Treated Visceral Leishmaniasis Patients Research Articles

Assessment of treatment outcomes of visceral leishmaniasis (VL) treated cases and impact of COVID-19 on VL management and control services in Bangladesh

BackgroundCOVID-19 has largely impacted the management of Visceral leishmaniasis (VL), like several other Neglected Tropical Diseases. The impact was particularly evident in Lower and Middle-Income countries where the already inadequate healthcare resources were diverted to managing the COVID-19 pandemic. Bangladesh achieved the elimination target for VL in 2016. To sustain this success, early diagnosis and treatment, effective vector control, and periodic surveillance are paramount. However, the specific control measures for VL in Bangladesh that were hampered during COVID-19 and their extent are unknown. MethodsThis study aimed at identifying the gaps and challenges in the follow-up of treated VL patients by interviewing both the treated VL cases and their health service providers. We followed VL cases treated between 2019 and 2020 in five VL endemic subdistricts (upazilas) both retrospectively and prospectively to monitor clinical improvement, relapse, or other consequences. Moreover, interviews were conducted with the health service providers to assess the impact of COVID-19 on VL case detection, treatment, reporting, vector control operations, and logistic supply chain management. ResultsThere was no added delay for VL diagnosis; however, VL treatment initiation and reporting time increased almost two-fold due to COVID-19. Indoor Residual Spraying activity was significantly hampered due to a shortage of insecticides. Out of 44 enrolled and treated VL patients, two relapsed (4.5 %), two developed Para Kala-Azar Dermal Leishmaniasis (4.5 %), and three (6.8 %) Post Kala-Azar Dermal Leishmaniasis (PKDL). The health service providers highlighted patients` unwillingness to visit the hospital, financial constraints, and distance from the hospitals as the main reasons for missed follow-up visits (20.5 %). Building good communication in the community, awareness schemes, and incentive-based approaches were suggested as possible solutions to mitigate these problems. ConclusionLong-term follow-up is required for the early detection and management of VL relapse and PKDL cases. Effective vector control measures, capacity development, and identification of new VL hotspots are pivotal in the VL endemic regions to sustain the elimination goal.

CD4+ T cells show a cytolytic phenotype functions in human visceral leishmaniasis

Abstract Control of Visceral leishmaniasis (VL), a parasitic disease caused by L. donovani, requires robust CD4 +T cells response to control the parasite replication by IFN-γ production and activation of macrophages. Our recent study on transcriptional signature of CD4 +T cell isolated from peripheral blood of active VL patients showed enhanced expression of genes related to cytotoxicity. In present study, we investigated expression of these cytotoxic molecules including granzyme B (GZMB), Granulysin, Perforin and NKG7 on different CD4 +T cell subsets to establish the role of GZMB in regulating CD4 +T cells functional capacity. PBMCs cells were isolated from active and post treated VL patients and endemic controls, and flow cytometry was performed. LAMP assay (CD107b expression) was performed to study the degranulation capacity of CD4 +T cells. Whole blood assay followed by ELISA was performed to measure antigen specific cytokine / GZMB production by CD4 +T cells. We found that activated and degranulating CD4 +T cells (CD38 +CD107 +) had higher expression of GZMB, Granulysin, perforin and NKG-7. Similarly, GZMB level in plasma and antigen stimulated whole blood culture supernatant were significantly elevated in active VL patient compared to post treatment and EC. There was no change is IFN-γ secretion when whole blood culture was stimulated with soluble leishmania antigen in presence of Con-A but a significant decline in IL-6 production was observed. In summary, VL CD4 +T cells show a cytolytic phenotype and further investigations are required to understand their differentiation and function, particularly for promoting anti-parasitic immunity for host protection and effective intervention or therapy. Extramural Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health (TMRC grant number U19AI074321)

Recombinant endonuclease III protein from Leishmania infantum associated with Th1-type adjuvants is immunogenic and induces protection against visceral leishmaniasis.

Vaccination against visceral leishmaniasis (VL) should be considered as a safe and effective measure to disease control; however, few vaccines are available against canine VL and there is no an approved human vaccine. In this context, in the present study, we evaluated the endonuclease III (ENDO) protein, which was recently showed to be antigenic for human disease, as a vaccine candidate against Leishmania infantum infection. The recombinant protein (rENDO) was administered in BALB/c mice alone or associated with saponin (rENDO/Sap) or micelles (rENDO/Mic) as adjuvants. Controls received saline, saponin or empty micelles. Results showed that both rENDO/Sap and rENDO/Mic compositions induced higher levels of IFN-γ, IL-12, TNF-α, and GM-CSF cytokines, besides nitrite and IgG2a isotype antibodies, before and after challenge infection, which were related to both CD4+ and CD8+ T cell subtypes. The immunological results contributed to significant reductions in the parasite load found in the spleens, livers, bone marrows and draining lymph nodes of the vaccinated animals. In general, mice immunized with rENDO/Mic presented a slightly higher Th1-type cellular and humoral immune response, as compared to those receiving rENDO/Sap. In addition, saponin caused a slight to moderate inflammatory edema in their vaccinated footpads, which was not observed when micelles were used with rENDO. In addition, a preliminary analysis showed that the recombinant protein was immunogenic to human cells cultures, since PBMCs from treated VL patients and healthy subjects showed higher lymphoproliferation and IFN-γ production in the culture supernatants. In conclusion, data suggest that rENDO could be considered as a candidate to be evaluated in future studies as vaccine to protect against VL.

Leishmania infantum (syn. L. chagasi) parasites affect the release of soluble CD14 by infected macrophages.

Functionally, cluster of differentiation 14 (CD14) is a co-receptor of the complex formed by lipopolysaccharide (LPS) and LPS-binding protein expressed on the membrane of a variety of cells. However, CD14 can be shed from the cell membrane into the circulation as soluble CD14 (sCD14) upon cell activation. Previously, our group reported that elevated sCD14 serum levels were associated with the clinical and laboratory findings in the context of visceral leishmaniasis (VL), but not in the context of LPS stimulation or bacterial infection. In the present study, we investigated the secretion dynamics of sCD14 in the context of Leishmania infantum (syn. L. chagasi) in vitro infection. Macrophages from treated VL patients and delayed-type hypersensitivity positive (DTH+) subjects were infected with L. infantum (syn. L. chagasi) promastigotes, and the infection index was evaluated (number of amastigotes per 100 infected macrophages). Additionally, the levels of sCD14, Inteleukin (IL)10, IL-6 and tumour necrosis factor alpha (TNF-α) were measured in the culture supernatants using the Luminex assay. Interestingly, the release of sCD14 was inversely correlated with the L. infantum (syn. L. chagasi) infection index. Of note, the release of sCD14 was upregulated and downregulated in the context of infected macrophages from DTH+ subjects and treated VL patients, respectively. Additionally, we also observed that the levels of sCD14 in the culture supernatants were positively correlated with the levels of TNF-α, IL-6 and IL-10. Therefore, our data suggest that macrophages from treated VL patients and DTH+ subjects respond differently to L. infantum (syn. L. chagasi) infection in the context of the release of sCD14; therefore, the release of sCD14 may be associated with the outcome of VL.

A Leishmania amastigote-specific hypothetical protein evaluated as recombinant protein plus Th1 adjuvant or DNA plasmid-based vaccine to protect against visceral leishmaniasis

Most studies evaluating vaccine candidates against visceral leishmaniasis (VL) have used parasite promastigote-expressed antigens; however, Leishmania proteins expressed in the amastigote forms should be considered, since few hours after infection this stage comes into contact with the host immune system and is responsible for the development of the disease. In this context, in the present study, a Leishmania amastigote-specific hypothetical protein, called LiHyJ, was evaluated as a recombinant protein plus saponin as an adjuvant or DNA vaccine to protect against VL. The vaccine effect was evaluated by means of the evaluation of immunological and parasitological analyses performed in BALB/c mice against Leishmania infantum infection. Results showed that rLiHyJ/saponin and DNA LiHyJ induced significantly higher levels of anti-protein and anti-parasite IFN-γ, IL-12, GM-CSF, and IgG2a isotype antibodies, which were associated with a low presence of IL-4 and IL-10. DNA vaccination induced higher IFN-γ production, mainly by CD8+ T cells, while rLiHyJ/saponin stimulated the production of this cytokine, mainly by CD4+ T cells. The parasite load evaluated in distinct organs showed that both immunization schedules significantly reduced organic parasitism, when compared to the controls. Similar results were found in the immunological and parasitological assays when using the recombinant protein or DNA, although the vaccination with rLiHyJ plus saponin induced a slightly higher Th1 response and lower parasite load, when compared to the use of DNA plasmid. The protein also proved to be immunogenic when peripheral blood mononuclear cells of treated VL patients and healthy subjects were in vitro stimulated, since higher IFN-γ and lower IL-4 and IL-10 levels were found in the culture supernatants. In conclusion, LiHyJ should be considered in future studies as a vaccine candidate to protect against VL.

Liposomal Formulation of ChimeraT, a Multiple T-Cell Epitope-Containing Recombinant Protein, Is a Candidate Vaccine for Human Visceral Leishmaniasis.

Background: Leishmaniases are neglected diseases caused by infection with Leishmania parasites and there are no human vaccines in use routinely. The purpose of this study was to examine the immunogenicity of ChimeraT, a novel synthetic recombinant vaccine against visceral leishmaniasis (VL), incorporated into a human-compatible liposome formulation. Methods: BALB/c mice were immunized subcutaneously with ChimeraT/liposome vaccine, ChimeraT/saponin adjuvant, or ChimeraT/saline and immune responses examined in vitro and in vivo. Results: Immunization with the ChimeraT/liposome formulation induced a polarized Th1-type response and significant protection against L. infantum infection. ChimeraT/liposome vaccine stimulated significantly high levels of interferon (IFN)-γ, interleukin (IL)-12, and granulocyte macrophage-colony stimulating factor (GM-CSF) cytokines by both CD4 and CD8 T-cells, with correspondingly lower levels of IL-4 and IL-10 cytokines. Induced antibodies were predominantly IgG2a isotype, and homologous antigen-stimulated spleen cells produced significant nitrite as a proxy for nitric oxide (NO). Furthermore, we examined a small number of treated VL patients and found higher levels of circulating anti-ChimeraT protein IgG2 antibodies, compared to IgG1 levels. Conclusions: Overall, the liposomal formulation of ChimeraT induced a protective Th1-type immune response and thus could be considered in future studies as a vaccine candidate against human VL.

Leishmania infantum amastin protein incorporated in distinct adjuvant systems induces protection against visceral leishmaniasis

The control measures against visceral leishmaniasis (VL) include a precise diagnosis of disease, the treatment of human cases, and reservoir and vector controls. However, these are insufficient to avoid the spread of the disease in specific countries worldwide. As a consequence, prophylactic vaccination could be interesting, although no effective candidate against human disease is available. In the present study, the Leishmania infantum amastin protein was evaluated regarding its immunogenicity and protective efficacy against experimental VL. BALB/c mice immunized with subcutaneous injections of the recombinant protein with or without liposome/saponin (Lip/Sap) as an adjuvant. After immunization, half of the animals per group were euthanized and immunological evaluations were performed, while the others were challenged with L. infantum promastigotes. Forty-five days after infection, the animals were euthanized and parasitological and immunological evaluations were performed. Results showed the development of a Th1-type immune response in rAmastin-Lip and rAmastin-Sap/vaccinated mice, before and after infection, which was based on the production of protein and parasite-specific IFN-γ, IL-12, GM-CSF, and nitrite, as well as the IgG2a isotype antibody. CD4+ T cells were mainly responsible for IFN-γ production in vaccinated mice, which also presented significant reductions in parasitism in their liver, spleen, draining lymph nodes, and bone marrow. In addition, PBMC cultures of treated VL patients and healthy subjects stimulated with rAmastin showed lymphoproliferation and higher IFN-γ production. In conclusion, the present study shows the first case of an L. infantum amastin protein associated with distinct delivery systems inducing protection against L. infantum infection and demonstrates an immunogenic effect of this protein in human cells.

Diagnostic evaluation of the amastin protein from Leishmania infantum in canine and human visceral leishmaniasis and immunogenicity in human cells derived from patients and healthy controls

The diagnosis of visceral leishmaniasis (VL) presents problems due to the toxicity and/or high cost of drugs. In addition, no vaccine exists to protect against human disease. In this study, the antigenicity and immunogenicity of amastin protein were evaluated in L. infantum–infected dogs and humans. For the diagnosis, besides the recombinant protein, 1 linear B-cell epitope was synthetized and evaluated in serological assays. Results showed high sensitivity and specificity values to detect the disease when both antigens were employed against a canine and human serological panel. By contrast, when using rA2 and a soluble Leishmania antigenic preparation, sensitivity and specificity values proved to be lower. A preliminary immunogenicity study showed that the amastin protein induced high IFN-γ and low IL-10 production in stimulated PBMC derived from treated VL patients and healthy subjects, thus suggesting a potential use of this protein as an immunogen to protect against human disease.

Evaluation of a Leishmania hypothetical protein administered as DNA vaccine or recombinant protein against Leishmania infantum infection and its immunogenicity in humans

Visceral leishmaniasis (VL) is a fatal disease when acute and untreated. The treatment against this disease is long and presents toxicity and/or high costs. Moreover, parasite resistance has been increasing. Therefore, alternative control measures to avoid the spread of disease should be considered. It is accepted that the development of the T helper (Th)1 immune response, based on the production of pro-inflammatory cytokines, is required for the control of parasites. Although recombinant protein-based vaccines have been tested against VL, they require supplementation with immune adjuvants. In addition, there is a scarcity of studies that comparatively evaluate the efficacy of the immunogens when administered by different delivery systems in mammalian hosts. In the present study, a Leishmania hypothetical protein, LiHyR, was cloned and evaluated by immunization as a plasmid deoxyribonucleic acid (DNA) vaccine or in a recombinant format plus saponin against Leishmania infantum infection. Results showed that both vaccination regimens induced a Th1 cell-based immunity, since high levels of interferon-gamma (IFN-γ), interleukin (IL)-2, IL-12, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor alpha (TNF-α) were found, and were associated with the low production of IL-4, IL-10, and anti-parasite immunoglobulin (IgG)1 isotype. In addition, significant reductions in the parasite load were found in the evaluated organs of the DNA LiHyR or rLiHyR/saponin-vaccinated animals. No significant difference was achieved between groups vaccinated with DNA or the recombinant protein. The antigen proved to be also immunogenic in human peripheral blood mononuclear cells (PBMCs) collected from healthy subjects and from untreated and treated VL patients. A higher IgG2 isotype was also found in sera samples of these subjects, thus demonstrating its possible use as a human vaccine. This study demonstrates the protective efficacy of a new Leishmania protein against VL, when it is administered as a DNA vaccine or a recombinant protein plus saponin, and points out its use as a human vaccine against disease.

Recombinant prohibitin protein of Leishmania infantum acts as a vaccine candidate and diagnostic marker against visceral leishmaniasis

Visceral leishmaniasis (VL) represents a serious public health problem, as Leishmania infantum is one of main disease causative agents in the Americas. In a previous immunoproteomic study, the prohibitin (PHB) protein was identified in L. infantum promastigote and amastigote extracts by antibodies in asymptomatic and symptomatic VL dog sera. This protein was found to be highly conserved between different Leishmania spp., but it presented a low identity with amino acid sequences of other organisms. The aim of the present study was to evaluate the cellular response induced by the recombinant PHB (rPHB) protein in BALB/c mice, as well as in PBMCs purified from untreated and treated VL patients, as well as to evaluate its protective efficacy against an infection by L. infantum promastigotes. Our data showed that there was a Th1 cellular response to rPHB, based on high levels of IFN-γ, IL-12, and GM-CSF in the immunized animals, as well as a proliferative response specific to the protein and higher IFN-γ levels induced in PBMCs from individuals who had recovered from the disease. The protection was represented by significant reductions in the parasite load in the animals’ spleen, liver, bone marrow, and draining lymph nodes, as compared to results found in the control groups. In addition, an anti-rPHB serology, using a canine and human serological panel, showed a high performance of this protein when diagnosing VL based on high sensitivity and specificity values, as compared to results found for the rA2 antigen and the soluble Leishmania antigenic extract. Our data suggest that PHB has a potential application for the diagnosis of canine and human VL through antibody detection, as well as an application as a vaccine candidate to protect against disease.

Chronic Arsenic Exposure and Risk of Post Kala-azar Dermal Leishmaniasis Development in India: A Retrospective Cohort Study.

BackgroundVisceral leishmaniasis (VL), with the squeal of Post-kala-azar dermal leishmaniasis (PKDL), is a global threat for health. Studies have shown sodium stibogluconate (SSG) resistance in VL patients with chronic arsenic exposure. Here, we assessed the association between arsenic exposure and risk of developing PKDL in treated VL patients.MethodsIn this retrospective study, PKDL patients (n = 139), earlier treated with SSG or any other drug during VL, were selected from the study cohort. Trained physicians, unaware of arsenic exposure, interviewed them and collected relevant data in a questionnaire format. All probable water sources were identified around the patient’s house and water was collected for evaluation of arsenic concentration. A GIS-based village-level digital database of PKDL cases and arsenic concentration in groundwater was developed and individual point location of PKDL cases were overlaid on an integrated GIS map. We used multivariate logistic regression analysis to assess odds ratios (ORs) for association between arsenic exposure and PKDL development.ResultsOut of the 429 water samples tested, 403 had arsenic content of over 10 μg/L, with highest level of 432 μg/L among the seven study villages. Multivariate adjusted ORs for risk of PKDL development in comparison of arsenic concentrations of 10.1–200 μg/L and 200.1–432.0 μg/L were 1.85 (1.13–3.03) and 2.31 (1.39–3.8) respectively. Interestingly, similar results were found for daily dose of arsenic and total arsenic concentration in urine sample of the individual. The multivariate-adjusted OR for comparison of high baseline arsenic exposure to low baseline arsenic exposure of the individuals in the study cohort was 1.66 (95% CI 1.02–2.7; p = 0.04).ConclusionOur findings indicate the need to consider environmental factors, like long time arsenic exposure, as an additional influence on treated VL patients towards risk of PKDL development in Bihar.

Misdiagnosis and mistreatment of post-kala-azar dermal leishmaniasis.

Post-kala-azar dermal leishmaniasis (PKDL) is a known complication of visceral leishmaniasis (VL) caused by L. donovani. It is rare in VL caused by L. infantum and L. chagasi. In Sudan, it occurs with a frequency of 58% among successfully treated VL patients. In the majority of cases, PKDL can be diagnosed on the basis of clinical appearance, distribution of the lesions, and past history of treated VL. The ideal diagnostic method is to demonstrate the parasite in smears, by culture or PCR. Diagnosis is particularly difficult in patients who develop PKDL in the absence of previous history of visceral leishmaniasis. We describe a case of cutaneous leishmaniasis misdiagnosed as PKDL and 3 cases of PKDL who were either misdiagnosed or mistreated as other dermatoses. This caused exacerbation of their disease leading to high parasite loads in the lesions and dissemination to internal organs in one of the patients, who was also diabetic. The latter patient had L. major infection. A fourth patient with papulonodular lesions on the face and arms of 17-year duration and who was misdiagnosed as having PKDL is also described. He turned out to have cutaneous leishmaniasis due to L. major. Fortunately, he was not treated with steroids. He was cured with intravenous sodium stibogluconate.

Serological Monitoring of Paediatric Visceral Leishmaniasis By IFA and ELISA Methods

Visceral leishmaniasis (VL) is endemic in all Mediterranean countries including Turkey, and children are at greater risk than adults in endemic areas. In VL patients, serological assays are considered to be sensitive for the diagnosis and/or follow up. The aim of this study was to assess the usefulness of serology for following up of childhood VL in Turkey. Sera obtained from twenty parasitologically confirmed children with VL were tested using IFAT and ELISA. The patients were monitored clinically and serologically (range: 20-500 days) during and after treatment. All VL patients were treated with meglumine antimonate. Anti-Leishmania antibodies in successfully treated VL patients showed a steep decline but, in three patients who had relapsed, an increase was detected. Significantly lower values were observed after treatment with both serological techniques. Mean ELISA optical density values before and after treatment were: 0.78 ± 0.36 (0.26-1.76) and 0.38 ± 0.24 (0.09-0.83) respectively, (p < 0.001) and mean IFAT values (log10 transformed titers) before and after treatment were: 3.02 ± 0.90 (1.81-4.51) and 2.16 ± 0.75 (1.20-3.90) respectively, (p < 0.001). ELISA and IFAT are valuable not only for diagnosis but also for monitoring of drug therapy in childhood visceral leishmaniasis as rapid and non-invasive techniques.

Evaluation of rapid diagnostic tests: visceral leishmaniasis

Visceral leishmaniasis (VL) is a severe infectious disease caused by a protozoan parasite: Leishmania donovani in East Africa and the Indian subcontinent and Leishmania infantum in Latin America and the Mediterranean basin. Not all leishmanial infections lead to overt clinical disease, but in those infected persons who do develop the disease, multiplication of the parasite in the reticulo-endothelial system causes prolonged fever, anaemia, hepatosplenomegaly and weight loss. VL is fatal if it is not adequately treated. The drugs currently used to treat VL can have severe side effects and the clinical presentation of VL is not sufficiently specific to guide treatment. Highly accurate (both sensitive and specific), cheap and simple rapid diagnostic tests (RDTs) are therefore crucial for case-management of VL. Early case detection followed by adequate treatment is also central to control of VL because, as yet, no vaccine is available and the long-term impact of vector control is unclear. Although the need for accurate VL diagnostics is obvious, innovation in this field has been slow. Since the 1980s, the main objective of VL diagnostics development has been to replace the direct demonstration of parasites in tissue smears, a technique that is invasive and requires considerable expertise, by a ‘field test’ that is more appropriate for use in a VL-endemic context. Several serological tests have been developed, but none are specific for VL disease as such, although they have proved useful in combination with a clinical case definition. New diagnostic tools are needed for more than just the confirmation of VL disease. No alternatives to parasitological methods are yet available to establish test of cure in treated VL patients. Clinicians do not have the tools to distinguish re-infection from relapse in cases of recurrence, and control programmes do not have validated assays for the surveillance of drug resistance in parasites. Furthermore, in the context of the VL elimination initiative, it would be desirable to have better markers of leishmanial infection at the population level. Any evaluation of a new diagnostic device should carefully identify its intended purpose. Too often developers and researchers confuse a device for the detection of leishmanial infection with a device for the confirmation of VL disease, and this is particularly the case for nucleic-acidbased assays. PCR is usually highly sensitive for detection of leishmanial infection, but this does not mean PCR will be useful for the confirmation of acute VL disease in patients in endemic areas, as many carriers of the infection in these areas will be PCRpositive without developing VL disease. This article will focus specifically on the evaluation of RDTs for confirmation of VL disease.

The economic impact of visceral leishmaniasis on households in Bangladesh

To explore current patterns of diagnosis and treatment, quantify household economic impact and identify household strategies to cover the costs of visceral leishmaniasis (VL) care in rural Bangladesh. Structured interviews with 113 VL patients from 87 households documenting all provider visits and expenditures for health care for VL, and the ways in which the expenditures were covered. Patients paid a median of 7 visits to six different providers before beginning VL treatment. All visited the subdistrict government hospital at least once. While health care, including antileishmanial drug therapy, is officially available free of charge at government facilities, 79% of patients reported making informal payments for provider access, diagnostics and drug administration; only 14% of patients received their full drug course from this source. For the 58% of patients who purchased the full treatment course, drug cost constituted 34% of direct expenditure. Median direct expenditure for one VL patient was US$87 and median income lost was $40; median total expenditure was 1.2 times annual per capita income of our study population. Households employed multiple coping strategies to cover expenditures, most commonly sale or rental of assets (62%) and taking out loans (64%). Visceral leishmaniasis treatment causes a major economic burden in affected families. Control strategies for VL should facilitate timely, affordable diagnosis and treatment of patients to decrease the infection reservoir and to alleviate the economic burden of VL on households.

T cell subset and cytokine profiles in human visceral leishmaniasis during active and asymptomatic or sub-clinical infection with Leishmania donovani

During an epidemiological study of visceral leishmaniasis (VL) in south-west Ethiopia, 33 VL patients, 9 treated VL patients, 14 individuals with sub-clinical infection, 34 individuals with asymptomatic infection, and 19 healthy controls were studied for T cell subsets and cytokine profiles. Negative leishmanin skin test, CD3 and CD4 lymphocytopenia, and significantly reduced numbers of memory CD4 + T cells were found in VL patients compared to treated VL patients or persons with self-limiting asymptomatic infection. The proportion of CD4 + and CD8 + T cells that produced IFN-γ and IL-4 after stimulation with PMA (Phorbol 12-myristate 13-acetate) and ionomycin was significantly reduced in VL patients compared to sub-clinical and asymptomatic infections or healthy controls. Plasma concentrations of IFN-γ and IL-10 were elevated in VL.

IL-10 AND IL-12 ARE THE MAIN REGULATORY CYTOKINES IN VISCERAL LEISHMANIASIS

Visceral leishmaniasis (VL) is characterized by the absence of cytokines such as IFN-γ and IL-12. Cure of VL is associated with a restoration of the ability to make these cytokines. The aim of the present study was to evaluate the role of IL-12 in the recovery of the ability to produce IFN-γ and to test whether or not IL-4 IL-10 and/or TGF-β could suppress IFN-γ production by PBMC from treated VL patients. High stimulation index (SI) of proliferation was observed in PBMC from subjects stimulated with Leishmania chagasi antigen (181±83). Neutralizing IL-12 inhibited lymphoproliferation [stimulation index (SI) of 210±114 to 1±0.6 (P<0.01)] and/or the production of IFN-γ [2792±402pg/ml to 407±449pg/ml (P<0.01)]. Recombinant IL-10 abrogated the lymphoproliferation (SI=2±3) while recombinant IL-4 or TGF-β had no effect on this response (147±22 and 194±12 respectively). IFN-γ was high when PBMCs were stimulated with L. chagasi (873±400pg/ml) and this was abrogated by the addition of IL-10 (5±2pg/ml). In contrast neither IL-4 or TGF-β suppressed IFN-γ production (837±244pg/ml and 759±523pg/ml). These results indicate that IL-12 plays an important role in the ability of treated VL patients to make IFN-γ and that IL-10 but not IL-4 or TGF-β inhibits this response.

Evaluation of the direct agglutination test (DAT) using freeze-dried antigen for the detection of anti- Leishmania antibodies in stored sera from various patient groups in Ethiopia

Tests on a panel of 421 serum samples from Ethiopia and 11 from Europe with the direct agglutination test (DAT) and freeze-dried antigen ( Leishmania donovani 1S) showed that a cut-off titre of 1:200 or 1:400 could be employed. Using a cut-off titre of 1:400, 181 (88·3%) of 205 serum samples from visceral leishmaniasis (VL) patients tested positive, as did 5 (13·9%) of 36 sera from patients with localized cutaneous leishmaniasis, 5 (71·4%) sera from 7 patients with diffuse cutaneous leishmaniasis and 8 (89·9%) sera from 9 HIV-positive VL patients. We also found that 19 (57·6%) sera from 33 treated VL patients were still positive in the DAT at 1·5 to 5 years after treatment.

Visceral leishmaniasis: use of the polymerase chain reaction in an epidemiological study in Baringo district, Kenya

The polymerase chain reaction was applied to capillary blood spots dried on filter paper from 20 parasitologically proved cases of visceral leishmaniasis (VL), 21 subclinical cases, and 11 healthy controls in a longitudinal study of anthroponotic VL in Baringo District, Kenya. Leishmania deoxyribonucleic acid (DNA) was detected 10·5 months before diagnosis and up to 3 years after diagnosis and apparently successful treatment. Subclinical cases can have detectable circulating parasite DNA in their blood. These findings may indicate that subclinical cases can be a reservoir and formerly treated VL patients can remain a reservoir for a long time. Xenodiagnosis should be performed on subclinical cases and former VL patients to establish their role in transmission of VL in Kenya.

IL-12 enhances Th1-type responses in human Leishmania donovani infections.

IL-12 is a pluripotent cytokine that interacts with NK and T cells to play a central role in the initiation and maintenance of Th1 responses and IFN-gamma production. Because of the interactive relationship between IL-12 and IFN-gamma response to infectious organisms, a study was undertaken to examine the role of IL-12 in the immune regulation of human visceral leishmaniasis (VL). Human (Hu) VL is associated with immune dysfunction and the appearance of IL-10 mRNA, not present in healed individuals. We found that PBMC from treated VL patients produced both IL-12 p40 and IFN-gamma in response to in vitro stimulation with Leishmania donovani. The production of both IL-12 p40 and IFN-gamma were interdependent and were abrogated by the addition of exogenous Hu rIL-10. In contrast, PBMC from active VL patients did not produce IL-12 p40 or IFN-gamma in response to L. donovani lysate. Neutralizing anti-IL-10 mAb led to the enhancement of IFN-gamma production by active VL PBMC cultured with L. donovani lysate, and this enhanced IFN-gamma production was blocked by anti-IL-12 mAb. The addition of exogenous Hu rIL-12 to PBMC from active VL patients resulted in the augmentation of IFN-gamma in response to L. donovani lysate. Therefore, treatment of active VL patient PBMC with anti-IL-10 or IL-12 shifted the response toward a Th1-type response with the production of IFN-gamma. These results indicate that IL-12 may play an important role in the regulation of the cellular immune responses in Hu VL.