Evaluation of enzyme-linked immunosorbent assay for diagnosis of post-kala-azar dermal leishmaniasis with crude or recombinant k39 antigen

The diagnosis of post-kala-azar dermal leishmaniasis (PKDL), a dermatosis that provides the only known reservoir for the parasite Leishmania donovani in India, remains a problem. Timely recognition and treatment of PKDL would contribute significantly to the control of kala-azar. We evaluated here the potential of the enzyme-linked immunosorbent assay (ELISA) as a diagnostic tool for PKDL. Antigen prepared from promastigotes and axenic amastigotes with parasite isolates that were derived from skin lesions of a PKDL patient gave sensitivities of 86.36 and 92%, respectively, in the 88 PKDL cases examined. The specificity of the ELISA test was examined by testing groups of patients with other skin disorders (leprosy and vitiligo) or coendemic infections (malaria and tuberculosis), as well as healthy controls from areas where this disease is endemic or is not endemic. A false-positive reaction was obtained in 14 of 144 (9.8%) of the controls with the promastigote antigen and in 14 of 145 (9.7%) of the controls with the amastigote antigen. Evaluation of the serodiagnostic potential of recombinant k39 by ELISA revealed a higher sensitivity (94.5%) and specificity (93.7%) compared to the other two antigens used. The data demonstrate that ELISA with crude or recombinant antigen k39 provides a relatively simple and less-invasive test for the reliable diagnosis of PKDL.     

Parasite detection in patients with post kala-azar dermal leishmaniasis in India: a comparison between molecular and immunological methods

AIMS: To evaluate the sensitivity and specificity of serological, immunohistochemical, and molecular methods in the diagnosis of post kala-azar dermal leishmaniasis (PKDL). METHODS: Twenty five patients with confirmed PKDL and 25 controls were included in the study. G2D10, a monoclonal antibody against Leishmania, was used for the immunohistochemical (IHC) staining of lesion sections to visualise anti-Leishmania donovani antibodies. The diagnostic usefulness of IHC was compared with enzyme linked immunosorbent assay (ELISA) with a recombinant (rk39) antigen, and a species specific polymerase chain reaction (PCR) assay, amplifying a kinetoplast minicircle DNA sequence. RESULTS: IHC detected 22 of 25 PKDL cases, giving a sensitivity of 88%. The diagnostic sensitivity of both the ELISA and PCR tests was higher (96%). All of the 25 controls examined were negative in PCR, indicating 100% specificity of the test, whereas ELISA showed 96% specificity. CONCLUSIONS: IHC with G2D10 significantly enhances the sensitivity of detection of PKDL over routine haematoxylin and eosin staining. ELISA with a recombinant antigen is an economical and practical assay. PCR is the most sensitive and specific diagnostic method for PKDL. The tests described would facilitate the recognition of patients with PKDL, enabling timely treatment, which would contribute greatly to the control of kala-azar.     

Potential of Direct Agglutination Test Based on Promastigote and Amastigote Antigens for Serodiagnosis of Post-Kala-Azar Dermal Leishmaniasis

Post-kala-azar dermal leishmaniasis (PKDL) is a dermal complication, a sequel to kala-azar. Diagnosis of PKDL presents a challenge due to the low parasite burden in the lesions. The direct agglutination test (DAT) based on promastigote and amastigote antigens of Leishmania donovani of indigenous isolates was developed to diagnose PKDL, and the results were compared with those of the rk39 strip test. The sensitivities of DAT for antileishmanial antibody detection, based on promastigote and amastigote antigens at a cutoff titer of 1:800 were 98.5% and 100%, respectively, with corresponding specificities of 96.5% and 100%. DAT could correctly detect 100% polymorphic cases and 95.4% macular PKDL cases. In comparison, the rk39 strip test was able to correctly diagnose 95.6% of polymorphic and 86.0% macular PKDL cases. DAT based on axenic amastigote antigen provided 100% sensitivity and specificity, making it particularly useful for macular PKDL cases, which are often missed by the rk39 strip test. Thus, DAT provides a simple, reliable, and inexpensive test for PKDL diagnosis with potential applicability in field conditions.     

Diagnosis of visceral leishmaniasis: comparative potential of amastigote antigen,recombinant antigen and PCR

Development of simple, economical and non-invasive tests for the early diagnosis of visceral leishmaniasis (VL) or kala-azar (KA) remains a challenge, and serological studies based on antigen prepared from the amastigote stage of Leishmania donovani, the stage that causes infection, are lacking. In the present study, circulating antibodies to total antigen isolated from the promastigote and amastigote stages of the parasite, as well as to recombinant K39 (rK39) antigen, are measured by enzyme-linked immunosorbent assay (ELISA) and the results compared with a polymerase chain reaction (PCR) test for KA diagnosis. In 116 samples of KA examined, the amastigote antigen gave significantly higher mean absorbance values in ELISA than did the promastigote antigen. The sensitivity for KA detection was significantly higher using the amastigote antigen (94%) than the promastigote antigen (90.5%). Analysis in 91 controls showed that specificity was higher with amastigote antigen (92.3%) than with promastigote antigen (86.8-89.0%). Reliability of ELISA diagnosis with amastigote antigen was only marginally lower than that with rK39 ELISA or with the PCR test. Easy availability and low cost of indigenous amastigote antigen, together with the simplicity of ELISA compared with PCR, make ELISA based on amastigote antigen a promising choice for the diagnosis of KA.     

Potential of direct agglutination test based on promastigote and amastigote antigens for serodiagnosis of post-kala-azar dermal leishmaniasis

Post-kala-azar dermal leishmaniasis (PKDL) is a dermal complication, a sequel to kala-azar. Diagnosis of PKDL presents a challenge due to the low parasite burden in the lesions. The direct agglutination test (DAT) based on promastigote and amastigote antigens of Leishmania donovani of indigenous isolates was developed to diagnose PKDL, and the results were compared with those of the rk39 strip test. The sensitivities of DAT for antileishmanial antibody detection, based on promastigote and amastigote antigens at a cutoff titer of 1:800 were 98.5% and 100%, respectively, with corresponding specificities of 96.5% and 100%. DAT could correctly detect 100% polymorphic cases and 95.4% macular PKDL cases. In comparison, the rk39 strip test was able to correctly diagnose 95.6% of polymorphic and 86.0% macular PKDL cases. DAT based on axenic amastigote antigen provided 100% sensitivity and specificity, making it particularly useful for macular PKDL cases, which are often missed by the rk39 strip test. Thus, DAT provides a simple, reliable, and inexpensive test for PKDL diagnosis with potential applicability in field conditions.     

Development of a species-specific PCR assay for detection of Leishmania donovani in clinical samples from patients with kala-azar and post-kala-azar dermal leishmaniasis

We have developed a PCR assay that is capable of amplifying kinetoplast DNA (kDNA) of Leishmania donovani in a species-specific manner among Old World leishmanias. With Indian strains and isolates of L. donovani the assay was sensitive enough to detect kDNA in an amount equivalent to a single parasite or less. The extreme sensitivity of the assay was reflected in its ability to detect parasite DNA from small volumes of peripheral blood of patients with kala-azar (KA) and from skin lesions from patients with post-KA dermal leishmaniasis (PKDL). A total of 107 clinical leishmaniasis samples were analyzed. Of these 102 (95.3%) were positive by PCR. The test provided a diagnosis of KA with 96% sensitivity using patient whole-blood samples instead of bone marrow or spleen aspirates that are obtained by invasive procedures. The assay was also successful in the diagnosis of 45 of 48 PKDL cases (93.8%). Cross-reactions with pathogens prevalent in the area of endemicity, viz., Mycobacterium tuberculosis, Mycobacterium leprae, and Plasmodium spp., could be ruled out. Eighty-one control samples, including dermal scrapings from healthy portions of skin from patients with PKDL were all negative. Two of twenty controls from the area of endemicity were found positive by PCR assay; however, there was a good possibility that these two were asymptomatic carriers since they were serologically positive for KA. Thus, this PCR assay represents a tool for the diagnosis of KA and PKDL in Indian patients in a noninvasive manner, with simultaneous species identification of parasites in clinical samples.     

Molecular Expression of a Cysteine Proteinase of Clonorchis sinensis and Its Application to an Enzyme-Linked Immunosorbent Assay for Immunodiagnosis of Clonorchiasis

We produced a recombinant cysteine proteinase of Clonorchis sinensis and tested its value as an antigen for serologic diagnosis of C. sinensis infections. The predicted amino acid sequence of the cysteine proteinase of C. sinensis was 58, 48, and 40% identical to those of cathepsin L cysteine proteinases from Paragonimus westermani, Schistosoma japonicum, and Fasciola hepatica, respectively. Western blotting analysis showed that sera from patients infected with C. sinensis strongly reacted with the recombinant protein and that sera from patients infected with S. japonicum weakly reacted with the recombinant protein. Antibody against the recombinant protein stained proteins migrating at about 37 and 28 kDa in C. sinensis adult worm crude extracts. Immunostaining revealed that the cysteine proteinase of C. sinensis was located in the intestinal epithelial cells of the adult parasite and in intrauterine eggs. The specificity and sensitivity of the recombinant antigen or C. sinensis adult worm crude extracts were assessed by an enzyme-linked immunosorbent assay (ELISA) using serum samples from humans infected with different parasites, including 50 patients with clonorchiasis, and negative controls. The sensitivities of the ELISA with the recombinant antigen and C. sinensis adult worm crude extracts were 96 and 88%, respectively. The specificities of the ELISA with the recombinant antigen and C. sinensis adult worm crude extracts were 96.2 and 100%, respectively. The results suggested that the recombinant cysteine proteinase-based ELISA could provide a highly sensitive and specific assay for diagnosis of clonorchiasis.     

rK39 Enzyme-Linked Immunosorbent Assay for Diagnosis of Leishmania donovani Infection

The rK39 enzyme-linked immunosorbent assay (ELISA) was compared with the direct agglutination test (DAT) for Leishmania donovani infection in the Sudan. rK39 ELISA proved more sensitive than DAT in diagnosis of kala-azar (93 and 80%, respectively); both tests may remain positive up to 24 months after treatment. For patients with post-kala-azar dermal leishmaniasis and individuals with subclinical infection, rK39 ELISA performed as well as DAT but could detect infection 6 months earlier in ~40% of patients. Conversion in DAT and rK39 ELISA also occurred in leishmanin skin test (LST)-positive individuals, suggesting active parasite replication (rK39 is an amastigote antigen) in these presumably immune individuals. In contrast to DAT, rK39 ELISA also detected infection in randomly selected LST-positive individuals (in four of six) and endemicity (LST-negative) controls (in one of five). rK39 ELISA appears more sensitive than DAT and may prove an important tool in epidemiological studies.     

Comparison of Two Recombinant Major Outer Membrane Proteins of the Human Granulocytic Ehrlichiosis Agent for Use in an Enzyme-Linked Immunosorbent Assay

Enzyme-linked immunosorbent assay (ELISA) for human granulocytic ehrlichiosis (HGE) using two different recombinant P44 proteins (rP44 and rP44-2hv) of the HGE agent as antigens was evaluated. Sera from a total of 72 healthy humans both from regions where HGE is nonendemic and regions where HGE is endemic were used as negative controls to determine the cutoff value for ELISA. Sera from a total of 14 patients (nine from whom the HGE agent was isolated and five who were HGE-PCR positive) were used as positive controls. One hundred nine sera from 72 patients in an area where HGE is endemic who were suspected of having HGE were examined by ELISA and indirect immunofluorescence assay (IFA). All IFA-negative sera were negative by both ELISAs. Of 39 sera that were IFA positive, 35 and 27 were positive by ELISA using rP44 and rP44-2hv, respectively, indicating that the use of rP44 is more sensitive. Western blot analysis of the four rP44-ELISA-negative IFA-positive sera using whole HGE agent as antigen suggests that these four sera were false IFA positive. There was no difference in results with or without the preabsorption of sera with Escherichia coli or with or without the cleavage of the fused protein derived from the vector. There was a significant positive correlation between IFA titers and optical densities of ELISAs. Four Ehrlichia chaffeensis-positive and 10 Borrelia burgdorferi-positive sera were negative by ELISA. However, two Babesia microti-positive sera showed strong cross-reactivity to the fused vector protein, which was eliminated after cleavage of the protein. Thus, ELISA using rP44 nonfusion protein would provide a simple, specific, and objective HGE serologic test which can be easily automated.     

Nested PCR assay for detection of Leishmania donovani in slit aspirates from post-kala-azar dermal Leishmaniasis Lesions

A nested PCR assay to detect parasite DNA in slit aspirates from skin lesions of patients with post-kala-azar dermal lesihmaniasis (PKDL) is described. PCR results were positive in 27 of 29 (93%) samples by nested PCR assay, while only 20 of 29 (69%) were positive in a primary PCR assay. The nested PCR assay allowed reliable diagnosis of PKDL in a noninvasive manner.     

Use of a recombinantDictyocaulus viviparus antigen in an enzyme-linked immunosorbent assay for immunodiagnosis of bovine dictyocaulosis

A specific recombinant antigen was evaluated for its immunodiagnostic potential in an enzyme-linked immunosorbent assay (ELISA). The antigen was used as a glutathione S-transferase (GST) fusion protein (DvGST3-14) or as a pure recombinant parasite protein (Dv3-14). A total of 55 sera from cattle experimentally infected withDictyocaulus viviparus, 24 sera from naturally infected cattle and 25 sera from helminth-free cattle were examined. ELISA results obtained for the sera from experimental infections showed a calculated specificity of >99% for both antigen preparations had a sensitivity of 93% (DvGST3-14) and 91% (Dv3-14). For field sera from natural infections, the specificity was calculated to be 90% (DvGST3-14) and >99% (Dv3-14) and the sensitivity, 85% (DvGST3-14) and 70% (Dv3-14).Dedicated to Prof. Dr. K.T. Friedhoff on the occasion of his 60th birthdayThis work was supported by grant Az Schn 267/4-1 from the Deutsche Forschungsgemeinschaft     

Evaluation of rK28 antigen for serodiagnosis of visceral Leishmaniasis in India

Antibody detection is a safely applied method at the wide scale in diagnosis of visceral Leishmaniasis (VL). In order to further advance serodiagnosis, the rK28 antigen has been recently introduced as a candidate for diagnosis of VL. We evaluated the sensitivity and specificity of the rK28 antigen in a micro-ELISA format in comparison to the rk39 antigen. The test was conducted on 252 parasitologically confirmed VL cases, 103 endemic healthy controls, 95 non-endemic healthy controls, 88 other infectious disease and 53 follow-up cases. Of 252 parasitologically confirmed VL cases, 251 cases were reported positive by rK28 antigen, yielding 99.6% sensitivity (95% CI, 0.97–0.99), which was similar to the sensitivity of rK39 ELISA (99.6%) (95% CI, 0.97–0.99). Specificity of the rK28 antigen in non-endemic and endemic healthy controls was 100% (95% CI 0.96–1) and 94.17% (95% CI, 0.88–0.97), respectively. In 88 different diseases, specificity was 95.45% (95% CI, 0.84–0.96). With the rK39 antigen, specificity of non-endemic and endemic controls and different diseases was 100% (95% CI 0.96–1), 92.23% (95% CI 0.85–0.96) and 96.59% (95% CI 0.90–0.98), respectively. Our results show that rK39 and rK28 antigens have similar sensitivity and specificity and rK28 can also be used as a serodiagnostic tool in the endemic population of Bihar.     

Use of PCR for Diagnosis of Post-Kala-Azar Dermal Leishmaniasis

Microscopy and PCR were compared for use in the diagnosis of post-kala-azar dermal leishmaniasis (PKDL) in 63 patients. Aspirates of lymph nodes (samples from 52 patients), skin (23 samples), and bone marrow (18 samples) were used. For 11 patients lymph node aspiration could be repeated 6 months after they recovered from PKDL. During active PKDL, PCR was positive for 42 of 52 (80.8%) lymph node aspirates and 19 of 23 (82.7%) skin aspirates, whereas microscopy was positive for only 9 of 52 (17.3%) lymph node aspirates and 7 of 23 (30.4%) skin aspirates. PCR was always positive when parasites were seen by microscopy. When the results obtained with lymph node and skin aspirates from the same patient (n = 16) were compared, there was complete agreement. Bone marrow samples were negative by microscopy and PCR for 16 patients and positive by both methods for 1 patient; for one sample only the PCR was positive. PCR confirmed the co-occurrence of visceral leishmaniasis and PKDL in one patient and confirmed the suspicion of this co-occurrence in the other patient. After recovery, no parasites were found by microscopy, but 2 of 11 (18.2%) samples were still positive by PCR. Thirty negative controls were all found to be PCR negative, and 15 positive controls were all PCR positive. Cross-reactions with Mycobacterium leprae could be ruled out. In conclusion, PCR with inguinal lymph node or skin aspirates is suitable for confirming the clinical diagnosis of PKDL. In some patients, lymph node aspirates are probably preferred because aspiration of material from the skin may leave scars.     

Molecular expression of a cysteine proteinase of Clonorchis sinensis and its application to an enzyme-linked immunosorbent assay for immunodiagnosis of clonorchiasis

We produced a recombinant cysteine proteinase of Clonorchis sinensis and tested its value as an antigen for serologic diagnosis of C. sinensis infections. The predicted amino acid sequence of the cysteine proteinase of C. sinensis was 58, 48, and 40% identical to those of cathepsin L cysteine proteinases from Paragonimus westermani, Schistosoma japonicum, and Fasciola hepatica, respectively. Western blotting analysis showed that sera from patients infected with C. sinensis strongly reacted with the recombinant protein and that sera from patients infected with S. japonicum weakly reacted with the recombinant protein. Antibody against the recombinant protein stained proteins migrating at about 37 and 28 kDa in C. sinensis adult worm crude extracts. Immunostaining revealed that the cysteine proteinase of C. sinensis was located in the intestinal epithelial cells of the adult parasite and in intrauterine eggs. The specificity and sensitivity of the recombinant antigen or C. sinensis adult worm crude extracts were assessed by an enzyme-linked immunosorbent assay (ELISA) using serum samples from humans infected with different parasites, including 50 patients with clonorchiasis, and negative controls. The sensitivities of the ELISA with the recombinant antigen and C. sinensis adult worm crude extracts were 96 and 88%, respectively. The specificities of the ELISA with the recombinant antigen and C. sinensis adult worm crude extracts were 96.2 and 100%, respectively. The results suggested that the recombinant cysteine proteinase-based ELISA could provide a highly sensitive and specific assay for diagnosis of clonorchiasis.     

Use of recombinant calreticulin and cercarial transformation fluid (CTF) in the serodiagnosis of Schistosoma mansoni

Schistosomiasis is traditionally diagnosed by microscopic detection of ova in stool samples, but this method is labour intensive and its sensitivity is limited by low and variable egg secretion in many patients. An alternative is an ELISA using Schistosoma mansoni soluble egg antigen (SEA) to detect anti-schistosome antibody in patient samples. SEA is a good diagnostic marker in non-endemic regions but is of limited value in endemic regions, mainly because of its high cost and limited specificity. Here we assess seven novel antigens for the detection of S. mansoni antibody in an endemic region (the Northern Nile Delta). Using recombinant S. mansoni calreticulin (CRT) and fragments thereof, anti-CRT antibodies were detected in the majority of 97 patients sera. The diagnostic value of some of these antigens was, however, limited by the presence of cross-reacting antibody in the healthy controls, even those recruited in non-endemic areas. Cercarial transformation fluid (CTF), a supernatant that contains soluble material released by the cercariae upon transformation to the schistosomula, is cheaper and easier to produce than SEA. An ELISA using CTF as the detection antigen had a sensitivity of 89.7% and an estimated specificity of 100% when used in non-endemic regions, matching the performance of the established SEA ELISA. CTF was substantially more specific than SEA for diagnosis in the endemic region, and less susceptible than SEA to cross-reacting antibody in the sera of controls with other protozoan and metazoan infections.     

Diagnostic and Prognostic Potential of a Competitive Enzyme-Linked Immunosorbent Assay for Leishmaniasis in India

A Leishmania donovani species-specific monoclonal antibody (monoclonal antibody D2) was evaluated for its diagnostic and prognostic potential by a competitive enzyme-linked immunosorbent assay (C-ELISA) in sera from Indian patients with visceral leishmaniasis (VL) and seven patients with post-kala-azar dermal leishmaniasis (PKDL). These results were compared with those obtained by microscopy with Giemsa-stained tissue smears and a direct enzyme-linked immunosorbent assay (direct ELISA) with crude parasite antigen. Of 121 patients with clinically diagnosed VL examined, 103 (85.1%) were positive and 11 (9.1%) were negative by all three methods. An additional 7 (5.8%) who were negative by microscopy were positive by both C-ELISA and direct ELISA. Seven PKDL patients were also examined and were found to be positive by all three methods. Analysis of the chemotherapeutic response to sodium antimony gluconate of these 110 serologically positive VL patients showed that 57 (51.8%) were drug responsive and 53 (48.2%) were drug resistant. The C-ELISA with sera from 20 longitudinally monitored VL patients before and after chemotherapy showed a significant decrease in percent inhibition of monoclonal antibody D2 in drug-responsive patients. However, in drug-unresponsive patients, the percent inhibition of D2 was unchanged or was slightly increased. Our results therefore indicate (i) the applicability of L. donovani species-specific monoclonal antibody D2 for sensitive and specific serodiagnosis by C-ELISA, (ii) that the C-ELISA is more sensitive than microscopy, especially for early diagnosis, (iii) that L. donovani is still the main causative agent of VL, irrespective of the chemotherapeutic response, and (iv) that the C-ELISA can be used to evaluate the success of drug treatment.     

Antibodies against whole sonicated Borrelia burgdorferi spirochetes, 41-kilodalton flagellin, and P39 protein in patients with PCR- or culture-proven late Lyme borreliosis.

The sensitivities and specificities of three enzyme-linked immunosorbent assays (ELISAs) for Borrelia burgdorferi antibodies were compared for 41 patients presenting with symptoms compatible with late Lyme borreliosis (LB) and 37 healthy controls. All subjects were living in southwestern Finland, where LB is endemic. Only patients with culture- or PCR-proven disease were enrolled in the study. The antigens of the ELISAs consisted of sonicated spirochetes, 41-kDa flagellin, and recombinant P39 protein of B. burgdorferi. Fifteen patients had strongly or moderately positive results in the serological assay(s), 19 patients had only weakly positive or borderline antibody levels, and the remaining 7 patients were seronegative by ELISA. The sensitivities of the ELISAs were 78.0% with sonicate antigen, 41.5% with 41-kDa flagellin, and 14.6% with P39 protein. The specificities of the tests were 89.2, 86.5, and 94.6%, respectively. The sonicate antigen ELISA seems to be an effective screening method. These results show that antibodies to B. burgdorferi may be present in low levels or even absent in patients with culture- or PCR-proven late LB. Therefore, in addition to serological testing, the use of PCR and cultivation is recommended in the diagnosis of LB.     

Kinetics and diagnostic and prognostic potential of quantitative Western blot analysis and antigen-specific enzyme-linked immunosorbent assay in experimental canine leishmaniasis

Quantitative computerized Western blot analysis of antibody responses during experimental canine Leishmania infantum infection distinguished between immunodominant and nonimmunodominant protein bands. Six infected beagles, positive by both PCR and parasite culture, were monitored over 75 weeks postinfection and during a 12-week allopurinol treatment course. All dogs were symptomatic at the time of treatment. Of 12 antigenic bands examined, the immunodominant bands (12, 14, 24, 29, 48, and 68 kDa) showed significantly increased intensities (P<0.01) and higher frequencies of recognition than the nonimmunodominant bands at all time points. Detection of the former bands at 6 weeks postinfection preceded seroconversion by enzyme-linked immunosorbent assay (ELISA) both on crude Leishmania antigen or the recombinant proteins rK39 and HSP70. Reactivity with the 14-, 48-, and 68-kDa bands signified early infection, whereas increased reactivity with the 14-, 24-, and 29-kDa bands was associated with posttreatment parasite persistence and potential unfavorable prognosis. Total lane intensity (TLI) emerged as a sensitive marker for early infection and increased as early as 4 weeks postinfection. TLI had a significantly higher (P<0.01) relative increase rate than crude Leishmania antigen or HSP70 or rK39 ELISA at all time points. These immunodominant antigens and TLI, as determined by quantitative Western blotting, will be valuable for early detection and treatment evaluation of canine leishmaniasis.     

Immunopathology of post kala-azar dermal leishmaniasis (PKDL): T-cell phenotypes and cytokine profile

In Sudan, post kala-azar dermal leishmaniasis (PKDL) caused by Leishmania donovani develops in half of the patients treated for visceral leishmaniasis (kala-azar). In most patients lesions heal spontaneously, but in others symptoms are severe and persist for years. This study examined the immunological response in lesions of PKDL patients by immunohistochemistry and compared the findings with results obtained using peripheral blood mononuclear cells (PBMCs). In all lesions, parasites or parasite antigen were present and provoked the formation of an inflammatory infiltrate consisting of a mixture of macrophages, lymphocytes, and plasma cells. In patients who had high interferon-gamma (IFNgamma) responses to Leishmania antigen in vitro, compact epithelioid granulomas were formed. The inflammatory cells were mainly CD3(+) and interleukin-10 (IL10) was the most prominent cytokine in the lesions. However, IFNgamma was found in all and IL4 in most lesions, in varying amounts. PBMCs from all patients responded to Leishmania antigen by IFNgamma production or proliferation. The results indicate that PKDL develops as a result of an influx of immunocompetent cells into skin, which harbours parasites. The inflammatory response to the parasites is complex. It involves several cell types and cytokines, of which some are antagonistic. It is conceivable that the balance between these cytokines determines the outcome of the disease.