mucocutaneous leishmaniasis often occurs in new world mostly due to leishmania(l ) braziliensis and less commonly l. panamensis and l. amazonensis(1 ) .
there are also several reports of mucocutaneous leishmaniasis in old world ( 24 ) . recently a case of mucocutaneous leishmaniasis due to l. major has been reported from iran ( 1 ) . in leishmaniasis
, laryngeal involvement often occurs in active cigarette smokers and in patients with chronic respiratory diseases ( 5 ) . a case of visceral leishmaniasis caused by l. major with skin and oral mucosal lesions in a man who was suffering from hoarseness and weight loss
a 28-year - old otherwise healthy , non - smoking man with the chief complaint of hoarseness from southeastern part of iran was referred to our dermatology center in may 2014 .
he had a few indurated , erythematous plaques on his face ( chin and upper lip ) and legs , a violaceous plaque on his hard palate , and complained of hoarseness for the past 7 years ( fig . 1 and 2 ) .
he also complained of dysphagia to solid diet , and about 20 kilograms weight loss in the last year .
hoarseness was the presenting symptom and lasted for about 5 years during the time that the cutaneous lesions were present .
skin lesions : erythematous plaque on chin and upper lip oral mucosal lesion : an erythematous plaque on the palate near the opening of the pharynx the study was approved by the local ethics committee and an informed consent was taken from the patient .
routine laboratory tests were normal except for a mild normocytic normochromic anemia , which was justified with chronic disease anemia .
erythrocyte sedimentation rate , complement levels ( c3  c4  ch50 ) , antinuclear antibody ( ana ) , and angiotensin converting enzyme ( ace ) tests were all within normal limits .
wright , 2-mercaptoethanol ( 2me ) , and widal tests were done for his constitutional symptoms and low back pain , which all came back negative .
vdrl and viral markers ( hivab  hbsag  hcvab ) were negative , and a ppd skin test was negative .
abdominal sonography showed mild hepatomegaly and a 6 * 8 mm lymph node in retroperitoneal near the superior mesenteric artery origin .
sonography of the neck showed bilateral internal jugular and submandibular lymphadenopathy ( 9 * 17 mm  9 * 13 mm ) .
a biopsy from his oral lesion was done with the differential diagnosis of lymphoma , tuberculosis , leishmaniasis , wegener s granulomatosis , sarcoidosis , squamous cell carcinoma , granular cell tumor and histiocytosis .
the hematoxylin and eosin stain ( h&e ) preparation of histopathologic slides of the mucosal and skin lesions showed loose granuloma formation with lymphoplasma cells . in specimen of mucosal biopsy leishman bodies
his cutaneous and mucosal lesions were examined by direct microscopy for acid - fast bacilli and the test was negative .
lastly , pcr was performed on the specimens of skin , bone marrow , mucosa and saliva .
the result of pcr was positive , and for the determination of pathogenic agent , amplification and sequencing analysis were performed as follow . in order to determine the possibility of leishmania infection ,
external primers csb2xf and csb1xr in the first round of pcr , and internal primers 13z and lir in the second round of pcr were utilized ( invitrogen , uk ) . as a positive control , genomic dnas of standard l. major ( mrhom / ir/75/er ) and l. tropica ( mohm / ir/09/khamesipour - mashhad ) were used in parallel .
nested pcr amplification of kdna extracted from skin , bone marrow , mucosa and saliva of the patient which then confirmed by sequence analyses 

 lane 1 : molecular weight marker 100bp 

 lane 2 : l. tropica 

 lane 3 : patient sample 

 lane 4 : l. major 

 
 lane 1 : molecular weight marker 100bp 
 
 lane 3 : patient sample 
  as it is shown , the nested pcr analysis of the patient sample was l. major . for confirmation of the species identification
, the 560bp pcr product of kdnas from the patient as well as standard l. major were eluted by promega wizard  sv gel and pcr clean up system ( promega , usa )
. then the eluted dnas were ligated into the topo vector pcr2.1 ( invitrogen , usa ) , transformed in top 10 e. coli , and plated in lb agar containing 100g / ml ampicillin . the positive colonies were first isolated and screened with ecori ( roche , germany ) as the restriction enzyme . after digestion ,
plasmid dna was purified ( qiagen , plasmid mini kit ) and sequenced with the m13f and m13r as flanking primers using the dideoxy chain termination method on an automated sequencer .
the sequence of kdna of the patient sample was the same as the iranian strain of l. major ( mrhom / ir/75/er ) .
pcr of skin , mucosa , saliva and bone marrow revealed l. major . with regard to diagnosis of leishmaniasis and probable visceral involvement ,
the direct agglutination test ( dat ) ( 7 ) with a remarkable high titer ( 1:102400 ) was positive , and the indirect fluorescent antibody test ( ifat ) for leishmania infection was also positive ( 1:800 ) . based on the histopathologic findings , the high titer of dat and ifat , and the results of pcr , a diagnosis of leishmaniasis with visceral involvement were confirmed .
since miltefosine has been used for visceral involvement and l. major infections , the following combination was used to treat the patient ( 8) .
miltefosine with the dose of 50 mg orally thrice daily was applied in combination with meglumine antimoniate ( glucantime ) 20 mg / kilograms / day for 28 days .
the only side effect of miltefosine was nausea , which was controlled with the ingestion of metoclopramide a half hour before taking miltefosine .
the oral and cutaneous lesions , as well his hoarseness , improved to a significant extent .
in order to determine the possibility of leishmania infection , nested pcr method was applied .
the primers were already designed by noyes ( 6 ) . external primers csb2xf and csb1xr in the first round of pcr , and internal primers 13z and lir in the second round of pcr were utilized ( invitrogen , uk ) . as a positive control , genomic dnas of standard l. major ( mrhom / ir/75/er ) and l. tropica ( mohm / ir/09/khamesipour - mashhad )
nested pcr amplification of kdna extracted from skin , bone marrow , mucosa and saliva of the patient which then confirmed by sequence analyses 

 lane 1 : molecular weight marker 100bp 

 lane 2 : l. tropica 

 lane 3 : patient sample 

 lane 4 : l. major 

 
 lane 1 : molecular weight marker 100bp 
 
 lane 3 : patient sample 
  as it is shown , the nested pcr analysis of the patient sample was l. major . for confirmation of the species identification
, the 560bp pcr product of kdnas from the patient as well as standard l. major were eluted by promega wizard  sv gel and pcr clean up system ( promega , usa )
. then the eluted dnas were ligated into the topo vector pcr2.1 ( invitrogen , usa ) , transformed in top 10 e. coli , and plated in lb agar containing 100g / ml ampicillin .
the positive colonies were first isolated and screened with ecori ( roche , germany ) as the restriction enzyme .
after digestion , plasmid dna was purified ( qiagen , plasmid mini kit ) and sequenced with the m13f and m13r as flanking primers using the dideoxy chain termination method on an automated sequencer .
the sequence of kdna of the patient sample was the same as the iranian strain of l. major ( mrhom / ir/75/er ) .
pcr of skin , mucosa , saliva and bone marrow revealed l. major . with regard to diagnosis of leishmaniasis and probable visceral involvement ,
the direct agglutination test ( dat ) ( 7 ) with a remarkable high titer ( 1:102400 ) was positive , and the indirect fluorescent antibody test ( ifat ) for leishmania infection was also positive ( 1:800 ) . based on the histopathologic findings , the high titer of dat and ifat , and the results of pcr , a diagnosis of leishmaniasis with visceral involvement were confirmed .
since miltefosine has been used for visceral involvement and l. major infections , the following combination was used to treat the patient ( 8) .
miltefosine with the dose of 50 mg orally thrice daily was applied in combination with meglumine antimoniate ( glucantime ) 20 mg / kilograms / day for 28 days .
the only side effect of miltefosine was nausea , which was controlled with the ingestion of metoclopramide a half hour before taking miltefosine .
the oral and cutaneous lesions , as well his hoarseness , improved to a significant extent .
to the best of our knowledge through internet search , the first report of mucosal involvement of leishmania in iran dated back to 1968 when ziai et al . reported a case of leishmaniasis with mucosal involvement from shiraz , iran ( 2 ) . since then there have been several additional reports of mucosal involvement of leishmaniasis from iran ( 3 , 4 ) .
according to a recent report , l. tropica , l. major , and l. infantum may be the causative agents , but l. major was the pathogenic agent in most of the patients ( 3 ) .
the case presented herein with the high titers of anti - leishmania antibodies using dat and ifat were in favor of visceral involvement .
dat , which is positive when the titer is higher than 1/3200 , is used for the diagnosis and seroepidemiology of visceral leishmaniasis in endemic areas ( 9 ) . in the current patient ,
these findings and the involvement of bone marrow confirmed the diagnosis of visceral leishmaniasis in the patient .
pcr findings of the cutaneous , bone marrow , mucosa and saliva samples were in favor of l. major as a causative agent . recently ,
karamian et al . have reported l. major as a causative agent of visceral leishmaniasis in the south of iran ( 10 ) . according to search in the literature
, there are a few reports of the coexistence of visceral leishmaniasis with cutaneous lesions in patients with hiv infection ( 1113 ) .
there is a report from iran in which a patient showed visceral and cutaneous lesions due to leishmania but , immunological tests in this case were negative and diagnostic splenectomy revealed the leishmania as the causative agent ( 14 ) .
our patient also presented with simultaneous visceral and cutaneous leishmaniasis , an hiv test was negative .
the case presented herein was a non - smoker , otherwise healthy man , with visceral leishmaniasis , who had a history of a long period of hoarseness , which can be a symptom of visceral leishmaniasis in smokers or people with chronic respiratory disease ( 5 ) .
this patient was presented as a rare case of visceral leishmaniasis with the presenting symptom of hoarseness , l. major as the causative agent , and simultaneous muco - cutaneous lesions .
treatment with the combination of miltefosine and meglumine antimoniate was effective and the dat titer decreased to the level of 1/52000 one month after the patient was discharged from the dermatology ward .
 herein , a 28-year - old man with hoarseness , skin and oral lesions is presented . at the time of admission , the patient had an erythematous plaque on his chin near his lower lip and an erythematous - violaceous plaque on his palate near the opening of the pharynx and 20 kg weight lost in last one year . 
 the biopsy of his skin lesions by hematoxylin and eosin staining revealed an infiltration of the dermis by lymphoplasma and histiocytic cells with a loose granuloma formation suggestive of leishmaniasis . 
 biopsy of mucosal lesions revealed leishman bodies in dermis . 
 pcr was performed on the specimens of skin , bone marrow , mucosa , and saliva , the results were positive . 
 the pathogenic agent was identified as leishmania major by the nested pcr . 
 serologic tests including direct agglutination test ( dat ) and indirect immunofluorescence test ( ifat ) were positive with high titers of anti - l . 
 infantum antibodies ( 1:102400 versus 1:800 , respectively ) , indicative of visceral involvement . 
 the patient responded to a combination of miltefosine and meglumine antimoniate ( glucantime ) . 
 visceral involvement due to l. major is rarely reported . to the best of our knowledge , 
 probably hoarseness due to l. major has not been previously reported from iran . 
Introduction
Case Report
Amplification and sequencing analysis
Discussion