Cutaneous leishmaniasis responds to daylight-activated photodynamic therapy: proof of concept for a novel self-administered therapeutic modality.

BACKGROUND: Cutaneous leishmaniasis (CL) is endemic in Israel, with hundreds of new cases reported in recent years. Photodynamic therapy (PDT) is highly effective for treatment of CL, but requires equipment available only at specialized centres. Daylight-activated PDT (DA-PDT) abolishes the need for artificial light sources and allows the patient to administer the treatment with no professional assistance. OBJECTIVES: The objective of this single-centre, open study was to establish proof of concept for the efficacy of DA-PDT in the treatment of CL using clinical, microbiological and molecular clearance as outcome measures. METHODS: Thirty-one patients with CL (11 Leishmania major and 20 Leishmania tropica) underwent DA-PDT. Fourteen patients were treated in the hospital garden under professional supervision and 17 patients underwent DA-PDT as a self-administered treatment modality at home. Following application of a thick layer of 16% methyl aminolaevulinate and 30-min occlusion, the lesions were exposed to daylight for 2·5 h. Treatment sessions were repeated at weekly intervals until clinical and microbiological cure. Control lesions were either treated with cryotherapy or left untreated. RESULTS: The overall cure rate for DA-PDT was 89% (intention-to-treat cure rate 77%); this was 86% for the hospital-based treatment group and 92% for the self-administered group. CONCLUSIONS: DA-PDT proved to be effective in the treatment of CL caused by L. major and L. tropica. More patients were treated according to a self-administered protocol, suggesting that DA-PDT can be adopted even in technologically deprived countries where the majority of Leishmania infections are encountered.

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.

Cutaneous involvement in a rare case of adult visceral leishmaniasis acquired in Israel.

Skin lesions are rare in visceral leishmaniasis, especially in Mediterranean countries. We describe an unusual case of visceral leishmaniasis in a 41-year-old man that began with a skin lesion. The parasites isolated from both the skin lesion and the bone marrow were typed as Leishmania donovani sensu stricto. This pathogen is not endemic in Israel or neighboring countries; its contribution to adult visceral leishmaniasis in Israel is summarized.

Early response gene expression during differentiation of cultured Leishmania donovani.

The promastigote form of the unicellular parasite, Leishmania donovani, must differentiate into the amastigote form to establish an infection in a mammalian host. Identification of genes whose expression changes during differentiation could help reveal mechanisms of Leishmania gene regulation and identify targets for controlling the diseases caused by this human pathogen. Two genomic clones were isolated, P9 that is more highly expressed in promastigotes than in axenic amastigotes and A14 that is preferentially expressed in axenic amastigotes. Analysis of the DNA sequences revealed open reading frames that would encode 55.5 kDa and 100 kDa proteins, respectively, with no homology to known proteins. The mRNA level for these genes during 24 h time courses of parasite differentiation in culture was compared to two genes known to be differentially expressed, c-lpk2 and mkk. Changes in RNA level occurred within 2 h for each gene and continued in advance of morphological changes. The expression levels of these four genes in axenic amastigotes correlated with results from animal-derived parasites.

Identification and cloning of Lmairk, a member of the Aurora/Ipl1p protein kinase family, from the human protozoan parasite Leishmania.

Lmairk, a gene encoding a member of the Aurora/Ipl1p family of protein kinases (AIRK), was cloned from the protozoan parasite Leishmania major. Aurora kinases are key enzymes involved in the regulation of normal chromosome segregation during mitosis and cytokenesis of eukaryotic cells. This single-copy gene located on L. major chromosome 28 encodes a 301 amino acid polypeptide. All 11 conserved eukaryotic protein kinase catalytic subdomains are present and the proposed AIRK signature sequence was identified in the activation loop between subdomains VII and VIII. Lmairk is expressed, as an approximately 2.4 kb message, in at least three different species of Leishmania. This report represents the first identification of an AIRK from the trypanosomatid family of early divergent eukaryotes.

Hemophagocytic lymphohistiocytosis associated with visceral leishmaniasis.

A 2-year-old child presented with fever and hepatosplenomegaly. Laboratory findings showed pancytopenia, hypertriglyceridemia, hyperferritinemia, and high levels of soluble-IL2 receptors. Initial bone marrow aspiration and biopsy revealed mild hemophagocytosis. A diagnosis of hemophagocytic lymphohistiocytosis was made and appropriate treatment was begun. Repeated marrow aspiration performed because of lack of clinical response revealed Leishmania amastigotes in macrophages in addition to active hemophagocytosis. Treatment with liposomal amphotericin resulted with rapid recovery. Visceral leishmaniasis should be considered in the differential diagnosis of hemophagocytic syndrome.

Antigenic analysis of gamonts of Hepatozoon canis purified from leukocytes.

Hepatozoon canis is a tick-borne apicomplexan parasite of dogs that infects neutrophils and parenchymal tissues. To study the antigenic characteristics of this parasite, a technique was devised for the purification of gamonts from peripheral blood neutrophils. White blood cells were separated on Ficoll-Hypaque density gradients and the gamonts were released from the host neutrophils by nitrogen cavitation. The blood used for purification originated from dogs with natural or experimental infections of H. canis with a parasitemia of 1.4-33%. The number of parasites collected ranged from 1.5 X 10(6) to 4.2 X 10(7). Portions of purified gamonts were separated and examined under phase and scanning electron microscopy, and the remaining purified parasites were then used as a source of antigens to characterize the humoral immune response by western blot analysis. Serum antibodies from infected dogs recognized more than 15 gamont antigens, and the antigenic patterns observed with sera from naturally and experimentally infected dogs were nearly similar. Four immunodominant protein bands of relative molecular weights of 107, 88, 63, and 28 kDa were recognized by all of the sera examined. The technique applied here for the isolation of host cell-free gamonts will facilitate studies on antigenic composition and immune responses against H. canis and on antigenic relationships between Hepatozoon from different host species and geographic regions.

A survey on canine leishmaniasis in western Turkey by parasite, DNA and antibody detection assays.

Human visceral leishmaniasis (VL) caused by Leishmania infantum is found throughout the Mediterranean Region, including Turkey, where dogs are considered to be the main reservoir host for this parasite. In the district of Manisa, western Turkey, 37 human VL cases were reported from June 1993-August 1997. Twenty-four villages in this district were chosen for a survey of disease prevalence in dogs. The dogs, 490 in total, were examined using either the indirect immunofluoresence assay (IFAT) or direct agglutination test (DAT). Anti-Leishmania antibodies were found by at least one test in 5.3% (26/490) of the dogs. Infections were confirmed by parasitological examination of or polymerase chain reaction (PCR) on lymph node aspirates in 65% (13/20) and 76.4% (13/17) of the seropositive dogs tested, respectively. The confirmation rate was 85% by combining the results of PCR and microscopy. Our results demonstrate that canine VL is wide spread in western Turkey where human VL is endemic, and that serodiagnosis is a valuable tool for monitoring the infection.

Analysis of immune responses in dogs with canine visceral leishmaniasis before, and after, drug treatment.

The incidence of canine visceral leishmaniasis (CVL) is increasing in the Mediterranean region. Many drugs have been tested for treatment of CVL, but little is known regarding their effect on test immune responses. In our study, three dogs naturally infected with Leishmania infantum and five dogs experimentally infected with the same strain, were treated with dimethasulfonate pentamidine (Lomidine) and the immune response evaluated before, and after, treatment. After the last injection, animals began to gain weight and the major clinical signs disappeared. Antibody titers gradually decreased to low levels, six months after treatment. At the same time, antigen specific lymphoproliferation reappeared in the sampled animals. This study shows that, after treatment, immune cellular responses to leishmanial antigens, involved in protection against Leishmania infection, were established.

Diagnostic and prognostic potential of antibodies against O-acetylated sialic acids in canine visceral leishmaniasis.

Employing bovine submaxillary mucin (BSM) as the coating agent, an enzyme-linked immunosorbent assay (BSM-ELISA) was developed to detect antibodies directed against O-acetylated sialic acids (O-AcSA) in canine visceral leishmaniasis (CVL). Serum samples were collected from 50 dogs previously screened by a parasite-ELISA to detect anti-leishmanial antibodies and designated as seropositive (n = 30) and seronegative (n = 20). The BSM-ELISA detected anti-O-AcSA antibodies in 29 out of 30 seropositive dogs and was negative in 15 out of 20 seronegative dogs; the sensitivity and specificity of the assay being 96.6% and 75%, respectively. Seven dogs from an endemic area in central Israel were longitudinally monitored for 15 months clinically, serologically and cultured for parasite. The levels of antibodies directed against O-AcSA increased with the appearance of clinical symptoms and/or seropositivity, disappeared when the disease was self-limiting as also with chemotherapeutic response and reappeared with relapse. The BSM-ELISA, therefore, represents a valuable tool for assessment of disease progression.

Immune responses in vaccinated dogs with autoclaved Leishmania major promastigotes.

A comparative study was undertaken on the immunogen power of autoclaved Leishmania major promastigotes (ALM) vaccines given simultaneously with either BCG or saponin against canine leishmaniasis. The humoral immune response was assessed by ELISA and western blotting. The cellular immune response was evaluated by the lymphocyte transformation test. Dogs vaccinated simultaneously with ALM and saponin showed high antibody titres to crude L. infantum antigens after the first vaccine booster and reacted with several antigens, with molecular weights from 26 to 108 kDa by western blotting. However, the lymphocyte proliferation of these dogs to the crude L. infantum antigen was not significantly different from the control group. In contrast, in dogs vaccinated simultaneously with ALM and BCG, the antibody titres to crude antigen were low. Their sera reacted with the same proteins recognised by sera from dogs vaccinated simultaneously with ALM and saponin by western blotting. However, the 85-kDa protein was only identified by sera taken from dogs vaccinated simultaneously with ALM and BCG. These latter exhibited specific lymphocyte proliferation to the L. infantum antigen. This cell proliferation was observed for approximately 9 months after the first dose of the vaccine. This study indicates that a combination of ALM as the vaccine and BCG as the adjuvant, in the dog model, was successful in inducing a cell immune response, which is implicated in protection of dogs against a Leishmania infection.

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.

Leishmania: identification of Old World species using a permissively primed intergenic polymorphic-polymerase chain reaction.

We have developed a permissively primed intergenic polymorphic-polymerase chain reaction (PPIP-PCR) which distinguishes between the Old World Leishmania complexes L. major, L. tropica, L. donovani, and L. aethiopica. This technique pairs one parasite-specific and one nonspecific oligonucleotide primer for the PCR. The specific primer was chosen from a unique leishmanial DNA sequence, clone pDOG 2, isolated from a L. donovani chagasi genomic DNA expression library. This sequence has a high DNA homology to the intergenic region of the L. major B/C genes which belong to the polymorphic LmcDNA16 gene family. The specific intergenic primer contains a high GC content, a stem-loop, and a 3'-CG residue. The nonspecific primer was selected from within the pBluescript (SK) plasmid. Using PPIP-PCR, parasites belonging to the L. major, L. tropica, L. donovani, and L. aethiopica complexes could be easily identified directly following agarose gel electrophoresis by the simple profiles of their PCR products. In addition, it was possible to discriminate between strains of L. major or L. donovani from distant geographical regions. Amplification of genomic DNA isolated from several nonleishmanial kinetoplastids yielded either no PCR products or unique bands which were distinct from the leishmanial profiles. Genomic DNA from nonkinetoplastid parasites, plants, or mammals was not amplified by PPIP-PCR. This technique is a rapid and reproducible method for the characterization of Old World Leishmania.

Emergence of visceral leishmaniasis in central Israel.

In 1994-1995, a child and five dogs from villages located between Jerusalem and Tel-Aviv, Israel were diagnosed with visceral leishmaniasis (VL). Based on these findings, the distribution of VL in domestic and wild canids in central Israel was examined. In the two villages where canine index cases were identified, a substantial proportion (11.5%, 14 of 122) of the dogs examined were seropositive. However, the rate of infection in five neighboring villages was only 1% (1 of 99). Parasites were cultured from 92% (12 of 13) of the seropositive dogs biopsied and the strains were characterized as Leishmania infantum by a clamped polymorphic-polymerase chain reaction, monoclonal antibodies, and/or excreted factor serology. The discovery of VL close to major urban centers is an important public health issue. The disease appears to have emerged recently in this area, and it is unclear whether the parasite was re-introduced or was continuously present at low levels in this region. The presence of seropositive wild canids, jackals (7.6%, 4 of 53) and red foxes (5%, 1 of 20), in central Israel, and the reappearance of the jackal population after near extinction suggests that wild canids may play a role in spreading this disease.

Species-specific detection of Leishmania in sandflies using an enzyme-linked immunosorbent assay.

A direct enzyme-linked immunosorbent assay (ELISA) using species-specific monoclonal antibodies was developed for the detection and identification of Leishmania in sandflies. A titration of mock-infected Phlebotomus papatasi showed that fewer than 2000 L. major promastigotes could be detected. The percentage of infected P. papatasi collected in the field, as determined by dissection, was compared to that revealed by the ELISA. Both methods gave similar results, irrespective of whether the flies were caught by sticky papers or light-traps. The percentage of infected flies determined by either method was also similar in experimentally infected colony reared sandflies. The ELISA can be carried out using multiple species-specific antibodies, and is as accurate as identification of infected sandflies by microscopical examination. The technique should be useful for identifying sandfly species involved in transmitting different species of Leishmania, and for rapid assessment of leishmanial infection rates in endemic regions.

Effect of protein kinase inhibitors on the growth, morphology, and infectivity of Leishmania promastigotes.

The effect of two protein kinase inhibitors, staurosporine and H-7, on the growth, morphology and infectivity of Leishmania major and Leishmania amazonensis promastigotes was examined. Incubation with H-7 (600 microM) for up to one hour had no effect on parasite growth, morphology or infectivity. Staurosporine, however, was cytotoxic for promastigotes and incubation for 1, 5 or 15 minutes with 10 microM inhibitor killed 19, 34 and 59%, respectively, of the parasites. Longer incubations, up to one hour, at this concentration did not increase parasite killing. However, treatment with 25 microM staurosporine for one hour was highly toxic, only 4% of the promastigotes surviving after 72 h. Lower concentrations of staurosporine, 0.25 and 2.5 microM, had only minor effects on parasite growth. Incubation of either L. major or L. amazonensis with staurosporine (10 microM for 10 minutes) caused marked morphological changes in the size and appearance of the flagellar pocket, and/or cytoplasm of the viable parasites. Treated parasites were still capable of infecting mouse peritoneal macrophages and causing disease in BALB/c mice, though the treated parasites were less virulent than control promastigotes. These results indicate that staurosporine, while inhibiting promastigote growth, does not prevent differentiation to amastigotes and amastigote replication.

Release of ecto-protein kinases by the protozoan parasite Leishmania major.

Leishmania major promastigotes have externally oriented ecto-protein kinases (PK) that are capable of phosphorylating both endogenous membrane substrates and foreign proteins. Live parasites phosphorylate protamine sulfate, casein, and phosvitin but not bovine serum albumin. Addition of exogenous PK substrates, such as phosvitin or casein, induced the shedding of ecto-PK that are capable of phosphorylating protamine sulfate. No phosphorylation of protamine sulfate was seen when cell-free supernatants from promastigotes incubated with either buffer alone or bovine serum albumin were used. A second enzyme, a constitutively released PK that phosphorylates casein or phosvitin and not protamine sulfate or mixed histones, was identified and characterized. This PK is inhibited by 5 microM staurosporine, 50 microg/ml heparin, and 75 microM CKI-7, concentrations typical of the IC50 found for other eukaryotic casein kinases (CK). The constitutively shed ecto-PK specifically phosphorylated a peptide substrate for CK1 but not for CK2, suggesting that this shed PK is similar to CK1.

Cloning from Leishmania major of a developmentally regulated gene, c-lpk2, for the catalytic subunit of the cAMP-dependent protein kinase.

Protein kinases are important in the regulation of cellular processes including growth and differentiation. Using the polymerase chain reaction with oligonucleotide primers derived from conserved regions of cAMP-dependent protein kinases (PKAs), three different DNA fragments were amplified from leishmanial genomic DNA. One fragment was used to isolate a stage specific gene, c-lpk2, from a Leishmania major genomic library. This gene shows high homology to other eukaryotic PKAs, and the open reading frame encodes a 332 amino acid protein with a predicted molecular mass of 38.2 kDa. When aligned with other PKAs the leishmanial enzyme has a unique eight amino acid extension at the carboxy terminus. The c-lpk2 gene is present as a single copy in L. major, L. donovani and L. amazonensis. The 5'-flanking region contains a polypyrimidine rich tract upstream from the predicted ATG start codon. The gene is highly expressed in promastigotes and barely detectable in amastigotes of L. major. Temperature increase was shown to rapidly down-regulate c-lpk2 expression. Transfer of L. amazonensis promastigotes to 35 degrees C resulted in the rapid disappearance of c-lpk2 mRNA (> 70% in 1 h), while at 26 degrees C the mRNA was more stable. The strict temperature dependence of mRNA degradation rate suggests that PKA expression is regulated post-transcriptionally.