Evaluation of cellular immunological responses in mono- and polymorphic clinical forms of post-kala-azar dermal leishmaniasis in India.

Post-kala-azar dermal leishmaniasis (PKDL) is a chronic dermal complication that occurs usually after recovery from visceral leishmaniasis (VL). The disease manifests into macular, papular and/or nodular clinical types with mono- or polymorphic presentations. Here, we investigated differences in immunological response between these two distinct clinical forms in Indian PKDL patients. Peripheral blood mononuclear cells of PKDL and naive individuals were exposed in vitro to total soluble Leishmania antigen (TSLA). The proliferation index was evaluated using an enzyme-linked immunosorbent assay (ELISA)-based lymphoproliferative assay. Cytokines and granzyme B levels were determined by cytometric bead array. Parasite load in tissue biopsy samples of PKDL was quantified by quantitative polymerase chain reaction (qPCR). The proportion of different lymphoid subsets in peripheral blood and the activated T cell population were estimated using flow cytometry. The study demonstrated heightened cellular immune responses in the polymorphic PKDL group compared to the naive group. The polymorphic group showed significantly higher lymphoproliferation, increased cytokines and granzyme B levels upon TSLA stimulation, and a raised proportion of circulating natural killer (NK) T cells against naive controls. Furthermore, the polymorphic group showed a significantly elevated proportion of activated CD4(+) and CD8(+) T cells upon in-vitro TSLA stimulation. Thus, the polymorphic variants showed pronounced cellular immunity while the monomorphic form demonstrated a comparatively lower cellular response. Additionally, the elevated level of both activated CD4(+) and CD8(+) T cells, coupled with high granzyme B secretion upon in-vitro TSLA stimulation, indicated the role of cytotoxic cells in resistance to L. donovani infection in polymorphic PKDL.

Long-term efficacy of single-dose radiofrequency-induced heat therapy vs. intralesional antimonials for cutaneous leishmaniasis in India.

BACKGROUND: Radiofrequency-induced heat therapy (RFHT) has been found to be safe and effective against cutaneous leishmaniasis (CL) in the short term, but its long-term efficacy is unclear. OBJECTIVES: To compare the long-term efficacy of RFHT vs. intralesional sodium stibogluconate (SSG) injections in the treatment of CL in India. METHODS: One hundred patients with a confirmed diagnosis of CL were randomly assigned in a 1 : 1 ratio to receive topical RFHT for 30-60 s or seven intralesional injections of SSG (50 mg cm(-2) of lesion). Improvement and recurrence were monitored every 15 days after the initiation of treatment for 4 months and then at 5, 6, 9, 12 and 18 months post-treatment; the rates of complete cure were compared. RESULTS: Lesions were healed in 47 out of 50 patients (94%) in the RFHT group and in 46 out of 50 patients (92%) in the SSG group at week 12. Time to complete healing was comparable in the two groups. At 6 months post-treatment, cure rates in the RFHT and SSG groups were 98% [95% confidence interval (CI) 94-100%] and 94% (95% CI 86-100%), respectively. Age, sex and lesion size or number had no effect on cure rates. No relapse of infection was recorded in cured patients in either group up to 12-18 months after initiation of treatment. Skin biopsies of cured lesions in eight out of eight (100%) patients from the RFHT group and three of three from the SSG group at 12 months showed minimal fibrosis and were negative for Leishmania tropica by polymerase chain reaction test. CONCLUSIONS: A single application of RFHT is safe, cosmetically acceptable and effective in inducing a long-term cure of CL.

Miltefosine as an effective choice in the treatment of post-kala-azar dermal leishmaniasis.

BACKGROUND: Post-kala-azar dermal leishmaniasis (PKDL) constitutes a parasite reservoir important in the transmission of visceral leishmaniasis (VL). Unacceptable treatment regimens and increasing drug resistance blight control programmes. The success of oral miltefosine in VL prompted a clinical, histopathological and parasitological study of this drug in PKDL. OBJECTIVES: To define the dose and duration of miltefosine for treatment of PKDL. METHODS: Twenty-six patients confirmed by slit-skin smear, histopathology and molecular tests were enrolled in the study. They received miltefosine capsules 50 mg thrice daily after food. Treatment was for 60 days with a provision to increase by 30 days if a responder had not attained a cure. Cure was ascertained by clinical and histopathological examination, and measuring parasite burden using real-time polymerase chain reaction. RESULTS: Twenty-four patients with a wide range of parasite burden completed the study. Twenty-three achieved a cure giving an initial cure rate of 96% (95% confidence interval 79-99%). Sixteen patients were cured with 50 mg thrice daily, 13 in 60 days and three within 90 days. In seven cases, miltefosine had to be reduced, because of gastrointestinal intolerance, to 50 mg twice daily to a total of 180 capsules. Lesional parasites were undetectable at 1 month post-treatment. Treatment was safe with no relapses at 1-year follow-up. CONCLUSION: Oral miltefosine, 50mg thrice daily for 60 days or twice daily for 90 days, could be an effective treatment for PKDL.

Evidence for involvement of TNFR1 and TIMPs in pathogenesis of post-kala-azar dermal leishmaniasis.

Semi-quantitative RT-PCR was exploited to analyse the intralesional cytokine gene expression in 14 post-kala-azar dermal leishmaniasis (PKDL) and 10 kala-azar (KA) patients. The data provided evidence for both inflammatory and non-inflammatory responses, as reflected by elevated tumour necrosis factor (TNF)-alpha and interleukin (IL)-10 in PKDL lesions compared with normal skin tissue (n = 6). The ratio of TNF-alpha : IL-10 message was 2.66 in PKDL cases, substantially higher than in KA (1.18). Investigation of TNF-alpha receptors (TNFR1 and TNFR2) revealed a significant down-regulation of TNFR1 transcript in both PKDL and KA compared with control. In the presence of elevated levels of TNF-alpha transcript, interference with type 1 effector activity in PKDL may be due to minimal expression of the TNFR1 gene. Investigation of matrix metalloproteinases, known to be induced by TNF-alpha, and the tissue inhibitors of matrix metalloproteinases (TIMPs), provided evidence for the roles of TIMP-1 and TIMP-3 in the pathogenesis of PKDL.

Elevated levels of IgG3 and IgG4 subclass in paediatric cases of kala azar.

Visceral leishmaniasis (VL) or Kala azar (KA) is a systemic disease caused by the parasites of the Leishmania donovani complex. Control measures rely on treatment with antileishmanial agents, however, fraught with problems such as toxicity or drug resistance. The incidence rate is on the rise for children, for reasons yet undefined. Previously we have shown significantly elevated level of IL-10 in children compared to adult KA cases. Here, antileishmanial antibody and C-reactive protein (CRP) levels were investigated in paediatrics and adult patients of KA and post-KA dermal leishmaniasis (PKDL). Level of IgG4 was significantly elevated in PKDL compared to KA, although total IgG and IgG1 were significantly lower. The antileishmania antibody levels of subclass IgG3 and IgG4 were found significantly elevated in paediatrics, however, levels of IgG, IgG1, IgG2 and CRP were comparable in paediatric and adult KA cases. In case of PKDL, levels of IgG and it subclass were similar in the two groups. No significant difference in antileishmanial antibody level was noticed between macular or polymorphic cases of PKDL. The differential antibody intensity in paediatric cases, together with significant level of circulating IL-10, could be considered as a marker of differential disease susceptibility.

Genetic fingerprinting and identification of differentially expressed genes in isolates of Leishmania donovani from Indian patients of post-kala-azar dermal leishmaniasis.

Post-kala-azar dermal leishmaniasis (PKDL) is an unusual dermatosis that develops as a sequel in 5-15% of cured cases of kala-azar (KA) after months or years of treatment in India. Molecular differences are reported to exist between the KA and PKDL isolates which may underlie the diversity in clinical manifestations of the disease. Here, arbitrary primed-PCR (AP-PCR) has been used for genetic fingerprinting of parasite isolates from dermal lesions of PKDL patients (n=14) and compared with bone-marrow derived parasites from KA patients (n=3). All isolates showed an identical AP-PCR pattern with 4 arbitrary primers. Further, AP-PCR was exploited to identify the stage regulated genes of the parasite. Six polymorphic fragments were identified in PKDL in comparison with KA isolates, and were subjected to Northern blot analysis. Five polymorphic fragments represented transcribed sequences; 4 out of 5 drew differential expression in pro- and amastigote stages, although the expression was comparable between PKDL and KA isolates. The study led to the identification of genes, which exhibit stage-regulated expression in Leishmania donovani derived from PKDL or KA patients, including a putative phosphodiesterase, DEAD box RNA helicase, iron superoxide dismutase b (fesodb) and a hypothetical protein. Demonstration of transcripts of DEAD box RNA helicase in PKDL and KA diseased tissues implicates its role in disease pathogenesis.

Transcriptome analysis during the process of in vitro differentiation of Leishmania donovani using genomic microarrays.

Leishmania donovani causes visceral disease (kala-azar), a major health problem throughout the tropics with 500 000 new cases every year. Leishmania differentiates from the promastigote to the amastigote form to establish infection in a mammalian host. To understand the process of differentiation, we assessed the global variation in gene expression in promastigotes, an intermediate stage of differentiation (PA24) and axenic amastigotes in culture using an L. donovani genomic microarray with 4224 clones printed in triplicate. During an intermediate stage of differentiation 24 h after shifting the promastigotes into amastigotes (PA24), there were 41 (approximately 1%) clones with expression > or = 2.0-fold higher than promastigotes, whereas in terminally differentiated amastigotes there were 130 (approximately 3%) such clones. Of particular interest were certain genes that exhibited a transient increase or decrease in expression at the PA24 stage. Kinases showed a transient increase, and surface molecules, PSA and amino acid permease, were prominent clones among those showing a brief decrease at the PA24 stage. The microarray results have been validated using Northern blots or RT-PCR. In summary, our results provide important clues about the genes involved in the differentiation process of L. donovani that may contribute to virulence.

Evaluation of PCR for diagnosis of Indian kala-azar and assessment of cure.

This study was done to evaluate PCR with Ld1 primers for the diagnosis of Indian visceral leishmaniasis (VL) and to assess its role in prediction of the disease outcome. The PCR assay was performed with DNA isolated from the peripheral blood of parasitologically confirmed cases of VL before the initiation of treatment, just after the end of treatment, and at 3 and 6 months of follow-up. The pretreatment PCR result was positive for 100 of 101 patients (sensitivity, 99%; confidence interval [CI], 94 to 100%). None of the 150 negative controls tested were PCR positive (specificity, 100%; CI, 96.8 to 100%). Of 60 patients who were treated at our center, 51 (85%; CI, 73 to 93%) became negative immediately after treatment and continued to be negative at 3 and 6 months of follow-up. At the 3-month follow-up, two of the remaining nine patients were PCR positive, making 58 (96.7%; CI, 87 to 100%) patients PCR negative. At the 6-month follow-up, all patients became PCR negative. One patient who was PCR negative immediately after the end of treatment relapsed 11 months later. This limited prospective study with VL patients suggests that the PCR assay is a highly sensitive and specific (99% and 100%, respectively) tool for the diagnosis of VL. In the majority of patients, it can identify a successful disease outcome; however, its translation into the field setting remains a major challenge.

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.

Antigenaemia and antibody response to Leishmania donovani stage-specific antigens and rk39 antigen in human immunodeficiency virus-infected patients.

In order to define the possible markers for the early diagnosis of asymptomatic visceral leishmaniasis in human immunodeficiency virus (HIV)-infected individuals, the antigenaemia and antibody response to stage-specific Leishmania donovani and rk39 antigens is assessed by enzyme-linked immunosorbent assay (ELISA) and immunoreactivity to stage-specific antigens analysed by Western blot. Serum samples from two out of 100 HIV-infected individuals were found positive for antigenaemia, antibody response to stage-specific L. donovani antigens and rk39 antigen, and one sample was also positive for antigenaemia and antibody response to L. donovani antigens, while antibody detection to rk39 antigen was not carried on this sample. Additionally, one sample was found positive for amastigote antigenaemia and antibody response to amastigote antigen, while in this patient promastigote antigenaemia and antibody response to promastigote L. donovani and rk39 antigen could not be detected. One sample was found positive for antigenaemia, antibody response to amastigote antigen and negative for antibody response to promastigote antigen, while in this patient response to rk39 antigen was borderline. Although antibody response to rk39 antigen could be detected in 9/88 (10%) HIV-infected individuals, in six of these nine patients neither antigenaemia nor antibody response to stage-specific L. donovani antigens could be detected. All 10 confirmed visceral leishmaniasis and HIV-negative control patients had positive antigenaemia and antibody response to L. donovani amastigote and promastigote antigens, while all the normal healthy individuals were negative. The study indicated that detection of antibody response to rk39 antigen, amastigote antigenaemia and antibody response to amastigote antigen may prove to be better markers than detection of promastigote antigenaemia, antibody response to promastigote antigen and immunoblot reactivity.

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.

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.

A simple and sensitive test for field diagnosis of post kala-azar dermal leishmaniasis.

BACKGROUND: Current methods for diagnosis of post kala-azar dermal leishmaniasis (PKDL) do not offer adequate sensitivity and specificity. OBJECTIVES: To develop a simple and sensitive test for field diagnosis of PKDL. METHODS: Immunochromatographic nitrocellulose strips precoated with recombinant k39 antigen were evaluated for the detection of circulating antibodies to leishmanial k39 in PKDL sera. A drop of serum applied to the strip followed by buffer led to the development of two visible bands indicating the presence of anti-k39 IgG. RESULTS: The strip test was able to detect cases of PKDL with 91% sensitivity. The specificity of the test was evaluated using controls with other skin diseases, other common infections and healthy persons from endemic and non-endemic regions. Of 125 controls examined, all were negative on the strip test, indicating 100% specificity of the test. CONCLUSIONS: The immunochromatographic nitrocellulose strips provide a non-invasive, rapid and accurate method for diagnosing PKDL.

Expression of a mutant form of Leishmania donovani centrin reduces the growth of the parasite.

Leishmania donovani, a protozoan parasite, causes visceral disease in humans. To identify genes that control growth, we have isolated for the first time in the order Kinetoplastida a gene encoding for centrin from L. donovani. Centrin is a calcium-binding cytoskeletal protein essential for centrosome duplication or segregation. Protein sequence similarity and immunoreactivity confirmed that Leishmania centrin is a homolog of human centrin 2. Immunofluorescence analysis localized the protein in the basal body. Calcium binding analysis revealed that its C-terminal Ca(2+) binding domain binds 16-fold more calcium than the N-terminal domain. Electrophoretic mobility shift of centrin treated with EGTA and abrogation of the shift in its mutants lacking a Ca(2+) binding site suggest that Ca(2+) binding to these regions may have a role in the protein conformation. The levels of centrin mRNA and protein were high during the exponential growth of the parasite in culture and declined to a low level in the stationary phase. Expression of N-terminal-deleted centrin in the parasite significantly reduces its growth rate, and it was found that significantly more cells are arrested in the G(2)/M stage than in control cells. These studies indicate that centrin may have a functional role in Leishmania growth.

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.

Heat-stress induced modulation of protein phosphorylation in virulent promastigotes of Leishmania donovani.

In parasites such as Leishmania, the study of molecular events induced in response to heat stress is of immense interest since temperature increase is an integral part of the life cycle. Protein phosphorylation is known to control major steps of proliferation and differentiation in eukaryotic cells. Studies on intracellular signaling systems in protozoa are relatively recent. We have examined the effect of heat shock on the protein phosphorylation status in promastigotes of Leishmania donovani. The patterns of total protein phosphorylation and specific phosphorylation at tyrosine residues were examined using [32P]-orthophosphate labelling of the parasites and immunoblotting with a monoclonal anti-phosphotyrosine antibody. The major proteins of L. donovani that were phosphorylated at 24 degrees C had apparent molecular weights of 110, 105, 66-68, 55, 36-40 and 20 kDa. Heat shock (from 24 to 37 degrees C) led to a significant decrease in phosphorylation of the majority of phosphoproteins in the virulent promastigotes. On the other hand, the avirulent promastigotes did not show any decrease in protein phosphorylation on exposure to heat stress. Predominant phosphorylation at tyrosine residues was detectable in proteins of putative size 105-110 kDa in both virulent and avirulent parasites. Heat shock led to a reduction in the level of phosphotyrosine in both these proteins in the case of virulent parasites, while no such reduction was detectable in avirulent parasites. Significant modifications in the phosphorylation status of proteins in response to heat stress including that of tyrosine containing proteins, observed exclusively in virulent parasites, suggest that modulation of protein phosphorylation/dephosphorylation may play a role in signal transduction pathways in the parasite upon heat shock encountered on entering the mammalian host.

Western blot analysis of humoral immune response to Leishmania donovani antigens in patients with post-kala-azar dermal leishmaniasis.

Sera from 32 Indian patients with post-kala-azar dermal leishmaniasis (PKDL) were examined for antibodies by immunoblot analysis using an antigen extract of Leishmania donovani. The study revealed that the humoral immune response in PKDL patients was quite distinct compared to that in kala-azar patients. Antibodies to 3 antigens of L. donovani (molecular sizes 110, 65 and 38-42 kDa) were predominant in a majority (78%) of PKDL patients. The most important finding was the consistent recognition of 2 parasite antigens (of 110 and 65 kDa) by PKDL sera; antibodies to the 110-kDa antigen were detectable in 97% of cases, while antibodies to the 65-kDa antigen were detectable in 100% of cases that were examined. None of the 18 cases of leprosy, 10 of vitiligo, or the 30 healthy persons included in the study showed antibodies to these 2 antigens. Thus Western blot analysis provided a highly sensitive test for PKDL patients. Further, it led to the identification of 2 parasite antigens (110 and 65 kDa) that elicit an antibody response in 97-100% of PKDL patients. Purified or recombinant versions of these proteins deserve consideration as potential target antigens in development of simpler, highly specific and sensitive serodiagnostic tests for PKDL.