Significantly Lower Anti-Leishmania IgG Responses in Sudanese versus Indian Visceral Leishmaniasis

Background

Visceral leishmaniasis (VL), a widely distributed systemic disease caused by infection with the Leishmania donovani complex (L. donovani and L. infantum), is almost always fatal if symptomatic and untreated. A rapid point-of-care diagnostic test for anti-Leishmania antibodies, the rK39-immunochromatographic test (rK39-ICT), has high sensitivity and specificity in South Asia but is less sensitive in East Africa. One of the underlying reasons may be continent-specific molecular diversity in the rK39 antigen within the L. donovani complex. However, a second reason may be differences in specific IgG anti-Leishmania levels in patients from different geographical regions, either due to variable antigenicity or immunological response.

Methodology/Principal Findings

We determined IgG titres of Indian and Sudanese VL patients against whole cell lysates of Indian and Sudanese L. donovani strains. Indian VL patients had significantly higher IgG titres against both L. donovani strains compared to Sudanese VL patients (p<0.0001). Mean reciprocal log₁₀ 50% end-point titres (1/log₁₀t₅₀) were i) 3.80 and 3.88 for Indian plasma and ii) 2.13 and 2.09 for Sudanese plasma against Indian and Sudanese antigen respectively (p<0.0001). Overall, the Indian VL patients therefore showed a 46.8–61.7 -fold higher mean ELISA titre than the Sudanese VL patients. The higher IgG titres occurred in children (<16 years old) and adults of either sex from India (mean 1/log₁₀t₅₀: 3.60–4.15) versus Sudan (mean 1/log₁₀t₅₀: 1.88–2.54). The greatest difference in IgG responses was between male Indian and Sudanese VL patients of ≥ 16 years old (mean 1/log₁₀t₅₀: 4.15 versus 1.99 = 144-fold (p<0.0001).

Conclusions/Significance

Anti-Leishmania IgG responses among VL patients in Sudan were significantly lower than in India; this may be due to chronic malnutrition with Zn²⁺ deficiency, or variable antigenicity and capacity to generate IgG responses to Leishmania antigens. Such differential anti-Leishmania IgG levels may contribute to lower sensitivity of the rK39-ICT in East Africa.     

Comparison of Visceral Leishmaniasis Diagnostic Antigens in African and Asian Leishmania donovani Reveals Extensive Diversity and Region-specific Polymorphisms

Background

Visceral leishmaniasis (VL), caused by infection with Leishmania donovani complex, remains a major public health problem in endemic regions of South Asia, East Africa, and Brazil. If untreated, symptomatic VL is usually fatal. Rapid field diagnosis relies principally on demonstration of anti-Leishmania antibodies in clinically suspect cases. The rK39 immunochromatographic rapid diagnostic test (RDT) is based on rK39, encoded by a fragment of a kinesin-related gene derived from a Brazilian L. chagasi, now recognised as L. infantum, originating from Europe. Despite its reliability in South Asia, the rK39 test is reported to have lower sensitivity in East Africa. A reason for this differential response may reside in the molecular diversity of the rK39 homologous sequences among East African L. donovani strains.

Methodology/Principal Findings

Coding sequences of rK39 homologues from East African L. donovani strains were amplified from genomic DNA, analysed for diversity from the rK39 sequence, and compared to South Asian sequences. East African sequences were revealed to display significant diversity from rK39. Most coding changes in the 5′ half of repeats were non-conservative, with multiple substitutions involving charge changes, whereas amino acid substitutions in the 3′ half of repeats were conservative. Specific polymorphisms were found between South Asian and East African strains. Diversity of HASPB1 and HASPB2 gene repeat sequences, used to flank sequences of a kinesin homologue in the synthetic antigen rK28 designed to reduce variable RDT performance, was also investigated. Non-canonical combination repeat arrangements were revealed for HASPB1 and HASPB2 gene products in strains producing unpredicted size amplicons.

Conclusions/Significance

We demonstrate that there is extensive kinesin genetic diversity among strains in East Africa and between East Africa and South Asia, with ample scope for influencing performance of rK39 diagnostic assays. We also show the importance of targeted comparative genomics in guiding optimisation of recombinant/synthetic diagnostic antigens.     

Low Parasite Load Estimated by qPCR in a Cohort of Children Living in Urban Area Endemic for Visceral Leishmaniasis in Brazil

Background

An important issue associated with the control of visceral leishmaniasis is the need to identify and understand the relevance of asymptomatic infection caused by Leishmania infantum. The aim of this study was to follow the course of asymptomatic L. infantum infection in children in an area of Brazil where it is endemic. The children were assessed twice during a 12-month period.

Methodology

In this population study, 1875 children, ranging from 6 months to 7 years of age, were assessed. Blood samples were collected on filter papers via finger prick and tested by ELISA (L. infantum soluble antigen and rk39). Seropositives samples (n = 317) and a number of seronegatives samples (n = 242) were subjected to qPCR. After 12 months, blood samples were collected from a subgroup of 199 children and tested for Leishmania spp. to follow the course of infection.

Principal Findings

At baseline qPCR testing identified 82 positive samples. The prevalence rate, as estimated for 1875 children based on the qPCR results, was 13.9%. The qPCR testing of whole blood samples collected from a cohort of children after 12 months (n = 199) yielded the following results: of the 44 (22.1%) children with positive qPCR results at baseline, only 10 (5.0%) remained positive, and 34 (17.1%) became negative; and of the 155 (77.9%) children with negative qPCR results, 131 (65.8%) remained negative, and 24 (12.1%) became positive at the follow-up measurement. The samples with positive findings at baseline (n = 82) had a mean of 56.5 parasites/mL of blood; and at follow-up the mean positive result was 7.8 parasites/mL.

Conclusions

The peripheral blood of asymptomatic children had a low and fluctuating quantity of Leishmania DNA and a significant decrease in parasitemia at 1-year follow-up. Quantitative PCR enables adequate monitoring of Leishmania infection.     

High-throughput analysis of synthetic peptides for the immunodiagnosis of canine visceral leishmaniasis

Background

Visceral leishmaniasis is the most severe form of leishmaniasis. Approximately 20% of zoonotic human visceral leishmaniasis worldwide is caused by Leishmania infantum, which is also known as Leishmania chagasi in Latin America, and disease incidence is increasing in urban and peri-urban areas of the tropics. In this form of disease, dogs are the main reservoirs. Diagnostic methods used to identify Leishmania infected animals are not able to detect all of the infected ones, which can compromise the effectiveness of disease control. Therefore, to contribute to the improvement of diagnostic methods for canine visceral leishmaniasis (CVL), we aimed to identify and test novel antigens using high-throughput analysis.

Methodology/Principal Findings

Immunodominant proteins from L. infantum were mapped in silico to predict B cell epitopes, and the 360 predicted peptides were synthesized on cellulose membranes. Immunoassays were used to select the most reactive peptides, which were then investigated with canine sera. Next, the 10 most reactive peptides were synthesized using solid phase peptide synthesis protocol and tested using ELISA. The sensitivity and specificity of these peptides were also compared to the EIE-LVC Bio-Manguinhos kit, which is recommended by the Brazilian Ministry of Health for use in leishmaniasis control programs. The sensitivity and specificity of the selected synthesized peptides was as high as 88.70% and 95.00%, respectively, whereas the EIE-LVC kit had a sensitivity of 13.08% and 100.00% of specificity. Although the tests based on synthetic peptides were able to diagnose up to 94.80% of asymptomatic dogs with leishmaniasis, the EIE-LVC kit failed to detect the disease in any of the infected asymptomatic dogs.

Conclusions/Significance

Our study shows that ELISA using synthetic peptides is a technique with great potential for diagnosing CVL; furthermore, the use of these peptides in other diagnostic methodologies, such as immunochromatographic tests, could be beneficial to CVL control programs.     

Development of a Fluorescent Based Immunosensor for the Serodiagnosis of Canine Leishmaniasis Combining Immunomagnetic Separation and Flow Cytometry

Background

An accurate diagnosis is essential for the control of infectious diseases. In the search for effective and efficient tests, biosensors have increasingly been exploited for the development of new and highly sensitive diagnostic methods. Here, we describe a new fluorescent based immunosensor comprising magnetic polymer microspheres coated with recombinant antigens to improve the detection of specific antibodies generated during an infectious disease. As a challenging model, we used canine leishmaniasis due to the unsatisfactory sensitivity associated with the detection of infection in asymptomatic animals where the levels of pathogen-specific antibodies are scarce.

Methodology

Ni-NTA magnetic microspheres with 1,7 µm and 8,07 µm were coated with the Leishmania recombinant proteins LicTXNPx and rK39, respectively. A mixture of equal proportions of both recombinant protein-coated microspheres was used to recognize and specifically bind anti-rK39 and anti-LicTNXPx antibodies present in serum samples of infected dogs. The microspheres were recovered by magnetic separation and the percentage of fluorescent positive microspheres was quantified by flow cytometry.

Principal Findings

A clinical evaluation carried out with 129 dog serum samples using the antigen combination demonstrated a sensitivity of 98,8% with a specificity of 94,4%. rK39 antigen alone demonstrated a higher sensitivity for symptomatic dogs (96,9%), while LicTXNPx antigen showed a higher sensitivity for asymptomatic (94,4%).

Conclusions

Overall, our results demonstrated the potential of a magnetic microsphere associated flow cytometry methodology as a viable tool for highly sensitive laboratorial serodiagnosis of both clinical and subclinical forms of canine leishmaniasis.     

Canine antibody response to Phlebotomus perniciosus bites negatively correlates with the risk of Leishmania infantum transmission

Background

Phlebotomine sand flies are blood-sucking insects that can transmit Leishmania parasites. Hosts bitten by sand flies develop an immune response against sand fly salivary antigens. Specific anti-saliva IgG indicate the exposure to the vector and may also help to estimate the risk of Leishmania spp. transmission. In this study, we examined the canine antibody response against the saliva of Phlebotomus perniciosus, the main vector of Leishmania infantum in the Mediterranean Basin, and characterized salivary antigens of this sand fly species.

Methodology/Principal Findings

Sera of dogs bitten by P. perniciosus under experimental conditions and dogs naturally exposed to sand flies in a L. infantum focus were tested by ELISA for the presence of anti-P. perniciosus antibodies. Antibody levels positively correlated with the number of blood-fed P. perniciosus females. In naturally exposed dogs the increase of specific IgG, IgG1 and IgG2 was observed during sand fly season. Importantly, Leishmania-positive dogs revealed significantly lower anti-P. perniciosus IgG2 compared to Leishmania-negative ones. Major P. perniciosus antigens were identified by western blot and mass spectrometry as yellow proteins, apyrases and antigen 5-related proteins.

Conclusions

Results suggest that monitoring canine antibody response to sand fly saliva in endemic foci could estimate the risk of L. infantum transmission. It may also help to control canine leishmaniasis by evaluating the effectiveness of anti-vector campaigns. Data from the field study where dogs from the Italian focus of L. infantum were naturally exposed to P. perniciosus bites indicates that the levels of anti-P. perniciosus saliva IgG2 negatively correlate with the risk of Leishmania transmission. Thus, specific IgG2 response is suggested as a risk marker of L. infantum transmission for dogs.     

rKLO8, a Novel Leishmania donovani – Derived Recombinant Immunodominant Protein for Sensitive Detection of Visceral Leishmaniasis in Sudan

Background

For effective control of visceral leishmaniasis (VL) in East Africa, new rapid diagnostic tests are required to replace current tests with low sensitivity. The aim of this study is to improve diagnosis of VL in East Africa by testing a new antigen from an autochthonous L. donovani strain in Sudan.

Methodology and Principle Findings

We cloned, expressed and purified a novel recombinant protein antigen of L. donovani from Sudan, designated rKLO8, that contains putative conserved domains with significant similarity to the immunodominant kinesin proteins of Leishmania. rKLO8 exhibited 93% and 88% amino acid identity with cloned kinesin proteins of L. infantum (synonymous L. chagasi) (K39) and L. donovani (KE16), respectively. We evaluated the diagnostic efficiency of the recombinant protein in ELISA for specific detection of VL patients from Sudan. Data were compared with a rK39 ELISA and two commercial kits, the rK39 strip test and the direct agglutination test (DAT). Of 106 parasitologically confirmed VL sera, 104 (98.1%) were tested positive by rKLO8 as compared to 102 (96.2%) by rK39. Importantly, the patients'' sera showed increased reactivity with rKLO8 than rK39. Specificity was 96.1% and 94.8% for rKLO8- and rK39 ELISAs, respectively. DAT showed 100% specificity and 94.3% sensitivity while rK39 strip test performed with 81.1% sensitivity and 98.7% specificity.

Conclusion

The increased reactivity of Sudanese VL sera with the rKLO8 makes this antigen a potential candidate for diagnosis of visceral leishmaniasis in Sudan. However, the suitability at the field level will depend on its performance in a rapid test format.     

Canine skin and conjunctival swab samples for the detection and quantification of Leishmania infantum DNA in an endemic urban area in Brazil

Background

We evaluated kDNA PCR/hybridization and quantitative real-time PCR (qPCR) targeting the gene of DNA polymerase of Leishmania infantum for CVL diagnosis and assessment of parasite load in clinical samples obtained invasively and non-invasively.

Methodology/Principal Findings

Eighty naturally infected dogs from an endemic urban area in Brazil were used. Animals were divided into two groups based on the presence or absence of CVL clinical sings. Skin biopsies, bone marrow, blood and conjunctival swabs samples were collected and submitted to L. infantum DNA detection. In addition, anti-Leishmania antibody titers were measured by Immunofluorescence antibody test. The symptomatic dogs had increased titers compared to asymptomatic dogs (P = 0.025). The frequencies of positive results obtained by kDNA PCR/hybridization for asymptomatic and symptomatic dogs, respectively, were as follows: right conjunctiva, 77.5% and 95.0%; left conjunctiva, 75.0% and 87.5%; skin, 45.0% and 75.0%; bone marrow, 50.0% and 77.5%; and blood, 27.5% and 22.5%. In both groups, the parasite load in the skin samples was the highest (P<0.0001). The parasite loads in the conjunctival swab and bone marrow samples were statistically equivalent within each group. The parasite burden in conjunctival swabs was higher in the dogs with clinical signs than in asymptomatic dogs (P = 0.028). This same relationship was also observed in the bone marrow samples (P = 0.002). No differences in amastigotes load in the skin were detected between the groups.

Conclusions

The conjunctival swab is a suitable clinical sample for qualitative molecular diagnosis of CVL. The highest parasite burdens were detected in skin regardless of the presence of VL-associated clinical signs. The qPCR results emphasized the role of dogs, particularly asymptomatic dogs, as reservoirs for CVL because of the high cutaneous parasite loads. These results may help to explain the maintenance of high transmission rates and numbers of CVL cases in endemic urban regions.     

Leishmania infantum Amastigotes Enhance HIV-1 Production in Cocultures of Human Dendritic Cells and CD4+ T Cells by Inducing Secretion of IL-6 and TNF-α

Background

Visceral leishmaniasis has emerged as an important opportunistic disease among patients infected with HIV-1. Both HIV-1 and the protozoan parasite Leishmania can productively infect cells of the macrophage-dendritic cell lineage.

Methodology/Principal Findings

Here we demonstrate that Leishmania infantum amastigotes increase HIV-1 production when human primary dendritic cells (DCs) are cocultured together with autologous CD4⁺ T cells. Interestingly, the promastigote form of the parasite does not modulate virus replication. Moreover, we report that amastigotes promote virus replication in both cell types. Our results indicate that this process is due to secretion of parasite-induced soluble factors by DCs. Luminex micro-beads array system analyses indicate that Leishmania infantum amastigotes induce a higher secretion of several cytokines (i.e. IL-1α, IL-2, IL-6, IL-10 and TNF-α) and chemokines (i.e. MIP-1α, MIP-1β and RANTES) in these cells. Studies conducted with pentoxifylline and neutralizing antibodies revealed that the Leishmania-dependent augmentation in HIV-1 replication is due to a higher secretion of IL-6 and TNF-α.

Conclusions/Significance

Altogether these findings suggest that the presence of Leishmania within DC/T-cell conjugates leads to an enhancement of virus production and demonstrate that HIV-1 and Leishmania can establish complex interactions in such a cellular microenvironment.     

Design,Development and Evaluation of rK28-Based Point-of-Care Tests for Improving Rapid Diagnosis of Visceral Leishmaniasis

Background

Visceral leishmaniasis (VL) is diagnosed by microscopic confirmation of the parasite in bone marrow, spleen or lymph node aspirates. These procedures are unsuitable for rapid diagnosis of VL in field settings. The development of rK39-based rapid diagnostic tests (RDT) revolutionized diagnosis of VL by offering high sensitivity and specificity in detecting disease in the Indian subcontinent; however, these tests have been less reliable in the African subcontinent (sensitivity range of 75–85%, specificity of 70–92%). We have addressed limitations of the rK39 with a new synthetic polyprotein, rK28, followed by development and evaluation of two new rK28-based RDT prototype platforms.

Methodology/Principal Findings

Evaluation of 62 VL-confirmed sera from Sudan provided sensitivities of 96.8% and 93.6% (95% CI = K28: 88.83–99.61%; K39: 84.30–98.21%) and specificities of 96.2% and 92.4% (95% CI = K28: 90.53–98.95%; K39: 85.54–96.65%) for rK28 and rK39, respectively. Of greater interest was the observation that individual VL sera with low rK39 reactivity often had much higher rK28 reactivity. This characteristic of the fusion protein was exploited in the development of rK28 rapid tests, which may prove to be crucial in detecting VL among patients with low rK39 antibody levels. Evaluation of two prototype lateral flow-based rK28 rapid tests on 53 VL patients in Sudan and 73 VL patients in Bangladesh provided promisingly high sensitivities (95.9% [95% CI = 88.46–99.1 in Sudan and 98.1% [95% CI = 89.93–99.95%] in Bangladesh) compared to the rK39 RDT (sensitivities of 86.3% [95% CI = 76.25–93.23%] in Sudan and 88.7% [95% CI = 76.97–95.73%] in Bangladesh).

Conclusions/Significance

Our study compares the diagnostic accuracy of rK39 and rK28 in detecting active VL cases and our findings indicate that rK28 polyprotein has great potential as a serodiagnostic tool. A new rK28-based RDT will prove to be a valuable asset in simplifying VL disease confirmation at the point-of-care.     

Diagnostic Accuracy of the Leishmania OligoC-TesT and NASBA-Oligochromatography for Diagnosis of Leishmaniasis in Sudan

Background

The Leishmania OligoC-TesT and NASBA-Oligochromatography (OC) were recently developed for simplified and standardised molecular detection of Leishmania parasites in clinical specimens. We here present the phase II evaluation of both tests for diagnosis of visceral leishmaniasis (VL), cutaneous leishmaniasis (CL) and post kala-azar dermal leishmaniasis (PKDL) in Sudan.

Methodology

The diagnostic accuracy of the tests was evaluated on 90 confirmed and 90 suspected VL cases, 7 confirmed and 8 suspected CL cases, 2 confirmed PKDL cases and 50 healthy endemic controls from Gedarif state and Khartoum state in Sudan.

Principal Findings

The OligoC-TesT as well as the NASBA-OC showed a sensitivity of 96.8% (95% CI: 83.8%–99.4%) on lymph node aspirates and of 96.2% (95% CI: 89.4%–98.7%) on blood from the confirmed VL cases. The sensitivity on bone marrow was 96.9% (95% CI: 89.3%–99.1%) and 95.3% (95% CI: 87.1%–98.4%) for the OligoC-TesT and NASBA-OC, respectively. All confirmed CL and PKDL cases were positive with both tests. On the suspected VL cases, we observed a positive OligoC-TesT and NASBA-OC result in 37.1% (95% CI: 23.2%–53.7%) and 34.3% (95% CI: 20.8%–50.9%) on lymph, in 72.7% (95% CI: 55.8%–84.9%) and 63.6% (95% CI: 46.6%–77.8%) on bone marrow and in 76.9% (95% CI: 49.7%–91.8%) and 69.2% (95% CI: 42.4%–87.3%) on blood. Seven out of 8 CL suspected cases were positive with both tests. The specificity on the healthy endemic controls was 90% (95% CI: 78.6%–95.7%) for the OligoC-TesT and 100% (95% CI: 92.9%–100.0%) for the NASBA-OC test.

Conclusions

Both tests showed high sensitivity on lymph, blood and tissue scrapings for diagnosis of VL, CL and PKDL in Sudan, but the specificity for clinical VL was significantly higher with NASBA-OC.     

Sphingolipid Degradation in Leishmania (Leishmania) amazonensis

Background

Human leishmaniasis is caused by more than 20 Leishmania species and has a wide range of symptoms. Our recent studies have demonstrated the essential role of sphingolipid degradation in the virulence of Leishmania (Leishmania) major, a species responsible for localized cutaneous leishmaniasis in the Old World. In this study, we investigated the function of sphingolipid degradation in Leishmania (Leishmania) amazonensis, an etiological agent of localized and diffuse cutaneous leishmaniasis in South America.

Methodology/Principal Findings

First, we identified the enzyme LaISCL which is responsible for sphingolipid degradation in L. amazonensis. Primarily localized in the mitochondrion, LaISCL shows increased expression as promastigotes progress from replicative log phase to non-replicative stationary phase. To study its function, null mutants of LaISCL (Laiscl⁻) were generated by targeted gene deletion and complemented through episomal gene add-back. In culture, loss of LaISCL leads to hypersensitivity to acidic pH and poor survival in murine macrophages. In animals, Laiscl⁻ mutants exhibit severely attenuated virulence towards C57BL6 mice but are fully infective towards BALB/c mice. This is drastically different from wild type L. amazonensis which cause severe pathology in both BALB/c and C57BL 6 mice.

Conclusions/Significance

A single enzyme LaISCL is responsible for the turnover of sphingolipids in L. amazonensis. LaISCL exhibits similar expression profile and biochemical property as its ortholog in L. major. Deletion of LaISCL reduces the virulence of L. amazonensis and the outcome of Laiscl⁻-infection is highly dependent on the host''s genetic background. Therefore, compared to L. major, the role of sphingolipid degradation in virulence is substantially different in L. amazonensis. Future studies may reveal whether sphingolipid degradation is required for L. amazonensis to cause diffuse cutaneous infections in humans.     

Gene expression profiling and molecular characterization of antimony resistance in Leishmania amazonensis

Background

Drug resistance is a major problem in leishmaniasis chemotherapy. RNA expression profiling using DNA microarrays is a suitable approach to study simultaneous events leading to a drug-resistance phenotype. Genomic analysis has been performed primarily with Old World Leishmania species and here we investigate molecular alterations in antimony resistance in the New World species L. amazonensis.

Methods/Principal Findings

We selected populations of L. amazonensis promastigotes for resistance to antimony by step-wise drug pressure. Gene expression of highly resistant mutants was studied using DNA microarrays. RNA expression profiling of antimony-resistant L. amazonensis revealed the overexpression of genes involved in drug resistance including the ABC transporter MRPA and several genes related to thiol metabolism. The MRPA overexpression was validated by quantitative real-time RT-PCR and further analysis revealed that this increased expression was correlated to gene amplification as part of extrachromosomal linear amplicons in some mutants and as part of supernumerary chromosomes in other mutants. The expression of several other genes encoding hypothetical proteins but also nucleobase and glucose transporter encoding genes were found to be modulated.

Conclusions/Significance

Mechanisms classically found in Old World antimony resistant Leishmania were also highlighted in New World antimony-resistant L. amazonensis. These studies were useful to the identification of resistance molecular markers.     

Neutrophils reduce the parasite burden in Leishmania (Leishmania) amazonensis-infected macrophages

Background

Studies on the role of neutrophils in Leishmania infection were mainly performed with L. (L) major, whereas less information is available for L. (L) amazonensis. Previous results from our laboratory showed a large infiltrate of neutrophils in the site of infection in a mouse strain resistant to L. (L.) amazonensis (C3H/HePas). In contrast, the susceptible strain (BALB/c) displayed a predominance of macrophages harboring a high number of amastigotes and very few neutrophils. These findings led us to investigate the interaction of inflammatory neutrophils with L. (L.) amazonensis-infected macrophages in vitro.

Methodology/Principal Findings

Mouse peritoneal macrophages infected with L. (L.) amazonensis were co-cultured with inflammatory neutrophils, and after four days, the infection was quantified microscopically. Data are representative of three experiments with similar results. The main findings were 1) intracellular parasites were efficiently destroyed in the co-cultures; 2) the leishmanicidal effect was similar when cells were obtained from mouse strains resistant (C3H/HePas) or susceptible (BALB/c) to L. (L.) amazonensis; 3) parasite destruction did not require contact between infected macrophages and neutrophils; 4) tumor necrosis factor alpha (TNF-α), neutrophil elastase and platelet activating factor (PAF) were involved with the leishmanicidal activity, and 5) destruction of the parasites did not depend on generation of oxygen or nitrogen radicals, indicating that parasite clearance did not involve the classical pathway of macrophage activation by TNF-α, as reported for other Leishmania species.

Conclusions/Significance

The present results provide evidence that neutrophils in concert with macrophages play a previously unrecognized leishmanicidal effect on L. (L.) amazonensis. We believe these findings may help to understand the mechanisms involved in innate immunity in cutaneous infection by this Leishmania species.     

UCP2 Deficiency Helps to Restrict the Pathogenesis of Experimental Cutaneous and Visceral Leishmaniosis in Mice

Background

Uncoupling protein 2 (UCP2) is a mitochondrial transporter that has been shown to lower the production of reactive oxygen species (ROS). Intracellular pathogens such as Leishmania upregulate UCP2 and thereby suppress ROS production in infected host tissues, allowing the multiplication of parasites within murine phagocytes. This makes host UCP2 and ROS production potential targets in the development of antileishmanial therapies. Here we explore how UCP2 affects the outcome of cutaneous leishmaniosis (CL) and visceral leishmaniosis (VL) in wild-type (WT) C57BL/6 mice and in C57BL/6 mice lacking the UCP2 gene (UCP2KO).

Methodology and Findings

To investigate the effects of host UCP2 deficiency on Leishmania infection, we evaluated parasite loads and cytokine production in target organs. Parasite loads were significantly lower in infected UCP2KO mice than in infected WT mice. We also found that UCP2KO mice produced significantly more interferon-γ (IFN-γ), IL-17 and IL-13 than WT mice (P<0.05), suggesting that UCP2KO mice are resistant to Leishmania infection.

Conclusions

In this way, UCP2KO mice were better able than their WT counterparts to overcome L. major and L. infantum infections. These findings suggest that upregulating host ROS levels, perhaps by inhibiting UPC2, may be an effective approach to preventing leishmaniosis.     

Improved canine and human visceral leishmaniasis immunodiagnosis using combinations of synthetic peptides in enzyme-linked immunosorbent assay

Background

Zoonotic visceral leishmaniasis (VL) is a severe infectious disease caused by protozoan parasites of the genus Leishmania and the domestic dogs are the main urban parasite reservoir hosts. In Brazil, indirect fluorescence antibody tests (IFAT) and indirect enzyme linked immunosorbent assay (ELISA) using promastigote extracts are widely used in epidemiological surveys. However, their sensitivity and specificity have often been compromised by the use of complex mixtures of antigens, which reduces their accuracy allowing the maintenance of infected animals that favors transmission to humans. In this context, the use of combinations of defined peptides appears favorable. Therefore, they were tested by combinations of five peptides derived from the previously described Leishmania diagnostic antigens A2, NH, LACK and K39.

Methodology/Principal Findings

Combinations of peptides derived A2, NH, LACK and K39 antigens were used in ELISA with sera from 44 human patients and 106 dogs. Improved sensitivities and specificities, close to 100%, were obtained for both sera of patients and dogs. Moreover, high sensitivity and specificity were observed even for canine sera presenting low IFAT anti-Leishmania antibody titers or from asymptomatic animals.

Conclusions/Significance

The use of combinations of B cell predicted synthetic peptides derived from antigens A2, NH, LACK and K39 may provide an alternative for improved sensitivities and specificities for immunodiagnostic assays of VL.     

A genomic-based approach combining in vivo selection in mice to identify a novel virulence gene in Leishmania

Background

Infection with Leishmania results in a broad spectrum of pathologies where L. infantum and L. donovani cause fatal visceral leishmaniasis and L. major causes destructive cutaneous lesions. The identification and characterization of Leishmania virulence genes may define the genetic basis for these different pathologies.

Methods and Findings

Comparison of the recently completed L. major and L. infantum genomes revealed a relatively small number of genes that are absent or present as pseudogenes in L. major and potentially encode proteins in L. infantum. To investigate the potential role of genetic differences between species in visceral infection, seven genes initially classified as absent in L. major but present in L. infantum were cloned from the closely related L. donovani genome and introduced into L. major. The transgenic L. major expressing the L. donovani genes were then introduced into BALB/c mice to select for parasites with increased virulence in the spleen to determine whether any of the L. donovani genes increased visceral infection levels. During the course of these experiments, one of the selected genes (LinJ32_V3.1040 (Li1040)) was reclassified as also present in the L. major genome. Interestingly, only the Li1040 gene significantly increased visceral infection in the L. major transfectants. The Li1040 gene encodes a protein containing a putative component of an endosomal protein sorting complex involved with protein transport.

Conclusions

These observations demonstrate that the levels of expression and sequence variations in genes ubiquitously shared between Leishmania species have the potential to significantly influence virulence and tissue tropism.     

IgG1 as a Potential Biomarker of Post-chemotherapeutic Relapse in Visceral Leishmaniasis,and Adaptation to a Rapid Diagnostic Test

Background

Visceral leishmaniasis (VL), caused by protozoa of the Leishmania donovani complex, is a widespread parasitic disease of great public health importance; without effective chemotherapy symptomatic VL is usually fatal. Distinction of asymptomatic carriage from progressive disease and the prediction of relapse following treatment are hampered by the lack of prognostic biomarkers for use at point of care.

Methodology/Principal Findings

All IgG subclass and IgG isotype antibody levels were determined using unpaired serum samples from Indian and Sudanese patients with differing clinical status of VL, which included pre-treatment active VL, post-treatment cured, post-treatment relapsed, and post kala-azar dermal leishmaniasis (PKDL), as well as seropositive (DAT and/or rK39) endemic healthy controls (EHCs) and seronegative EHCs. L. donovani antigen-specific IgG1 levels were significantly elevated in relapsed versus cured VL patients (p<0.0001). Using paired Indian VL sera, consistent with the known IgG1 half-life, IgG1 levels had not decreased significantly at day 30 after the start of treatment (p = 0.8304), but were dramatically decreased by 6 months compared to day 0 (p = 0.0032) or day 15 (p<0.0001) after start of treatment. Similarly, Sudanese sera taken soon after treatment did not show a significant change in the IgG1 levels (p = 0.3939). Two prototype lateral flow immunochromatographic rapid diagnostic tests (RDTs) were developed to detect IgG1 levels following VL treatment: more than 80% of the relapsed VL patients were IgG1 positive; at least 80% of the cured VL patients were IgG1 negative (p<0.0001).

Conclusions/Significance

Six months after treatment of active VL, elevated levels of specific IgG1 were associated with treatment failure and relapse, whereas no IgG1 or low levels were detected in cured VL patients. A lateral flow RDT was successfully developed to detect anti-Leishmania IgG1 as a potential biomarker of post-chemotherapeutic relapse.