Leishmaniasis in Bahia, Brazil: evidence that Leishmania amazonensis produces a wide spectrum of clinical disease

One hundred fourteen Leishmania isolates from patients with different clinical forms of leishmaniasis in the State of Bahia, Brazil, were characterized by indirect radioimmune binding assay using specific monoclonal antibodies (serodeme analysis). Seventy-five of these isolates were also analyzed by enzyme electrophoresis, based on 11 enzyme loci; parasite species were compared, according to their characteristic zymodemes, to those of WHO Leishmania reference strains. All isolates could be classified into one of three species: Leishmania amazonensis (n = 40), L. braziliensis (n = 39) or L. chagasi (n = 35). The most interesting information obtained from this study is the realization that L. amazonensis is capable of producing a wide spectrum of disease in humans. Infection with this parasite was associated with many different clinical presentations, including cutaneous leishmaniasis [CL] (20/49 cases), mucocutaneous leishmaniasis [MCL] (5/13 cases) and, of special note, visceral leishmaniasis [VL] (11/46 cases), as well as four cases of post kalaazar dermal leishmaniasis [PKDL]. In situ tissue parasite characterization, by immunoperoxidase assay and employing anti-L. amazonensis amastigote monoclonal antibodies, confirmed the infection with this species in two cases of CL, one case of DCL, one case of MCL and one case of PKDL. Our results also demonstrate the difficulty of parasite differentiation based on clinical grounds, since at least L. amazonensis infection can be associated with all types of leishmanial diseases, and different Leishmania species may be associated with indistinguishable clinical presentations. Since leishmanial parasites may vary in their biological behavior or in their response to treatment, it is important that their identification be made by reliable methods.     

Rapid clearance of circulating Leishmania kinetoplast DNA after treatment of visceral leishmaniasis

With the aim of evaluating the utility of the detection of Leishmania kDNA in peripheral blood for the cure assessment of visceral leishmaniasis (VL), a PCR based method was performed in patients with confirmed VL at three follow-up periods after specific chemotherapy with pentavalent antimonial. In 16 out of 17 (94.1%) patients with pre-treatment detectable kDNA that were clinically cured, the PCR turned negative up to 37 days after the initiation of treatment, remaining negative over 90 days after treatment. The clearance of Leishmania kDNA from peripheral blood of patients with VL hints to occur during or shortly after treatment concurring or preceding clinical recovery.     

Leishmaniasis among organ transplant recipients

Leishmaniasis is a rarely reported disease among transplant recipients; however, the number of published cases has quadrupled since the beginning of the 1990s. Most cases have been observed in patients living in countries of the Mediterranean basin. Leishmaniasis is most commonly associated with kidney transplantation (77%), and cases are also recorded among patients undergoing liver, heart, lung, pancreas, and bone marrow transplantation. Visceral leishmaniasis (VL) is the most frequently observed clinical presentation, followed by mucosal leishmaniasis and more rarely cutaneous leishmaniasis. Transplant recipients with VL develop the classic clinical form of the disease, which is a febrile hepatosplenic and pancytopenic syndrome. Immunodepression seems to predispose to development of mucosal leishmaniasis caused by viscerotropic strains. Early diagnosis of VL is crucial for patient therapy and outcome; however, this is frequently overlooked or delayed in transplant patients. Pentavalent antimonials are the most commom form of treatment for VL, but have a high incidence of toxicity (34%). Although used in fewer patients, liposomal amphotericin B seems to be better tolerated and should be considered as first-line therapy in transplant recipients.     

A simplified and standardized polymerase chain reaction format for the diagnosis of leishmaniasis

BACKGROUND: Definite diagnosis of Leishmania infections is based on demonstration of the parasite by microscopic analysis of tissue biopsy specimens or aspirate samples. However, microscopy generally shows low sensitivity and requires invasive sampling. METHODS: We describe here the development of a simple and rapid test for the detection of polymerase chain reaction-amplified Leishmania DNA. A phase 1 evaluation of the text was conducted in clinical samples from 60 nonendemic and 45 endemic control subjects and from 44 patients with confirmed cutaneous leishmaniasis (CL), 12 with mucocutaneous leishmaniasis (MCL), and 43 with visceral leishmaniasis (VL) from Peru, Kenya, and Sudan. RESULTS: The lower detection limits of the assay are 10 fg of Leishmania DNA and 1 parasite in 180 microL of blood. The specificity was 98.3% (95% confidence interval [CI], 91.1%-99.7%) and 95.6% (95% CI, 85.2%-98.8%) for nonendemic and endemic control samples, respectively, and the sensitivity was 93.2% (95% CI, 81.8%-97.7%), 91.7% (95% CI, 64.6%-98.5%), and 86% (95% CI, 72.7%-93.4%) for lesions from patients with CL or MCL and blood from patients with VL, respectively. CONCLUSIONS: The Leishmania OligoC-TesT showed high specificity and sensitivity in clinical samples and was able to detect the parasite in samples obtained by less invasive means, such as blood, lymph, and lesion scrapings. The assay is a promising new tool for simplified and standardized molecular detection of Leishmania parasites.     

Persistence of leishmania parasites in scars after clinical cure of American cutaneous leishmaniasis: is there a sterile cure?

BACKGROUND: It is uncertain whether Leishmania parasites ever disappear after clinical cure of American cutaneous leishmaniasis (ACL). Recently, sensitive molecular techniques have allowed the identification of Leishmania parasites directly in specimens from patients' scars. METHODS: Scars of 32 patients from northeastern Brazil who were treated and clinically cured of ACL were analyzed by use of polymerase chain reaction (PCR), culture, and histopathologic examination. RESULTS: DNA specific for Leishmania (Viannia) was detected in scars of 30 (93.7%) of 32 patients. In specimens from 3 of the scars, Leishmania parasites could be isolated by culture; PCR results also were positive for those 3 specimens. No parasites were found by histopathologic examination, and fibrotic alterations were present in all cases, with slight inflammatory foci observed in 4 of the cases studied. CONCLUSIONS: The results suggest that clinical cure of ACL is rarely associated with sterile cure. The implications of persistence of parasites for the clinical evolution, relapse, and transmission of leishmaniasis deserves further studies, particularly with the increasing incidence of coinfection with leishmaniasis and acquired immunodeficiency syndrome.     

Post-kala-azar dermal leishmaniasis

Post-kala-azar dermal leishmaniasis (PKDL) is a complication of visceral leishmaniasis (VL); it is characterised by a macular, maculopapular, and nodular rash in a patient who has recovered from VL and who is otherwise well. The rash usually starts around the mouth from where it spreads to other parts of the body depending on severity. It is mainly seen in Sudan and India where it follows treated VL in 50% and 5-10% of cases, respectively. Thus, it is largely restricted to areas where Leishmania donovani is the causative parasite. The interval at which PKDL follows VL is 0-6 months in Sudan and 2-3 years in India. PKDL probably has an important role in interepidemic periods of VL, acting as a reservoir for parasites. There is increasing evidence that the pathogenesis is largely immunologically mediated; high concentrations of interleukin 10 in the peripheral blood of VL patients predict the development of PKDL. During VL, interferon gamma is not produced by peripheral blood mononuclear cells (PBMC). After treatment of VL, PBMC start producing interferon gamma, which coincides with the appearance of PKDL lesions due to interferon-gamma-producing cells causing skin inflammation as a reaction to persisting parasites in the skin. Diagnosis is mainly clinical, but parasites can be seen by microscopy in smears with limited sensitivity. PCR and monoclonal antibodies may detect parasites in more than 80% of cases. Serological tests and the leishmanin skin test are of limited value. Treatment is always needed in Indian PKDL; in Sudan most cases will self cure but severe and chronic cases are treated. Sodium stibogluconate is given at 20 mg/kg for 2 months in Sudan and for 4 months in India. Liposomal amphotericine B seems effective; newer compounds such as miltefosine that can be administered orally or topically are of major potential interest. Although research has brought many new insights in pathogenesis and management of PKDL, several issues in particular in relation to control remain unsolved and deserve urgent attention.     

Visceral leishmaniasis in haemophiliac patients with HIV: four case reports

Visceral leishmaniasis (VL), an endemic parasitosis in Spain, is increasing as an important opportunistic infection in AIDS patients. We describe four cases of severe haemophilic patients with end-stage HIV disease who present with visceral leishmaniasis. We report the clinical course, methods of diagnosis and response to therapy. From the assessed clinical data it does not appear to be any difference with regard to VL either in other HIV risk groups or in immunocompetent groups. In contrast with previous reports, we have found that despite their immunodepressed state, positive serology for Leishmania was found in three of the four cases. A similar observation has been made in patients with VL who are immunodepressed because of other reasons. We also confirm previous reports of poor response and intolerance of antimony treatment, the deteriorating course of the disease in AIDS patients and that there is only a slight relationship between the disease and the cause of patient death. We agree with the proposition that this pathology ought to be included in the definition critera for AIDS.     

Immunity to a salivary protein of a sand fly vector protects against the fatal outcome of visceral leishmaniasis in a hamster model

Visceral leishmaniasis (VL) is a fatal disease for humans, and no vaccine is currently available. Sand fly salivary proteins have been associated with protection against cutaneous leishmaniasis. To test whether vector salivary proteins can protect against VL, a hamster model was developed involving intradermal inoculation in the ears of 100,000 Leishmania infantum chagasi parasites together with Lutzomyia longipalpis saliva to mimic natural transmission by sand flies. Hamsters developed classical signs of VL rapidly, culminating in a fatal outcome 5-6 months postinfection. Saliva had no effect on the course of infection in this model. Immunization with 16 DNA plasmids coding for salivary proteins of Lu. longipalpis resulted in the identification of LJM19, a novel 11-kDa protein, that protected hamsters against the fatal outcome of VL. LJM19-immunized hamsters maintained a low parasite load that correlated with an overall high IFN-gamma/TGF-beta ratio and inducible NOS expression in the spleen and liver up to 5 months postinfection. Importantly, a delayed-type hypersensitivity response with high expression of IFN-gamma was also noted in the skin of LJM19-immunized hamsters 48 h after exposure to uninfected sand fly bites. Induction of IFN-gamma at the site of bite could partly explain the protection observed in the viscera of LJM19-immunized hamsters through direct parasite killing and/or priming of anti-Leishmania immunity. We have shown that immunity to a defined salivary protein (LJM19) confers powerful protection against the fatal outcome of a parasitic disease, which reinforces the concept of using components of arthropod saliva in vaccine strategies against vector-borne diseases.     

Positive Montenegro skin test among patients with sporotrichosis in Rio De Janeiro

Visceral leishmaniasis (VL) or kala-azar continues to persist as one of the major public health problems in many tropical countries. However, no effective treatment for radical cure of the disease is yet available. Miltefosine, an alkyl phospholipid compound, is the first orally effective drug, which has shown 98% cure rate of VL patients during phase III clinical trial in India. Since this drug requires long course of treatment and has long half-life, there are fairly good chances of emergence of resistance. Furthermore, this drug has produced severe side-effects in some of the cases.We therefore examined the possibility of minimizing these effects by applying miltefosine in lower doses in combination with picrloviv, an immunomodulator against Leishmania donovani in hamsters (Mesocricetus auratus). The picroliv per se showed no antileishmanaial potential. However, when given with suboptimal dose of miltefosine, it enhanced efficacy of the latter from 45 to 86% on day 7 post treatment and from 32 to 64% on day 28 post treatment. Interestingly, the efficacy of this combination was as good as the curative dose of miltefosine alone. Thus, this combination appears to offer a fruitful strategy for treatment of VL.     

Use of purified parasite proteins from Leishmania donovani for the rapid serodiagnosis of visceral leishmaniasis

Serodiagnosis of visceral leishmaniasis (VL) due to Leishmania donovani by using crude parasite antigen is complicated in many endemic areas because of cross-reactions with sera from humans with Chagas' disease. We used affinity-purified parasite proteins to develop a direct dot-blot assay for VL. Double-blind tests were carried out on sera from 40 patients with well-documented VL, from controls in endemic areas, and from patients with other diseases. Using gp70-2, 36 (90.0%) of 40 sera from patients with kala azar were correctly diagnosed; 1 (1.2%) of 86 sera from patients without kala azar was misdiagnosed. With dp72, 21 (100%) of 21 sera from patients with VL were correctly identified; 5 (7.0%) of 71 negative sera were misdiagnosed. None of the 18 sera from patients with Chagas' disease reacted positively against gp70-2. Our assay is rapid, simple, and specific and represents a new method for reliably diagnosing and monitoring VL.     

Widespread atypical cutaneous Leishmaniasis caused by Leishmania (L.) Chagasi in Nicaragua.

Leishmania chagasi, the causative agent of visceral leishmaniasis (VL) in the Americas, has recently been associated with atypical cutaneous leishmaniasis (ACL) in Central America; however, little comprehensive information about this disease is available. Clinical, epidemiologic, and parasitologic characteristics of 252 ACL cases and 44 VL cases in Nicaragua were analyzed. Visceral leishmaniasis is primarily associated with malnourished children less than five years of age, whereas ACL is found predominantly in children greater than five years of age and young adults. Genetically similar parasites are associated with both disease manifestations. The sand fly Lutzomyia evansi, in addition to Lu. longipalpis, may be involved in transmission of L. chagasi to humans. Our results indicate that ACL is more prevalent than previously thought, affecting up to 10% of a local population. The fact that the same parasite appears to cause both ACL and the potentially fatal visceral disease suggests that the host immune response is critical in determining the outcome of L. chagasi infection. The public health implications of the wide-spread presence of L. chagasi are discussed.     

Adenosine deaminase activity in sera of patients with visceral leishmaniasis in Nepal

This longitudinal study was conducted in BP Koirala Institute of Health Sciences (BPKIHS), a Medical University situated in eastern Nepal, between May 2001 and December 2001. The main objective of the study was to identify the role of adenosine deaminase (ADA) activity in patients with visceral leishmaniasis (VL) for management. There was a significant increase in mean ADA activity in sera of 49 patients with VL (323.71+/-184.51 IU/L) compared with 50 samples of control groups (47.11+/-24.94 IU/L) from the same endemic area (P < 0.001). ADA activities were found to be significantly decreased (50.35+/-41.35 IU/L) in follow-up cases (n = 19) after 30 days with sodium stibogluconate treatment at a dose of 20 mg/kg/day intramuscularly. The fall in the level of ADAF (after treatment) in follow-up cases correlated with the cure of disease, as evident from improvement of vital signs and symptoms and the absence of Leishmania donavani bodies in the sera. The study therefore suggests the possibility of using human serum ADA as a convenient marker to evaluate the diagnosis of VL to support the clinical findings, especially in those settings where there is a lack of highly qualified personnel and diagnostic facilities.     

MMP-9 activity is induced by Leishmania braziliensis infection and correlates with mucosal leishmaniasis

Infection of humans with Leishmania braziliensis typically results in localized cutaneous leishmaniasis (LCL). Rarely, after months or years of apparent clinical cure, some patients develop the destructive mucosal leishmaniasis (ML). ML results from L. braziliensis dissemination, probably via phagocytic cells. As the preferred cells for Leishmania spp. colonization, macrophages are critical to infection control, and may contribute to parasite dissemination. However, the host factors that determine this outcome are unknown. Matrix metalloproteinase 9 (MMP-9) is known to be important for immune cell migration, macrophage recruitment, and effective granuloma formation. Moreover, MMP-9 has been involved in Mycobacterium tuberculosis dissemination. Here, we demonstrate that in vitro infection of human macrophages with L. braziliensis increased the secretion and activation of MMP-9. We also demonstrate that macrophages from healthy cured individuals with previous history of ML had increased MMP-9 activity compared to LCL cured individuals. These findings may represent a fundamental difference in host innate immunity that could contribute to the clinical leishmaniasis presentation.     

Utility of lymph node aspiration in the diagnosis of visceral leishmaniasis in Sudan

We evaluated lymph node aspiration (LNA) as a simple diagnostic procedure for visceral leishmaniasis (VL). Lymph node aspiration was compared with the direct agglutination test (DAT) using a diagnostic titer > or = 1:6,400 in 7,880 suspected VL patients in eastern Sudan. Compared with DAT, LNA had a sensitivity of 65.1% (95% confidence interval = 63.5-66.6%). Parasite density in LNA correlated strongly with DAT titers (P < 0.0001), and low parasite density accounted for 78.1% of positive LNA results with DAT titers < 1:6,400 (n = 782). Risk factors predictive of a positive LNA result were an age of 1-29 years, male sex, a hemoglobin level < 10.0 g/dL, a DAT titer > or = 1:800, and a location with a higher prevalence of VL. Lymph node and splenic aspirations were similarly accurate as tests of cure after treatment of 50 VL patients in southern Sudan. Pre-treatment LNA results were negative in 20 cases of severe post kala-azar dermal leishmaniasis.     

Accidental Leishmania mexicana infection in an immunosuppressed laboratory technician

A 30 year-old female laboratory technician under immunosuppressive treatment because of systemic lupus erythematosus (SLE) developed cutaneous leishmaniasis 8 months after accidental percutaneous inoculation of amastigote culture forms of Leishmania mexicana . Leishmania -specific PCR and restriction analysis patterns were identical for both the laboratory strain and the clinical specimen. The lesion was resistant to local paromomycin and oral ketoconazole, but responded to local application of meglumine antimonate. No signs of dissemination or visceralization occurred during the 5-month period of observation. However, a future recurrence cannot be excluded since a persistent infection even after clinical cure has always to be considered in leishmaniasis. Patients under immunosuppressive therapy are possibly at risk of clinical relapse or disseminating infection although there is no experience with regard to leishmaniasis mexicana . Serious infection may require interferon gamma as part of the treatment which may contribute to deterioration of concomitant diseases like SLE. In any case, the exposure of immunodeficient laboratory workers to Leishmania spp . should be avoided.     

Use of rK39 for diagnosis of post kala-azar dermal leishmaniasis in Nepal

A recently developed nitrocellulose-based dipstick test, rK39, has been widely used for the diagnosis of kala-azar. In this study, we evaluated its use for the diagnosis of post kala-azar dermal leishmaniasis (PKDL). We also investigated the time taken by patients to develop PKDL after apparent cure of kala-azar (visceral leishmaniasis, VL) and the time taken by patients to come to the hospital after the appearance of symptoms of PKDL. A majority of patients developed the disease within three years after the apparent cure of kala-azar (KA). A majority of patients sought treatment within five years after the onset of PKDL. The amastigotes of Leishmania donovani bodies (LDBs) were demonstrated in 70, 20, and 20% of slit-skin smears (SSS) prepared, respectively, from nodular, papular, and macular forms. The presence of highest density (6+) LDBs in the SSS of 20% of nodular PKDL patients indicated that they may have acted as reservoir in the community. Other reservoirs are not known in Nepal. Only 8% cases were detected by aldehyde test. Although this test is obsolete it is still used in rural parts of Nepal. The dipstick (rK39) was 96% sensitive and 100% specific to diagnose PKDL. Its positive predictive value, negative predictive value, and diagnostic efficacy were 100, 91, and 97% respectively. Due to the advantage of cost compared with the direct agglutination test (DAT), and being easy to use and store in field conditions, rK39 is a good tool to diagnose PKDL in rural situations. All the PKDL patients were cured of the disease after treatment by SAG.     

Increased levels of interleukin-10 and IgG3 are hallmarks of Indian post-kala-azar dermal leishmaniasis

BACKGROUND: Post-kala-azar dermal leishmaniasis (PKDL), an established sequela of visceral leishmaniasis (VL), is proposed to facilitate anthroponotic transmission of VL, especially during interepidemic periods. Immunopathological mechanisms responsible for Indian PKDL are still poorly defined. METHODS: Our study attempted to characterize the immune profiles of patients with PKDL or VL relative to that of healthy control subjects by immunophenotyping, intracellular cytokine staining of peripheral blood mononuclear cells, and enzyme-linked immunosorbent assay for serum cytokines and immunoglobulin G (IgG) subclasses. RESULTS: Patients with PKDL had significantly raised percentages of peripheral CD3+CD8+ cells compared with control subjects, a difference that persisted after cure. Patients with PKDL showed an intact response to phytohemagglutinin, with the percentages of lymphocytes expressing interferon (IFN)-gamma, interleukin (IL)-2, IL-4, and IL-10 being comparable to those in control subjects. Patients with VL had decreased IFN-gamma and IL-2 expression, which was restored after cure, and increased IL-10 expression, which persisted after cure. In their response to Leishmania donovani antigen, patients with PKDL showed a 9.6-fold increase in the percentage of IL-10-expressing CD3+CD8+ lymphocytes compared with control subjects, and this percentage decreased with treatment. Patients with PKDL had raised levels of IgG3 and IgG1 (surrogate markers for IL-10), concomitant with increased serum levels of IL-10. CONCLUSIONS: IL-10-producing CD3+CD8+ lymphocytes are important protagonists in the immunopathogenesis of Indian PKDL.     

Evaluation of In Vitro Production of IFN-gamma, IL-10, IL-12 and IL-13 by Blood Cells in Patients with Cutaneous Leishmaniasis Lesions

This study investigated the in vitro production of interferon-gamma, interleukin (IL)-10, IL-12, and IL-13, after antigenic stimulation of the cells (with Leishmania antigen and lipopolysaccharide) using whole blood from patients with cutaneous leishmaniasis lesions caused by Leishmania tropica and in normal volunteers with history of cutaneous leishmaniasis.ELISA results showed that the mean production of interferon-gamma by cells of whole blood in patients with lesions in response to Leishmania antigen was significantly lower than corresponding values in volunteers with history of cutaneous leishmaniasis (P< 0.05) and significantly higher levels of IL-10 production in patients with lesions were observed compared with cured volunteers of the disease (P<0.01). A similar level of IL-12, including p40 subunit of IL-12, was detected in both groups tested in this study in response to stimulation of parasite antigen. The levels of the IL-13 after stimulation with Leishmania antigen were significantly more in patients compared with volunteers with history of cutaneous leishmaniasis (P< 0.01). There was no significant difference in the mean production of IFN-gamma, IL-10, IL-12 and IL-13 by PHA or LPS stimulated cells from patients with lesions and volunteers with history of the disease, indicating that there was no qualitative defect in cytokine production in these patients.In this study, we have detected the decreased production of interferon- gamma by cells of patients with lesions of cutaneous leishmaniasis in response to parasite antigen and unbalanced production of regulatory cytokines such as IL-10 and IL-13 using the whole-blood stimulation assay technique. The required small volume of blood and the rapid set up time are the advantages in this assay technique. Using this assay for further immunodetection of cytokines may confirm its value for clinical investigation.     

Recent developments in leishmaniasis

PURPOSE OF REVIEW: The leishmaniases, caused by protozoan parasites of the genus Leishmania, are a significant health problem in many regions of the world. This review highlights the recent advances in the study of leishmaniasis related to parasite biology, disease pathogenesis, clinical evaluation and treatment, and prevention. RECENT FINDINGS: Genetic heterogeneity and clonal diversity is common among Leishmania strains. Gene knockout, overexpression, and re-introduction studies have identified a number of genes that play a role in parasite virulence. Surprisingly, the importance of the surface lipophosphoglycan in parasite virulence appears to differ among Leishmania spp. Studies in experimental animal models have further defined the roles of CD4 and CD8 T cells, IL-4, IL-10, and IL-12 in the control, maintenance, or progression of disease. The effect of Leishmania on dendritic cells and macrophage effector function has also been an important area of investigation. A number of new vaccine candidates have been identified through experimental animal studies. Clinical studies of leishmaniasis have focused on the host determinants of disease (most notably HIV co-infection), serological and DNA-based diagnostic assays, and treatment. Antimony-resistant cases of cutaneous and visceral leishmaniasis have become more common; liposomal amphotericin and oral miltefosine are promising alternative therapies. SUMMARY: Significant advances have been made in the areas of pathogenesis, host defence, and treatment of leishmaniasis. A number of new vaccine candidates and potential targets of drug therapy have been identified, but progress from preclinical studies to clinical trials has been slow. Translational research, built upon the solid foundation of existing and ongoing basic investigation, is a high priority.