tissue samples were taken from 27 patients with suspected cl who had consulted the health centers from march 2005 to march 2006 .
local reference laboratories evaluated all stained slides by light microscopy and positively identified leishmania amastigotes .
patients had no history of travel and were assumed to been infected in morocco ; all received free intralesional injections of meglumine antimoniate ( glucantime ; sanofi - aventis , bridgewater , nj , usa ) until total recovery , according to the protocol in the mmh leishmaniasis control manual .
samples were collected in areas of morocco known for high cl incidence : north ( sidi kacem ) , center ( beni mellal and boulemane ) , southeast ( errachidia ) , and southwest ( taroudant and ouarzazate ) ( figure 1 ) .
dna extraction and pcr analysis by amplification of the ribosomal internal transcribed spacer 1 ( its1 ) , using stained slides , was performed as described by schonian et al .
we used 0.6-nm primers and pcr - ready supreme mix ( syntezza bioscience , jerusalem , israel ) in 25 l of total reaction .
leishmania dna ( 10 ng / reaction ) from reference strains l. tropica ( mhom / az/1974/saf - k27 ) , l. major ( mhom / tm/1973/5askh ) , and l. infantum ( mhom / tn/1980/ipt1 ) were used as positive controls .
after amplification , the pcr product was digested with 1.5 l bsuri endonuclease ( mbi fermentas , burlington , ontario , canada ) , and all digested products were analyzed by agarose gel electrophoresis ( 12 ) .
all patients had classic symptoms of cl , from small erythematous papules to nodules and ulcerative lesions .
papular lesions , nodular lesions , or both were present in 30% of the cl patients ; ulcerative lesions , in 52% . neither the papular / nodular nor the ulcerative forms correlated with a particular leishmania species .
the erythematous clinical form was present in 18% of total case - patients and in 63% of case - patients from the sidi kacem region .
undigested its1 amplicons from the 27 slides produced a band of 300350 bp ( data not shown ) , which confirmed the presence of leishmania dna .
band patterns from the digested samples were compared with digested standards for each reference strain and identified the parasite species ( figure 2 ) as follows : l. major , 3 samples each from ouarzazate and errachidia ; l. tropica , 2 samples from taroudant , 4 from beni mellal , and 7 from boulemane ; l. infantum , 8 samples from sidi kacem .
comparison between endonuclease bsuri digestion patterns of internal transcribed spacer 1pcr products from clinical samples and reference leishmania strains .
clinical samples , lanes 16 : lane 1 , boulmane ; lane 2 , ouarzazate ; lanes 36 , sidi kacem .
reference strains , lanes 79 : lane 7 , l. tropica ( mhom / su/1974/saf - k27 ) ; lane 8 , l. major ( mhom / tm/1973/5askh ) ; lane 9 , l. infantum ( mhom / tn/1980/ipt1 ) .
cl caused by l. major or l. tropica and vl caused by l. infantum have been reported in morocco ( 59 ) .
pcr on archived tissue samples enabled us to investigate the epidemiology of cl in disease - endemic regions of morocco and identify those species responsible for this disease in several new foci ( table ) . our results , together with those of previous studies ( 59 ; unpub .
data from mmh , 2001 ) , indicate that cl caused by l. tropica is found throughout the center of the country in a band stretching from the atlantic ocean along the length of the atlas mountains almost to the mediterranean sea .
cl caused by l. major is present in the desert region south of the atlas mountains in a strip bordering the sahara desert ( figure 1 ) .
we report on a focus of cl in morocco caused by l. infantum , 8 samples from sidi kacem . in morocco ,
the only previous human cl case caused by l. infantum was reported in 1996 , within an active focus of vl ( 13 ) .
the northern coastal regions of morocco are endemic for human and canine vl . as in other vl - endemic regions surrounding the mediterranean sea , this disease is caused by l. infantum ( 3 ) .
although it is unusual for this parasite to cause cl , our finding is similar to a recent report from tunisia , where l. infantum was shown to cause sporadic cl in regions endemic for vl .
it appeared to have emerged in a new region of the country and was suggested to be more prevalent than originally indicated ( 14 ) .
cl and vl overlap in many provinces of central morocco ; anthroponotic foci of l. tropica cl are found in fes and taza ( 79 ) ( figure 1 ) , not far from existing vl foci including sidi kacem .
furthermore , several cases of canine vl caused by l. tropica have been reported in regions where canine vl is caused by l. infantum .
the nodular form of cl was caused by all 3 species ; ulcerative lesions were seen only with cl caused by l. tropica and l. major .
of the 8 patients in sidi kacem with l. infantum infection , 5 showed the atypical erythematous papular form .
the overlapping distribution of parasite species , causing diseases with similar clinical pictures , demonstrates the need for additional epidemiologic and ecologic studies of cl in conjunction with species identification .
this is especially important as traditional methods of determining infection from patient history and microscopic examination prove increasingly unreliable .
pcr can be performed rapidly on fresh or archived samples and does not require culturing of large amounts of parasites .
in addition , pcr costs have come down considerably , and costs can be further reduced by sending samples by regular mail to a central facility .
recent studies document the emergence of new leishmania foci and the coexistence of multiple leishmania species in the same geographic locale , including much of northern africa ( 14 ) .
we recommend that treatment protocols , particularly in areas of coexistence , be predicated on diagnosis of not only the clinical form 
cl versus vl  but additionally the disease - causing species . in morocco , local physicians and healthcare administrators
often do not realize that different species of leishmania require differential treatments , which can result in a failure to diagnose more serious disease .
however , recurrent failure of local treatments ( paromomycin and intralesional sodium stibogluconate ) against l. tropica was evident ( 11 ) .
because l. tropica , and now l. infantum , cause both vl and cl , a physician treating a cutaneous lesion may overlook visceral disease , and a host of costlier health problems may ensue . a simple , sensitive pcr test could easily reduce such risk .
further surveillance of cases and suspected cases from these foci should confirm the results of this limited study .
recent implementation of pcr - based diagnosis in an outbreak in northern algeria increased the positive diagnosis of cl by 69% over cases diagnosed by using microscopy alone ( 15 ) . furthermore
, anthroponotic cl caused by l. tropica is limited to parts of southern europe , asia , and africa ; diagnosis and treatment of the disease at its earliest stage is of paramount importance for reduction of the human reservoir .
failure to promptly diagnose and treat all cases will result in continued dissemination of the parasite .
 during the past 20 years , cutaneous leishmaniasis has emerged as a major public health threat in morocco . 
 we describe distribution of leishmania major and l. tropica in morocco and a new focus of cutaneous leishmaniasis due to l. infantum . 
 we recommend using molecular techniques to diagnose suspected leishmaniasis cases . 
The Study
Conclusions