Performance of immunoblotting in diagnosis of visceral Leishmaniasis in human immunodeficiency virus-Leishmania sp.-coinfected patients

This study evaluated the performance of immunoblotting with Leishmania infantum soluble antigens for the diagnosis of visceral leishmaniasis in human immunodeficiency virus (HIV)-infected and immunocompetent patients and assessed the humoral responses of patients coinfected with HIV and Leishmania. In this work, the results of the immunoblot analysis were compared to those of parasitological examination (Giemsa-stained smears and/or parasite isolation in Novy, Nicolle, and MacNeal medium from bone marrow) and indirect immunofluorescence and counterimmunoelectrophoresis techniques. Patients were considered to be infected if one or more of the comparison techniques gave a positive result. Immunoblotting was considered to be positive if at least one band was present. For 198 HIV-positive patients with a clinical suspicion of visceral leishmaniasis, immunoblot analysis had a sensitivity of 70.6%, a specificity of 73.2%, and an accuracy of 72.7%. For a separate group of 40 immunocompetent patients not infected with Leishmania, the immunoblot analysis was negative for all patients (100% specificity), and for a third group of 32 immunocompetent patients with confirmed visceral leishmaniasis, the immunoblot analysis was positive for all patients (100% sensitivity). Sera of coinfected patients recognized few bands and recognized bands at lower intensities compared with sera from immunocompetent patients. The most frequently detected band was 63 to 66 kDa (55.9%), with the difference being statistically significant compared to frequency of detection of the other bands. This study confirms that the humoral response in patients coinfected with HIV and Leishmania is much lower than that in immunocompetent patients and that the immunoblot method is a sensitive, noninvasive, and specific serological technique for the diagnosis of visceral leishmaniasis in immunocompromised patients.     

Molecular, cytological, and immunocytochemical study and kDNA sequencing of laryngeal Leishmania infantum infection

Mucosal leishmaniasis is a well-known clinical manifestation of infections mainly caused by New World Leishmania species, especially Leishmania braziliensis (Viannia) in Central and South America. It is extremely uncommon in the world, even in the endemic areas such as Fars Province, Southern Iran. Two male immunocompetent subjects who developed Leishmania mucosal lesion mimicking a laryngeal tumor presented with a several-months history of dysphonia, dyspnea, hoarseness, and odynophagia. Multiple smears from the lesions showed structures resembling the amastigote form of Leishmania. Nested PCR analysis to amplifying a fragment of Leishmania infantum kinetoplastid DNA from the Giemsa-stained smear resulted in a fragment of 680 bp. Sequence analysis of one of the strains showed 98 % similarity to L. infantum strain IranJWinf (GenBank accession no. AB678348.1) and 96 % similarity to L. infantum isolate MCAN/ES/98/10445 (GenBank accession no. EU437407.1), while another strain showed 97 % similarity with two L. infantum strains from kala-azar patient (GenBank accession nos. AJ223725.1 and AF027577.1). Immunocytochemical staining with anti-L. infantum mAb (D2) was positive. Primary mucosal leishmaniasis (ML) may occur in the immunocompetent patients who reside in or travel to endemic areas of leishmaniasis. Mucosal leishmaniasis contracted in endemic areas, such as Iran, has to be considered in the differential diagnosis of lesions in the other mucosa and may occur in previously healthy persons. Therefore, cytology, PCR, and immunocytochemistry-based methods with anti-Leishmania mAb are helpful in the diagnosis of ML.     

PCR detection of specific Leishmania-DNA in patients with periodontal disease

This study deals with the detection of Leishmania braziliensis DNA in gingival specimens from 10 individuals who all had suffered from cutaneous leishmaniasis 5-10 years prior to the examination and all had been treated with anti-leishmaniasis drugs. This preliminary study gives an interesting contribution to the oral microbiology of this disease, with the observation that inflamed periodontal tissues can serve as a factor affecting the dispersion of Leishmania parasites in individuals who had suffered from cutaneous leishmaniasis. These finding are corroborated by the results of polymerase chain reaction (PCR) which demonstrated the presence of Leishmania DNA in tissue samples of patients with periodontal diseases.     

Antiactin and antitubulin antibodies in canine visceral leishmaniasis

Visceral leishmaniasis, a chronic and often fatal disease, is caused by the protozoan parasite Leishmania donovani. Both specific and nonspecific antibodies are produced in the course of the disease, and autoantibodies may be involved in pathogenesis. Tubulin and actin have been found to be associated with L. donovani. To learn whether antiactin and antitubulin antibodies are present in visceral leishmaniasis, we tested sera from 263 infected dogs by enzyme-linked immunosorbent assay for antibodies to the antigens L. donovani, actin, and tubulin. All samples reacted positively with L. donovani, and a high percentage reacted positively with all three antigens. Sera from 202 uninfected dogs were also tested, none reacted with L. donovani antigen, although positive reactions were observed for 8 of the samples with actin or tubulin. It was found that the antibody-antigen reaction occurred at the Fab portion of the immunoglobulin molecule. Competitive enzyme immunoassays showed that the reaction was inhibited if the positive serum was first incubated with L. donovani antigen, actin, or tubulin and then tested by enzyme-linked immunosorbent assay. These results suggest that antiactin and antitubulin antibodies are present in the sera of dogs infected with visceral leishmaniasis.     

Role of PCR in diagnosis and prognosis of visceral leishmaniasis in patients coinfected with human immunodeficiency virus type 1

A group of 76 consecutive human immunodeficiency virus (HIV)-positive patients with fever of unknown origin (n = 52) or fever associated with pulmonary diseases was evaluated in order to assess the usefulness of PCR with peripheral blood in the diagnosis and follow-up of visceral leishmaniasis. We identified 10 cases of visceral leishmaniasis among the 52 patients with fever of unknown origin. At the time of diagnosis, all were parasitemic by PCR with peripheral blood. During follow-up, a progressive decline in parasitemia was observed under therapy, and all patients became PCR negative after a median of 5 weeks (range, 6 to 21 weeks). However, in eight of nine patients monitored for a median period of 88 weeks (range, 33 to 110 weeks), visceral leishmaniasis relapsed, with positive results by PCR with peripheral blood reappearing 1 to 2 weeks before the clinical onset of disease. Eight Leishmania infantum and two Leishmania donovani infections were identified by PCR-restriction fragment length polymorphism analysis. PCR with peripheral blood is a reliable method for diagnosis of visceral leishmaniasis in HIV-infected patients. During follow-up, it substantially reduces the need for traditional invasive tests to assess parasitological response, while a positive PCR result is predictive of clinical relapse.     

杜氏利什曼原虫酸性核糖体蛋白—1基因的克隆化与序列分析

根据杜氏利什曼原虫（Ｌ．ｄｏｎｏｖａｎｉ）与婴儿利什曼原虫（Ｌ．ｉｎｆａｎｔｕｍ）基因组核苷酸序列之间具有高度的同源性的特点，设计合成了酸性核糖体蛋白－１（ＡＲＰ－１）基因序列特异性的引物，应用多聚酶链反应（ＰＣＲ）技术，以杜氏利什曼原虫的基因组ＤＮＡ为模板，扩增获得了杜氏利什曼原虫的ＡＲＰ－１基因克隆。序列分析表明，杜氏利什曼原虫与婴儿型利什曼原虫ＡＲＰ－１基因的核苷酸序列之间的同源性为９３％，编码的氨基酸残基序列的同源性为９８．０％。     

Isolated leishmanial lymphadenopathy - A rare type of leishmaniasis in India: A Case Report

A patient presented with isolated, soft to firm, inguinal swelling since childhood clinically thought to be a benign lipomatous lesion. Fine‐needle aspiration of the swelling revealed amastigote form of Leishmania donovani in a background of reactive lymphoid hyperplasia. Excision of the swelling resulted in reversal of positive Aldehyde test. Isolated leishmanial lymphadenopathy in an immunocompetent person, is a rare manifestation of leishmaniasis in India. The possible role of transplacental transmission is discussed. Diagn. Cytopathol. 2012. © 2011 Wiley Periodicals, Inc.     

Mediterranean visceral leishmaniasis in immunocompetent children. Report of two cases relapsed after specific therapy

Visceral leishmaniasis (VL) is endemic in areas bordering the Mediterranean Sea (Spain, Italy, France, Greece, Morocco, Tunisia) where it is caused by Leishmania infantum and is transmitted by the bite of a hematophagous sandfly belonging to Phlebotomus spp.; the dog constitutes the main reservoir of infection. Two cases of VL in immunocompetent children are described. Both patients lived in endemic areas for leishmaniasis (Sicily) and at admission were febrile, pale and had splenomegaly. In both patients anti-leishmania antibodies were present and a definitive diagnosis was confirmed by demonstration of leishmania parasites by microscopy or polymerase chain reaction (PCR) in the bone marrow aspirates. The use of PCR performed on peripheral blood has been reported to be highly sensitive for the diagnosis and follow-up of children with VL. One patient was treated with N-dimethylglucamine, Glucantim, the other one with liposomal Amphotericin B (AmBisome). Both had symptomatic relapses 3 months later, and recovered following re-treatment with AmBisome administered intravenously at a dosage of 3 mg/Kg for ten consecutive days. The patients were monitored for one year after treatment was completed.     

Rapid identification of causative species in patients with Old World leishmaniasis.

Conventional methods for the identification of species of Leishmania parasite causing infections have limitations. By using a DNA-based alternative, the present study tries to develop a new tool for this purpose. Thirty-three patients living in Marseilles (in the south of France) were suffering from visceral or cutaneous leishmaniasis. DNA of the parasite in clinical samples (bone marrow, peripheral blood, or skin) from these patients were amplified by PCR and were directly sequenced. The sequences observed were compared to these of 30 strains of the genus causing Old World leishmaniasis collected in Europe, Africa, or Asia. In the analysis of the sequences of the strains, two different sequence patterns for Leishmania infantum, one sequence for Leishmania donovani, one sequence for Leishmania major, two sequences for Leishmania tropica, and one sequence for Leishmania aethiopica were obtained. Four sequences were observed among the strains from the patients: one was similar to the sequence for the L. major strains, two were identical to the sequences for the L. infantum strains, and the last sequence was not observed within the strains but had a high degree of homology with the sequences of the L. infantum and L. donovani strains. The L. infantum strains from all immunocompetent patients had the same sequence. The L. infantum strains from immunodeficient patients suffering from visceral leishmaniasis had three different sequences. This fact might signify that some variants of L. infantum acquire pathogenicity exclusively in immunocompromised patients. To dispense with the sequencing step, a restriction assay with HaeIII was used. Some restriction patterns might support genetic exchanges in members of the genus Leishmania.     

Use of PCR in Diagnosis of Human American Tegumentary Leishmaniasis in Rio de Janeiro, Brazil

In Brazil, the most common etiological agent of American tegumentary leishmaniasis is Leishmania (Viannia) braziliensis. In general, diagnostic techniques envisage the visualization of the parasite, but that technique has a low sensitivity. The main purpose of the present work was to evaluate the PCR as a routine tool for the diagnosis of leishmaniasis. Biopsy specimens from cutaneous or mucosal lesions were taken from 230 individuals from areas where Leishmania is endemic: 216 patients who had a clinical picture suggestive of leishmaniasis and 14 individuals with cutaneous lesions due to other causes. Each specimen was processed for histopathologic examination, culture, touch preparation, and DNA isolation. Oligonucleotides that amplify the conserved region of the minicircle molecules of Leishmania were used in a hot-start PCR. While at least one conventional technique was positive for Leishmania for 62% (134 of 216) of the patients, PCR coupled to hybridization was positive for 94% (203 of 216) of the patients. The 14 patients whose clinical picture was not suggestive of leishmaniasis had negative results by all techniques. The impact of the PCR was striking in mucosal disease. While the disease in only 17% (4 of 24) of the patients could be diagnosed by conventional techniques, PCR was positive for 71% (17 of 24) of the patients. Hybridization showed that all cases of disease were caused by parasites belonging to the Viannia subgenus. Altogether, the results indicate that PCR is a valuable tool for the diagnosis of leishmaniasis on a routine basis and is likely to provide valuable epidemiological information about the disease in countries where it is endemic.     

Detection of Leishmania in Immunocompromised Patients Using Peripheral Blood Spots on Filter Paper and the Polymerase Chain Reaction

 The aim of the present study was to investigate whether the polymerase chain reaction could be used to detect Leishmania infantum in peripheral blood spots of immunocompromised patients. Although visceral leishmaniasis in immunocompromised individuals is routinely diagnosed by direct microscopy or by culture of biopsy material, both methods have disadvantages. In order to evaluate an alternative method of diagnosis, blood spots were collected on filter paper from 24 immunocompromised individuals with visceral leishmaniasis diagnosed by bone marrow microscopy or culture. The samples were tested using the polymerase chain reaction. Leishmania DNA was detected in 15 of 20 patients who had not yet begun treatment for Leishmania infection and in two of four patients undergoing treatment. Using microscopy or culture, parasites were detected in 5 of 19 and 8 of 19 fresh blood samples, respectively. The results suggest that the polymerase chain reaction can be used with blood spots on filter paper as an initial screening method for immunocompromised patients suspected to have Leishmania infection.     

Evaluation of PCR assay in diagnosis and identification of cutaneous leishmaniasis: a comparison with the parasitological methods

The aims of this study are to identify Leishmania species and compare and validate internal transcribed spacers (ITS) polymerase chain reaction (PCR) assay against parasitological methods for diagnosing cutaneous leishmaniasis (CL). We used the ITS-PCR, parasite culture, and microscopic evaluation of stained smears on 155 specimens from suspected cases of (CL) patients who referred to Mashhad Health Centers (northeast Iran). The PCR indicated the sensitivity (98.8%), correctly diagnosing 86 of the 87 confirmed positive specimens. Microscopy and parasite culture alone showed 79.3% sensitivity (69/87 positive) and 86.2% sensitivity (75/87 positive), respectively, while microscopy and culture in combination improved sensitivity totally to 100% (87/87). The results also revealed that Leishmania tropica species is dominant (96.5%) in the studied regions. This study suggests that both the parasitological techniques reliably were used for the diagnosis of CL, and the ITS-PCR assay without using RFLP analysis is useful for identifying Leishmania species in the area.     

The development of post-kala-azar dermal leishmaniasis (PKDL) is associated with acquisition of Leishmania reactivity by peripheral blood mononuclear cells (PBMC)

PKDL develops in about 50% of Sudanese patients treated for visceral leishmaniasis (kala-azar). Patients with kala-azar were entered into this study and followed for a period of up to 2 years. During follow up 12 patients developed PKDL and eight did not. Proliferative responses and cytokine production to Leishmania donovani and control antigens were measured in vitro using PBMC isolated at the time of diagnosis of kala-azar, after treatment of visceral leishmaniasis, during follow up, and at the time of diagnosis of PKDL. Proliferative responses and interferon-gamma (IFN-gamma) production were low at diagnosis and increased after treatment of kala-azar in both patients who developed (group 1) and those who did not develop PKDL later (group 2). In group 1, development of PKDL was always associated by an increased PBMC response to Leishmania antigen in proliferation and IFN-gamma production assays. There were no differences in Leishmania antigen-induced production of IL-4, IL-5 and IL-10 between or within the two groups. We have previously shown that Leishmania parasites spread to the skin during visceral leishmaniasis and proposed that PKDL was the result of an immunological attack on parasites, which have survived in the skin despite the drug treatment. The finding that PKDL develops after treatment of kala-azar as Leishmania-reactive T cells start to circulate in peripheral blood in sufficient numbers to be detected in in vitro assays supports this hypothesis.     

Antibody response against a Leishmania donovani amastigote-stage-specific protein in patients with visceral leishmaniasis.

The antibody response against an amastigote-specific protein (A2) from Leishmania donovani was investigated. Sera from patients with trypanosomiasis and various forms of leishmaniasis were screened for anti-A2 antibodies. Sera from patients infected only with L. donovani or Leishmania mexicana specifically recognized the A2 recombinant protein. These results were consistent with karyotype analyses which revealed that the A2 gene is conserved in L. donovani and L. mexicana strains. The potential of this antigen in diagnosis was further explored by screening a series of sera obtained from patients in regions of the Sudan and India where L. donovani is endemic. The prevalence of anti-A2 antibodies was determined by Western blotting for all samples. Enzyme-linked immunosorbent assay (ELISA) and an immunoprecipitation assay were also performed on some of the samples. Anti-A2 antibodies were detected by ELISA in 82 and 60% of the samples from individuals with active visceral leishmaniasis (kala-azar) from the Sudan and India, respectively, while the immunoprecipitation assay detected the antibodies in 92% of the samples from India. These data suggest that the A2 protein may be a useful diagnostic antigen for visceral 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.     

Identification of an immunodominant 32-kilodalton membrane protein of Leishmania donovani infantum promastigotes suitable for specific diagnosis of Mediterranean visceral leishmaniasis.

Sera from 35 patients suffering from Mediterranean visceral leishmaniasis (caused by Leishmania donovani infantum) and 59 patients with various forms of cutaneous leishmaniasis prevalent in the sub-Mediterranean countries (caused by Leishmania major, L. donovani infantum, or Leishmania tropica) were tested by immunoblotting and enzyme-linked immunosorbent assay (ELISA) with both membrane and soluble antigens prepared from L. donovani infantum parasites. Control sera were from healthy children (n = 41), adults with nonleishmanial diseases (n = 40), and patients with Chagas' disease (n = 12). A P32 antigen present in the membrane preparation from L. donovani infantum parasites was recognized by 95% of serum specimens from patients with Mediterranean visceral leishmaniasis but not by serum specimens from patients with cutaneous leishmaniasis or sera from control individuals. An ELISA with electroeluted P32 antigen was found to have a specificity and sensitivity of 94% in the serodiagnosis of Mediterranean visceral leishmaniasis. Healthy children with asymptomatic Leishmania infection were seronegative for the P32 antigen by ELISA. These results suggest that antibodies to P32 antigen develop only in patients with visceral leishmaniasis and that the P32 ELISA may be useful in areas where the disease is endemic for discriminating between patients with this disease and those with other clinical conditions.     

Leishmania donovani-reactive Th1- and Th2-like T-cell clones from individuals who have recovered from visceral leishmaniasis.

Infections in humans by Leishmania donovani parasites can result in a fatal disease, visceral leishmaniasis (VL), or in a self-limiting asymptomatic infection. In murine models of the infection employing Leishmania major, the course of the disease can be directed into a VL-like syndrome by interleukin-4 (IL-4)-producing Th2 cells, or cure may result by Th1 cells secreting gamma interferon (IFN-gamma). The present study examined the potential of human T cells to generate Th1 or Th2 responses to L. donovani. The profiles of IFN-gamma, IL-4, and lymphotoxin secretion after antigen stimulation were analyzed in a panel of L. donovani-reactive CD4+ human T-cell clones generated from individuals who had recovered from VL after antimonial treatment. Two of the T-cell clones produced large amounts of IL-4 without production of IFN-gamma, seven clones produced both IFN-gamma and IL-4, and eight produced only IFN-gamma. This is the first report of a Th1- and Th2-type response in human leishmaniasis. These results suggest that in analogy with murine models, there is a dichotomy in the human T-cell response to L. donovani infections. Preferential activation of IL-4-producing Th2-like cells may be involved in the exacerbation of human VL, whereas activation of IFN-gamma-producing Th1 cells may protect the host from severe disease. Identification of leishmanial antigens activating one or the other type of T cells will be important in the development of vaccines against leishmaniasis.     

Comparison of microscopic examination,rK39, and PCR for visceral leishmaniasis diagnosis in Turkey

The laboratory diagnosis of visceral leishmaniasis is based on microscopic examination, culture, serological tests, and molecular methods. In this study, we examined 50 blood specimens from suspected visceral leishmaniasis patients by microscopic examination, recombinant antigen dipstick test (rK39), and polymerase chain reaction (PCR) in the University of Cukurova, Faculty of Medicine, Parasitology Department in Turkey. We calculated the sensitivity–specificity and positive–negative predictive values for these diagnostic tests. We found that positive predictive value of microscopy examination, rK39 dipstick test, and PCR were 20%, 24%, and 58% for visceral leishmaniasis, respectively. When we compared polymerase chain reaction, recombinant antigen dipstick test, and microscopic examination for visceral leishmaniasis diagnosis, the polymerase chain reaction is more sensitive (100%) than recombinant antigen dipstick test and microscopy examination.     

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.     

Heterogeneity of the genes encoding the major surface glycoprotein of Leishmania donovani.

The major surface glycoprotein of Leishmania (GP63) is present on all known species of Leishmania and likely plays an integral role during the infection of macrophages in the mammalian host. To identify regions of GP63 which may be of functional significance, the nucleotide sequence of a gene encoding GP63 of Leishmania donovani was determined and compared to the sequences reported for GP63 genes of Leishmania major and Leishmania chagasi. The GP63 nucleotide and predicted protein sequence was highly conserved among the 3 species despite their diverse geographical distribution. L. donovani GP63 is encoded by a multigene family and the gene locus contains at least 7 tandemly repeated genes and at least 3 genes which are dispersed from the tandem array. In addition, polymerase chain reaction and Southern blot analyses demonstrated that there was size heterogeneity within the pro-peptide coding regions of the multiple GP63 genes of L. donovani and that such genes were expressed concurrently in the promastigote life stage.