4 (A11) specific for L. tropica, and 64 XLVI-5B8B3 (T1) specific for L. major, each was used as a primary monoclonal antibody. 65 Sections of 3 μm in thickness were used for immunohistochemical (IHC) analysis. The slides 66 were deparaffinized in xylol, rehydrated, and treated with 3% hydrogen peroxide solution for 67 10 minutes at room temperature to quench endogenous peroxides. The antigen retrieval was 68 conducted by pre-treatment with microwaving (power 100 for 10 min and then power 20 for 69 20 min) using a 10-mmol/L concentration of citrate buffer (pH 6.0). The primary antibody 70 was applied for 1 hour (diluted 1:200). Detection of the immunoreaction was achieved. The 71 detection system used was Envision+(DakoCytomation) and developed with 72 diaminobenzidine (DakoCytomation). 3,3'-diaminobenzidine–hydrogen peroxide was applied 73 as the chromogen and hematoxylin was used as the counterstain. The nasal lesions showed 74 immunoreactivity for specific monoclonal antibodies (mAb) of L. major and the oral lesions 75 on A uust 0, 2017 by gest ht://jcm .sm .rg/ D ow nladed fom for L. tropica (Fig. 2C), while the IHC staining of the nasal lesions with L. tropica mAb and 76 IHC staining of the oral lesions for L. major was negative (Fig. 2D). 77 To identify the Leishmania-specific DNA, the entire smear was scraped off the slide with a 78 sterile scalpel, and as previously described, the phenol-chloroform-isoemil alcohol extraction 79 method was used to extract the DNAs (23). The DNA samples were dissolved in 50 μl 80 deionized distilled water and stored at 4 °C. Variable segments on the minicircles of the 81 kinetoplast DNA from the Leishmania species present in the smear scrapings were amplified 82 with two rounds of nested PCR. The primers for the first round were CSB1XR (ATT TTT 83 CGC GAT TTT CGC AGA ACG) and CSB2XF (CGA GTA GCA GAA ACT CCC GTT 84 CA) and for the second round were LiR (TCG CAG AAC GCC CCT) and 13Z (ACT GGG 85 GGT TGG TGT AAA ATAG) (23). The PCR products of the second-round of the PCR were 86 loaded onto a 1.5% agarose gel. As the positive controls, the DNA extracted from the 87 promastigote cultures of the reference strains of L. infantum (MCAN/IR/97/LON490) were 88 run on each gel. Extravasation oral mucocele from 10 patients were used as the negative 89 controls. The negative controls, ultrapure water replaced the template DNA, were also run. 90 The fragment of L. major specific kinetoplast mini-circle DNA, 560 bp in length, was 91 amplified from the nasal, whereas the fragment of L. ropica, 750 bp in length, was amplified 92 from the oral lesions by the second-round PCR assay, respectively (Fig. 3). 93 The patient was treated with intravenous infusion of Amphotericin B, 1mg/kg/day, for 14 94 days and the resolution of the lesions started one week after treatment started. 95 96 Ethics Statement 97 The Ethics Committee of the Faculty of Medical School, University of Shiraz Medical 98 Sciences and the authors' institutional review board of Dr. Daneshbod Laboratory approved 99 the study and the authors group collected written informed consent from the patient. 100 on A uust 0, 2017 by gest ht://jcm .sm .rg/ D ow nladed fom Discussion 101 Mucosal leishmaniasis is a rare disease in the world, even in endemic areas such as Iran 102 (6,25). The importance of ML is due to the severity of its clinical lesions, poor response to 103 traditional antimony therapy and destruction of the nasal architecture with gross facial 104 alterations (17). Mucosal leishmaniasis is a form of tegumentary leishmaniasis that has been 105 shown to be associated with L. braziliensis, L. panamensis and less frequently with L. 106 amazonensis, although it has been reported in infections caused by other new word 107 Leishmania species, such as L. guyanensis (13). A few patients with ML have been described 108 in the old world in infections caused by L. infantum, L. tropica, and L. major (16,18,21,25). 109 The patient in the present study had nasal lesions caused by L. major, but L. tropica was 110 isolated from his oral lesions too. The association between ML and previous or active skin 111 lesions is widely accepted, as both forms can be originated from a single species (13). It has 112 also been demonstrated that localization of the parasites in the mucous membrane of nasal, 113 oral, and pharyngeal areas occurs as a result of migration of Leishmania via lymphatic or it is 114 due to the hematogenous dissemination of the amastigotes from the skin of 5% of the patients 115 affected with CL (14). Oral involvement is unusual and in most cases it becomes evident after 116 several years of resolution of the original cutaneous lesions (22). 117 Sporadic L. major and L. tropica infections have occasionally been reported in patients with 118 ML in Afghanistan, Saudi Arabia and Sudan (5,9,11). Fars Province, a region in southern 119 Iran, is a classical focus of CL and the previous studies have consistently documented the 120 etiologic agent to be L. tropica and L. major in urban and rural areas, respectively (4,12). 121 Mixed infections by different Leishmania species could explain differences in the clinical 122 course of these infections as well as resistant cases (1). We presented the first report of co123 infection by L. major and L. tropica isolated from a patient with ML. In the sub-Andean 124 region of Bolivia, co-infection by L. amazonensis and L. infantum/L. chagasi have been 125 on A uust 0, 2017 by gest ht://jcm .sm .rg/ D ow nladed fom identified in a patient with diffuse cutaneous leishmaniasis (20). In the suburban district of 126 Campo Grande, Municipality of Rio de Janeiro (Brazil) L. donovani and L. braziliensis have 127 been isolated from bone marrow and forehead of a patient with concurrent asymptomatic VL 128 and typical CL (24). There are data indicating that concomitant natural infection with L. 129 donovani and L. major has occurred in humans with CL and VL (2,15). Mixed infections 130 have also been observed in sand flies and dogs (8,10). Antoniou et al. (2004) indicated that 131 the VL form may occur due to mixed infection by different strains of the L. infantum (3). 132 Such reservoirs are exposed to a large numbers of sand fly bites, which increases the 133 possibility of infection by different strains or species of the parasite. Moreover, mixed 134 infections of the same macrophage by different species of Leishmania have been shown to be 135 experimentally possible (1). Based on the results of PCR and IHC, concomitant or mixed 136 mucosal infection can occur in immunocompetent subjects with two Leishmania species. This 137 seems to be the first described case of concurrent or mixed infection of ML with L. major and 138 L. tropica. Leishmania major was isolated from the nasal lesion that occurred two months 139 earlier than the oral lesion from which L. tropica was isolated and this can indicate that, at 140 least in this patient, a previous infection with L. major did not protect against L. tropica. On 141 the other hand, it has previously been reported that L. tropica primary infection was not 142 efficient in reducing the parasite load of the spleen in the secondary L. major infection (19). 143 This result has important implications for development of vaccines against leishmaniases and 144 emphasizes attention to mixed ML infection in diagnosis and treatment. 145 146 Acknowledgements 147 The authors would like to thank the authorities of the Veterinary School, Shiraz University 148 and Medical School, Shiraz University of Medical Sciences, and Institute of Experimental 149 Pathology, University of Muenster for their support. They also would like to thank Drs. M. 150 on A uust 0, 2017 by gest ht://jcm .sm .rg/ D ow nladed fom Davarmanesh, M.M. Davarpanah, K. Daneshbod, M. Kalantari, and Prof. J. Brosius, Dr. T. 151 Rozhdestvensky, and G. Randau, Institute of Experimental Pathology, University of 152 Muenster, for their help and advice. 153