cl was diagnosed in patients as described elsewhere , according to characteristic lesion morphology , positive skin test results , seropositivity to leishmania antigen , and/or presence of parasites in the lesion .
a total of 58 patients with lcl due to l. braziliensis ( 27 males ; mean age [ sd ] , 29.6  2.3 years ) were recruited and treated in 2 outpatient clinics ( jequi and jiquiri - ba , ne brazil ) covering the same rural area .
ten patients with lcl due to l. amazonensis ( 6 males ; mean age [ sd ] , 38.8  6 years ) were recruited and treated at the professor edgar santos university hospital ( salvador - ba , ne brazil ) .
in addition , a paired sample was obtained from 13 patients at the time of definite clinical cure ( range , 68417 days ) .
patients with lcl ( due to either l. braziliensis or l. amazonensis ) received standard intravenous pentavalent antimony ( glucantime ; rhodia , 20 mg / kg / day for 20 days ) .
cure was defined by the complete scarring of lesions , without induration and relapse during 2 years of follow - up . although all l. amazonensis  infected patients were cured within 90 days following a single treatment cycle , l. braziliensis  infected patients needed a mean ( sd ) of 2.2  0.2 treatment cycles over 190.5  16.0 days to achieve cure , with treatment for 23 of 58 patients ( 39.7% ) classified as failing ( > 2 cycles ) .
eight patients with dcl ( 3 males ; mean age [ sd ] , 31.5  7.1 years ) infected with l. amazonensis were recruited and treated at the presidente dutra university hospital  hupd ( so lus - ma , ne brazil ) .
patients with dcl had a mean disease duration ( sd ) of 10.9  2.1 years , during which several different therapeutic schemes ( mean number [ sd ] per patient , 2.7  0.9 , including liposomal amphotericin b and combinations of glucantime , interferon  , and pentamidine ) were used , with no or only a transient clinical effect .
since dcl is a rare clinical manifestation , no samples from untreated patients ( at diagnosis ) were available for comparison . sod1
plasma levels were quantified using a human sod1 enzyme - linked immunosorbent assay kit ( calbiochem ) .
rna was extracted from lesion biopsy specimens from patients with lcl and those with dcl , as well as skin biopsy specimens from healthy controls , using trizol , followed by an additional purification using rneasy ( qiagen benelux , venlo , the netherlands ) . using in situ transcriptomics
, we simultaneously quantified host and parasite rnas in lesion biopsy specimens from patients with lcl and those with dcl , as well as skin biopsy specimens from healthy controls , by ncounter technology ( nanostring , seattle , wa ) , based on molecular bar coding of target rna transcripts and digital detection at the femtomolar range , using direct hybridization , without reverse transcription or amplification .
human sod1 , sod2 , sod3 , ifna1 , ifna2 , ifna4 , ifnb1 , and l. braziliensis and l. amazonensis sod1 - 5 messenger rna ( mrna ) levels were quantified in situ , in addition to mrnas of several housekeeping genes ( gusb , g6pd , gapdh , and hprt1 ) , for normalization , as well as leukocyte - specific genes ( cd3 , cd14 , cd19 , cd56 , and cd45 ) .
briefly , monocyte - derived human macrophages were infected with l. amazonensis ( mhom / br/87/ba125 ) and treated with sod1 ( sigma ) for 48 hours , followed by extensive washing , staining with hematoxylin / eosin , and counting of intracellular amastigotes ( 100 cells , with duplicate analysis for each sample ) .
parametric and nonparametric tests were performed according to kolmogorov - smirnov test for normality . for multiple comparisons , the kruskal 
wallis test with the dunn posttest and 1-way analysis of variance with a posttest for linear trend were used . for comparison between 2 groups , the f test , the mann 
whitney u test , the t test , or the wilcoxon test was used . to identify biomarkers , receiver operating characteristic ( roc )
all tests were 2 tailed , and differences were considered significant at p values of < .05 .
we measured plasma levels of sod1 in healthy controls , patients with lcl ( due to l. braziliensis or l. amazonensis ) , and patients with dcl ( due to l. amazonensis ) and investigated its potential role as a biomarker . the mean sod1 level ( sem ) among patients with lcl due to l. braziliensis (
90.6  8.0 pg / ml ) was 3-fold greater than that among healthy controls ( 30.2  3.9 pg / ml ; p < .01 , by the kruskal  wallis ; p < .001 , by the dunn posttest ) , whereas the mean level ( sem ) among patients with lcl due to l. amazonensis ( 93.1  24.7 pg / ml ) was 3.1-fold greater than that among healthy controls ( p < .01 , by the kruskal  wallis test ; p < .01 , by the dunn posttest ) . on the other hand ,
pg / ml ) were 4.4-fold greater than those among patients with lcl due to l. amazonensis ( p = .0005 , by the mann 
whitney u test ; figure 1a ) . to investigate whether increased sod1 levels during lcl is related to active disease , the sod1 level was also measured in paired samples of from 13 patients with lcl due to l. braziliensis before treatment ( at diagnosis ) and after treatment ( at definite clinical cure ) .
sod1 levels decreased 1.6-fold between pretreatment and posttreatment samples ( 99.2  20.81 pg / ml vs 60.4  5.2 pg / ml ; p = .027 , by the wilcoxon matched pairs test ; supplementary figure 1a ) .
since variances differed significantly between controls and patients with lcl due to l. braziliensis ( p < .0001 , by the f test ) , we hypothesized that heterogeneous sod1 levels in patients with lcl might reflect differences in clinical status and/or therapeutic response .
sod1 levels did not significantly correlate with age , sex , disease duration , lesion size , or healing time among patients with lcl ( data not shown ) .
however , sod1 plasma levels were significantly associated with therapeutic response , quantified as the number of treatment cycles required for cure ( p = .0069 , by the kruskal 
wallis test ; p < .001 , by the dunn posttest ; figure 1c ) . by use of roc curve analysis
, successful ( 1 cycle ) and failing ( > 2 cycles ) therapeutic response could be significantly discriminated through sod1 plasma levels ( area under the roc curve , 0.83 ; p = .00025 ) , authenticating its clinical utility as a biomarker ( figure 1d ) . above a cutoff of 122 pg / ml , 9 of 22 patients ( 40.9% ) in whom treatment was failing
could be identified with 100% specificity ( ie , 0 patients cured with 1 treatment cycle displayed sod1 levels of > 122 pg / ml ) . strikingly , at a similar cutoff of 112 pg / ml , patients with dcl could be discriminated from treatable patients with lcl ( due to l. amazonensis ) with 100% sensitivity and 90% specificity ( area under the roc curve , 0.95 ; p = .001 ; figure 1b ) . 

figure 1.superoxide dismutase 1 ( sod1 ) is a biomarker for therapeutic failure in cutaneous leishmaniasis . a , sod1 measurements in plasma samples from 20 healthy controls , 58 patients with localized cutaneous leishmaniasis ( lcl ) due to leishmania braziliensis ) , 10 patients with lcl due to leishmania amazonensis , and 8 patients with diffuse cutaneous leishmaniasis ( dcl ) due to l. amazonensis .
wallis test ; and * * * p < .001 , by the dunn posttest , for healthy controls vs patients with lcl due to l. braziliensis ; * * p < .01 , by the dunn posttest , for healthy controls vs patients with lcl due to l. amazonensis ; and * * * p < .001 , by the mann 
whitney u test , for patients with lcl due to l. amazonensis vs patients with dcl .
b , plasma sod1 level significantly discriminates patients with lcl due to l. amazonensis from patients with dcl due to l. amazonensis ( area under the receiving operating characteristic [ roc ] curve , 0.95 ; * * * p = .001 ) .
c , sod1 measurements in plasma samples from patients with lcl due to l. braziliensis , classified according to number of treatment cycles ( p = .0069 , by the kruskal 
d , plasma sod1 level significantly predicts therapeutic response in patients with lcl due to l. braziliensis ( success is defined as achievement of cure after 1 treatment cycle ; failure is defined as lack of cure after > 2 treatment cycles ; area under the receiving operating characteristic curve , 0.83 ; * * * p = .00025 , without outliers , as in panel c ) .
superoxide dismutase 1 ( sod1 ) is a biomarker for therapeutic failure in cutaneous leishmaniasis .
a , sod1 measurements in plasma samples from 20 healthy controls , 58 patients with localized cutaneous leishmaniasis ( lcl ) due to leishmania braziliensis ) , 10 patients with lcl due to leishmania amazonensis , and 8 patients with diffuse cutaneous leishmaniasis ( dcl ) due to l. amazonensis .
wallis test ; and * * * p < .001 , by the dunn posttest , for healthy controls vs patients with lcl due to l. braziliensis ; * * p < .01 , by the dunn posttest , for healthy controls vs patients with lcl due to l. amazonensis ; and * * * p < .001 , by the mann 
whitney u test , for patients with lcl due to l. amazonensis vs patients with dcl .
b , plasma sod1 level significantly discriminates patients with lcl due to l. amazonensis from patients with dcl due to l. amazonensis ( area under the receiving operating characteristic [ roc ] curve , 0.95 ; * * * p = .001 ) .
c , sod1 measurements in plasma samples from patients with lcl due to l. braziliensis , classified according to number of treatment cycles ( p = .0069 , by the kruskal 
d , plasma sod1 level significantly predicts therapeutic response in patients with lcl due to l. braziliensis ( success is defined as achievement of cure after 1 treatment cycle ; failure is defined as lack of cure after > 2 treatment cycles ; area under the receiving operating characteristic curve , 0.83 ; * * * p = .00025 , without outliers , as in panel c ) . to test whether these high plasma sod1 levels in patients with cl might be directly related to an increased parasite burden
, human macrophages from healthy donors were infected with l. amazonensis ( 5:1 ) and cultivated in the absence or presence of recombinant sod1 protein . as shown in figure 2a ,
sod1 treatment significantly increased parasite load ( p = .042 , by the t test ) .
moreover , infected human macrophages treated with recombinant sod1 protein presented cytomorphological features ( figure 2b ) strikingly similar to the vacuolized and highly parasitized macrophages from cutaneous lesions of patients with dcl ( figure 2c ) . 

figure 2.superoxide dismutase 1 ( sod1 ) correlates to parasite burden in vitro and in situ in diffuse cutaneous leishmaniasis ( dcl ) .
a and b , human macrophages were infected with leishmania amazonensis ( 5:1 ratio ) for 4 hours and then treated in the absence or presence of sod1 protein ( 175 u / ml ) for 48 hours .
a , parasite burden was quantified ; each bar represents the mean  standard error of the mean for 3 donors ( * p = .042 , by the t test ) .
b , infected macrophages ( 1000 magnification ) left untreated ( left panel ) or treated with sod1 ( right panel ) .
c , cutaneous lesion biopsy specimen from a representative patient with dcl stained with hematoxylin and eosin ( 1000 magnification ) .
d , positive correlation between human sod1 and parasite sod2 ( r = 0.98 , p = .019 ) and sod4 ( r = 0.99 , p = .0026 ) messenger rna ( mrna ) levels ( normalized to g6pd ) , quantified by ncounter in situ in biopsy specimens from patients with dcl ( n = 4 ) .
superoxide dismutase 1 ( sod1 ) correlates to parasite burden in vitro and in situ in diffuse cutaneous leishmaniasis ( dcl ) .
a and b , human macrophages were infected with leishmania amazonensis ( 5:1 ratio ) for 4 hours and then treated in the absence or presence of sod1 protein ( 175 u / ml ) for 48 hours .
a , parasite burden was quantified ; each bar represents the mean  standard error of the mean for 3 donors ( * p = .042 , by the t test ) .
b , infected macrophages ( 1000 magnification ) left untreated ( left panel ) or treated with sod1 ( right panel ) .
c , cutaneous lesion biopsy specimen from a representative patient with dcl stained with hematoxylin and eosin ( 1000 magnification ) .
d , positive correlation between human sod1 and parasite sod2 ( r = 0.98 , p = .019 ) and sod4 ( r = 0.99 , p = .0026 ) messenger rna ( mrna ) levels ( normalized to g6pd ) , quantified by ncounter in situ in biopsy specimens from patients with dcl ( n = 4 ) . finally , using in situ transcriptomics ( ncounter ) , we demonstrated a significant positive correlation between host sod1 mrna level and parasite sod2 ( p = .019 , r = 0.98 ) and sod4 ( p = .0026 , r = 0.99 ) mrna in dcl .
taken together , these data suggest that systemic sod1 might play a causal role in both the clinical and anatomopathological phenotype of dcl .
up to now , no noninvasive biomarker for therapeutic response to sb has been described in cl . in the present work ,
we demonstrate host sod1 as a biomarker for therapeutic failure of first - line sb in new world patients with cl .
sod1 plasma levels could significantly discriminate between successful and failing therapeutic response in patients with lcl ( due to l. braziliensis ) and between treatable patients with lcl ( due to l. amazonensis ) and refractory patients with dcl .
quantification of sod1 plasma level at diagnosis represents a rapid , sensitive , and inexpensive new tool in the clinical management of cl .
further research will be necessary to test its predictive value in cl caused by other leishmania species , since in peru failure of antimonial therapy is significantly associated with infections due to l. braziliensis and leishmania peruviana but not with infection due to leishmania guyanensis . by identifying , at the time of diagnosis , up to 40.9% of patients ( with 100% specificity ) in whom antimonial therapy will fail ( figure 1c ) , second - line treatment ( eg , amphotericin b or miltefosine ) could be provided as a first choice , thereby diminishing public health costs and avoiding unnecessary and prolonged exposure of the patients to the often severe side effects of intravenous pentavalent antimony therapy . although sod1 is an intracellular antioxidant enzyme , our results suggest a direct role for increased systemic ( plasma ) sod1 as a trigger of parasite replication in infected macrophages .
elsewhere , treatment of infected human macrophages with purified sod1 in vitro led to strongly increased parasite burden and the appearance of large parasitophorous vacuoles , reminiscent of typical nodular lesions in dcl .
moreover , this correlation between sod1 and parasite burden was recapitulated in situ in dcl lesions , as evidenced by a strong linear relationship ( r = 0980.99 ) between host ( human sod1 ) and parasite ( l. amazonensis sod2 and sod4 ) sods .
this parallel cross - regulation at the transcriptional level between superoxide - quenching enzymes that are hundreds of millions of years distant in evolution is a striking example of interkingdom signaling . to our knowledge , this is the first description of interkingdom signaling in protozoan - animal interaction . in keeping with our cytomorphological data ( figure 2b ) ,
this phenomenon is highly specific for cytosolic sod1 , since cross - kingdom correlations were not observed for mitochondrial sod2 or extracellular sod3 with any of the parasite mrnas .
the extremely long ( mean disease duration [ sd ] , 10.9  2.1 years ) uncontrolled parasite replication in patients with dcl might have led to selection of sod - overexpressing parasite clones [ 7 , 8 ] .
alternatively , increased systemic levels of sod1 in patients with lcl and those with dcl might reflect a parasite escape mechanism from host leishmanicidal activity mediated by type i ifn , as suggested by our previous work   and recently confirmed in murine and human macrophages . in agreement with this hypothesis
, we found a significant positive correlation between sod1 mrna and total ifn-/ mrna levels , using in situ transcriptomics ( ncounter ; supplementary figure 2a ) .
finally , since all of our patients with dcl also experienced failure of multiple other treatment schemes ( including pentamidine , amphotericin b , and ifn- , alone or in combination ) , an increased plasma sod1 level truly reflects broad therapeutic failure . the dramatic increase in dcl
also reinforces the idea that sod1 is not a mere surrogate marker but a key molecule in dcl pathogenesis , driving perennial parasite replication and systemic dissemination , as suggested by figure 2a  d .
hence , sod1 is also a candidate therapeutic target in cl , reinforcing our previous suggestion of pharmacologic sod1 inhibition by detc   as a viable therapeutic alternative in lcl and dcl in particular , for which no effective treatment options exist . in conclusion ,
our results indicate that host sod1 is a clinically useful biomarker for therapeutic failure , as well as a possible therapeutic target , in lcl and dcl .
supplementary materials consist of data provided by the author that are published to benefit the reader .
 we show that increased plasma superoxide dismutase 1 ( sod1 ) levels are statistically significant predictors of the failure of pentavalent antimony treatment for cutaneous leishmaniasis caused by leishmania braziliensis . in leishmania amazonensis  infected patients , host sod1 levels can be used to discriminate between localized and drug - resistant diffuse cutaneous leishmaniasis . using in situ transcriptomics ( ncounter ) , we demonstrate a significant positive correlation between host sod1 and interferon / messenger rna ( mrna ) levels , as well as interkingdom correlation between host sod1 and parasite sod2/4 mrna levels . in human macrophages , in vitro treatment with sod1 increases the parasite burden and induces a diffuse cutaneous leishmaniasis  like morphology . thus , sod1 is a clinically relevant biomarker and a therapeutic target in both localized and diffuse cutaneous leishmaniasis . 
PATIENTS AND METHODS
RESULTS
DISCUSSION
Supplementary Data