Apolipoprotein E Is Upregulated in Blood and Circulating Monocytes of Indian Patients With Visceral Leishmaniasis.

Apolipoprotein E (ApoE) has been associated with several diseases including Parkinson's disease, Alzheimer's and multiple sclerosis. ApoE also has documented immunomodulatory functions. We investigated gene expression in circulating monocytes and in bone marrows of patients with visceral leishmaniasis (VL) living in an endemic area in Bihar, India, and contrasted these with control healthy subjects or other diagnostic bone marrows from individuals in the same region. Samples from VL patients were obtained prior to initiating treatment. Our study revealed significant upregulated expression of the apoE transcript in patients with VL. Furthermore, the levels of ApoE protein were elevated in serum samples of subjects with VL compared with healthy endemic controls. These observations may provide clues regarding the complex interactions between lipid metabolism and immunoregulation of infectious and inflammatory diseases.

Simplified molecular diagnosis of visceral leishmaniasis: Laboratory evaluation of miniature direct-on-blood PCR nucleic acid lateral flow immunoassay.

BACKGROUND: Diagnosis of visceral leishmaniasis (VL) in resource-limited endemic regions is currently based on serological testing with rK39 immunochromatographic tests (ICTs). However, rK39 ICT frequently has suboptimal diagnostic accuracy. Furthermore, treatment monitoring and detection of VL relapses is reliant on insensitive and highly invasive tissue aspirate microscopy. Miniature direct-on-blood PCR nucleic acid lateral flow immunoassay (mini-dbPCR-NALFIA) is an innovative and user-friendly molecular tool which does not require DNA extraction and uses a lateral flow strip for result read-out. This assay could be an interesting candidate for more reliable VL diagnosis and safer test of cure at the point of care. METHODOLOGY/PRINCIPLE FINDINGS: The performance of mini-dbPCR-NALFIA for diagnosis of VL in blood was assessed in a laboratory evaluation and compared with the accuracy of rK39 ICTs Kalazar Detect in Spain and IT LEISH in East Africa. Limit of detection of mini-dbPCR-NALFIA was 650 and 500 parasites per mL of blood for Leishmania donovani and Leishmania infantum, respectively. In 146 blood samples from VL-suspected patients from Spain, mini-dbPCR-NALFIA had a sensitivity of 95.8% and specificity 97.2%, while Kalazar Detect had a sensitivity of 71.2% and specificity of 94.5%, compared to a nested PCR reference. For a sample set from 58 VL patients, 10 malaria patients and 68 healthy controls from Ethiopia and Kenya, mini-dbPCR-NALFIA had a pooled sensitivity of 87.9% and pooled specificity of 100% using quantitative PCR as reference standard. IT LEISH sensitivity and specificity in the East African samples were 87.9% and 97.4%, respectively. CONCLUSIONS/SIGNIFICANCE: Mini-dbPCR-NALFIA is a promising tool for simplified molecular diagnosis of VL and follow-up of treated patients in blood samples. Future studies should evaluate its use in endemic, resource-limited settings, where mini-dbPCR-NALFIA may provide an accurate and versatile alternative to rK39 ICTs and aspirate microscopy.

Diversity, Leishmania detection, and blood meal sources of sand flies from Iguatama, Minas Gerais, Brazil.

This study investigated the sand fly fauna of the municipality Iguatama, in the Midwest Region of Minas Gerais state, Brazil, including Leishmania infection rates and blood meal sources. Sand flies were collected during four periods over the course of a single year, encompassing both dry and rainy seasons, using CDC light traps placed in peridomiciles where dogs were seropositive for visceral leishmaniasis (VL). A total of 762 sand fly specimens, representing 12 species across seven genera, were collected. Lutzomyia longipalpis was the most abundant species, comprising 57.6% of the collected specimens, followed by Nyssomyia neivai (19.6%) and Nyssomyia whitmani (10.5%). Species richness and diversity varied among collection periods, with the highest diversity observed in January 2019. Molecular analysis detected Leishmania DNA in 12.5% of the sand fly specimens, with Le. infantum being the predominant species. Blood meal analysis revealed feeding on multiple vertebrate species, including humans, rats, dogs, and chickens. The presence of Leishmania DNA in sand flies, and the identification of human blood meals, highlight the potential role of these species in VL transmission. These findings underscore the importance of continued surveillance and control measures to prevent the spread of VL and reduce transmission risk in the region.

Persistent T cell unresponsiveness associated with chronic visceral leishmaniasis in HIV-coinfected patients.

A large proportion of HIV-coinfected visceral leishmaniasis (VL-HIV) patients exhibit chronic disease with frequent VL recurrence. However, knowledge on immunological determinants underlying the disease course is scarce. We longitudinally profiled the circulatory cellular immunity of an Ethiopian HIV cohort that included VL developers. We show that chronic VL-HIV patients exhibit high and persistent levels of TIGIT and PD-1 on CD8+/CD8- T cells, in addition to a lower frequency of IFN-γ+ TIGIT- CD8+/CD8- T cells, suggestive of impaired T cell functionality. At single T cell transcriptome and clonal resolution, the patients show CD4+ T cell anergy, characterised by a lack of T cell activation and lymphoproliferative response. These findings suggest that PD-1 and TIGIT play a pivotal role in VL-HIV chronicity, and may be further explored for patient risk stratification. Our findings provide a strong rationale for adjunctive immunotherapy for the treatment of chronic VL-HIV patients to break the recurrent disease cycle.

Impaired signaling pathways on Berardinelli-Seip congenital lipodystrophy macrophages during Leishmania infantum infection.

Berardinelli-Seip congenital lipodystrophy (CGL), a rare autosomal recessive disorder, is characterized by a lack of adipose tissue. Infections are one of the major causes of CGL individuals' premature death. The mechanisms that predispose to infections are poorly understood. We used Leishmania infantum as an in vitro model of intracellular infection to explore mechanisms underlying the CGL infection processes, and to understand the impact of host mutations on Leishmania survival, since this pathogen enters macrophages through specialized membrane lipid domains. The transcriptomic profiles of both uninfected and infected monocyte-derived macrophages (MDMs) from CGL (types 1 and 2) and controls were studied. MDMs infected with L. infantum showed significantly downregulated expression of genes associated with infection-response pathways (MHC-I, TCR-CD3, and granzymes). There was a transcriptomic signature in CGL cells associated with impaired membrane trafficking and signaling in response to infection, with concomitant changes in the expression of membrane-associated genes in parasites (e.g. δ-amastins). We identified pathways suggesting the lipid storage dysfunction led to changes in phospholipids expression and impaired responses to infection, including immune synapse (antigen presentation, IFN-γ signaling, JAK/STAT); endocytosis; NF-kappaB signaling; and phosphatidylinositol biosynthesis. In summary, lipid metabolism of the host plays an important role in determining antigen presentation pathways.

Meteorological, demographic, and environmental risk factors for Leishmania infantum in wildlife.

Leishmania infantum is an important and neglected vector-borne zoonotic protozoa endemic in the Mediterranean basin. Several wild and domestic mammals can contribute to maintaining its circulation but their importance as effective reservoirs is still under discussion and varies depending on local ecological communities. By combining environmental, climatic, and individual information, this study assessed the presence of L. infantum DNA in a set of wild species from Northwestern Italy and the potential ecological factors related to the risk of infection. From 2020 to 2022, 304 free-ranging wild animals were analyzed for the detection of L. infantum DNA in the spleen and popliteal lymph node (when available). The prevalence obtained in wild boar (Sus scrofa) and roe deer (Capreolus capreolus) was higher than those previously reported (% ± confidence interval 95%; 42.9 ± 18.4% and 27 ± 6.6% in wild boar and roe deer, respectively), and this is the first report of this parasite infecting the coypu Myocastor coypus (60 ± 34.7%). L. infantum DNA was detected in all the seasons including those free of adult sandflies and seasonal differences were minimal, suggesting a long course of infection. The models revealed that animals from rainy areas with higher greenness during the summer, highly populated by humans and predominantly covered by water surfaces had a higher risk of L. infantum. This study contributes to confirming previous findings on the existence of a sylvatic cycle for L. infantum in certain regions of Italy, as well as on the potential epidemiological role of roe deer for this parasite given the elevated prevalence found.

Leishmania donovani mitogen-activated protein kinases as a host-parasite interaction interface.

Leishmaniasis, a major globally re-emerging neglected tropical disease, has a restricted repertoire of chemotherapeutic options due to a narrow therapeutic index, drug resistance, or patient non-compliance due to toxicity. The disease is caused by the parasite Leishmania that resides in two different forms in two different environments: as sessile intracellular amastigotes within mammalian macrophages and as motile promastigotes in sandfly gut. As mitogen-activated protein kinases (MAPKs) play important roles in cellular differentiation and survival, we studied the expression of Leishmania donovani MAPKs (LdMAPKs). The homology studies by multiple sequence alignment show that excepting LdMAPK1 and LdMAPK2, all thirteen other LdMAPKs share homology with human ERK and p38 isoforms. Expression of LdMAPK4 and LdMAPK5 is less in avirulent promastigotes and amastigotes. Compared to miltefosine-sensitive L. donovani parasites, miltefosine-resistant parasites have higher LdMAPK1, LdMAPK3-5, LdMAPK7-11, LdMAPK13, and LdMAPK14 expression. IL-4-treatment of macrophages down-regulated LdMAPK11, in virulent amastigotes whereas up-regulated LdMAPK5, but down-regulated LdMAPK6, LdMAPK12-15, expression in avirulent amastigotes. IL-4 up-regulated LdMAPK1 expression in both virulent and avirulent amastigotes. IFN-γ-treatment down-regulated LdMAPK6, LdMAPK13, and LdMAPK15 in avirulent amastigotes but up-regulated in virulent amastigotes. This complex profile of LdMAPKs expression among virulent and avirulent parasites, drug-resistant parasites, and in amastigotes within IL-4 or IFN-γ-treated macrophages suggests that LdMAPKs are differentially controlled at the host-parasite interface regulating parasite survival and differentiation, and in the course of IL-4 or IFN-γ dominated immune response.

Long-term hematopoietic stem cells trigger quiescence in Leishmania parasites.

Addressing the challenges of quiescence and post-treatment relapse is of utmost importance in the microbiology field. This study shows that Leishmania infantum and L. donovani parasites rapidly enter into quiescence after an estimated 2-3 divisions in both human and mouse bone marrow stem cells. Interestingly, this behavior is not observed in macrophages, which are the primary host cells of the Leishmania parasite. Transcriptional comparison of the quiescent and non-quiescent metabolic states confirmed the overall decrease of gene expression as a hallmark of quiescence. Quiescent amastigotes display a reduced size and signs of a rapid evolutionary adaptation response with genetic alterations. Our study provides further evidence that this quiescent state significantly enhances resistance to treatment. Moreover, transitioning through quiescence is highly compatible with sand fly transmission and increases the potential of parasites to infect cells. Collectively, this work identified stem cells in the bone marrow as a niche where Leishmania quiescence occurs, with important implications for antiparasitic treatment and acquisition of virulence traits.

Transcriptional signatures in human macrophage-like cells infected by Leishmania infantum, Leishmania major and Leishmania tropica.

BACKGROUND: In the Mediterranean basin, three Leishmania species have been identified: L. infantum, L. major and L. tropica, causing zoonotic visceral leishmaniasis (VL), zoonotic cutaneous leishmaniasis (CL) and anthroponotic CL, respectively. Despite animal models and genomic/transcriptomic studies provided important insights, the pathogenic determinants modulating the development of VL and CL are still poorly understood. This work aimed to identify host transcriptional signatures shared by cells infected with L. infantum, L. major, and L. tropica, as well as specific transcriptional signatures elicited by parasites causing VL (i.e., L. infantum) and parasites involved in CL (i.e., L. major, L. tropica). METHODOLOGY/PRINCIPAL FINDINGS: U937 cells differentiated into macrophage-like cells were infected with L. infantum, L. major and L. tropica for 24h and 48h, and total RNA was extracted. RNA sequencing, performed on an Illumina NovaSeq 6000 platform, was used to evaluate the transcriptional signatures of infected cells with respect to non-infected cells at both time points. The EdgeR package was used to identify differentially expressed genes (fold change > 2 and FDR-adjusted p-values < 0.05). Then, functional enrichment analysis was employed to identify the enriched ontology terms in which these genes are involved. At 24h post-infection, a common signature of 463 dysregulated genes shared among all infection conditions was recognized, while at 48h post-infection the common signature was reduced to 120 genes. Aside from a common transcriptional response, we evidenced different upregulated functional pathways characterizing L. infantum-infected cells, such as VEGFA-VEGFR2 and NFE2L2-related pathways, indicating vascular remodeling and reduction of oxidative stress as potentially important factors for visceralization. CONCLUSIONS: The identification of pathways elicited by parasites causing VL or CL could lead to new therapeutic strategies for leishmaniasis, combining the canonical anti-leishmania compounds with host-directed therapy.

Phenotypic and functional characteristics of monocyte subsets in the blood and bone marrow of Indian subjects with Visceral Leishmaniasis.

Visceral leishmaniasis (VL) is a potentially fatal parasitic infection caused by Leishmania donovani in India. L. donovani is an obligate intracellular protozoan residing mostly in macrophages of the reticuloendothelial system throughout chronic infection. Monocytic phagocytes are critical in the pathogenesis of different forms of leishmaniasis. Subsets of monocytes are distinguished by their surface markers into CD14+CD16- classical monocytes, CD14+CD16+ intermediate monocytes, and CD16++CD14low non-classical monocyte subsets. During cutaneous leishmaniasis (CL), intermediate monocyte are reported to be a source of inflammatory cytokines IL-1ß and TNF, and they express CCR2 attracting them to sites of inflammatory pathology. We examined monocyte subsets in the blood and bone marrow of patients with VL from an endemic site in Bihar, India, and found these contrasted with the roles of monocytes in CL. During VL, intermediate and non-classical CD16+ monocyte subsets expressed instead a non-inflammatory phenotype with low CCR2, high CX3CR1 and low microbicidal oxidant generation, making them more similar to patrolling monocytes than inflammatory cells. Bone marrow CD16+ monocyte subsets expressed a phenotype that might be more similar to the inflammatory subsets of CL, although our inability to obtain bone marrow from healthy donors in the endemic region hampered this interpretation Overall the data suggest that CD16+ intermediate monocyte subsets in VL patients express a phenotypes that contributes to an immunosuppressed pathologic immune state, but in contrast to CL, these do not mediate localized inflammatory responses.

A panel of recombinant Leishmania donovani cell surface and secreted proteins identifies LdBPK_323600.1 as a serological marker of symptomatic infection.

Visceral leishmaniasis is a deadly infectious disease and is one of the world's major neglected health problems. Because the symptoms of infection are similar to other endemic diseases, accurate diagnosis is crucial for appropriate treatment. Definitive diagnosis using splenic or bone marrow aspirates is highly invasive, and so, serological assays are preferred, including the direct agglutination test (DAT) or rK39 strip test. These tests, however, are either difficult to perform in the field (DAT) or lack specificity in some endemic regions (rK39), making the development of new tests a research priority. The availability of Leishmania spp. genomes presents an opportunity to identify new diagnostic targets. Here, we use genome data and a mammalian protein expression system to create a panel of 93 proteins consisting of the extracellular ectodomains of the Leishmania donovani cell surface and secreted proteins. We use these panel and sera from murine experimental infection models and natural human and canine infections to identify new candidates for serological diagnosis. We observed a concordance between the most immunoreactive antigens in different host species and transmission settings. The antigen encoded by the LdBPK_323600.1 gene can diagnose Leishmania infections with high sensitivity and specificity in patient cohorts from different endemic regions including Bangladesh and Ethiopia. In longitudinal sampling of treated patients, we observed reductions in immunoreactivity to LdBPK_323600.1 suggesting it could be used to diagnose treatment success. In summary, we have identified new antigens that could contribute to improved serological diagnostic tests to help control the impact of this deadly tropical infectious disease. IMPORTANCE: Visceral leishmaniasis is fatal if left untreated with patients often displaying mild and non-specific symptoms during the early stages of infection making accurate diagnosis important. Current methods for diagnosis require highly trained medical staff to perform highly invasive biopsies of the liver or bone marrow which pose risks to the patient. Less invasive molecular tests are available but can suffer from regional variations in their ability to accurately diagnose an infection. To identify new diagnostic markers of visceral leishmaniasis, we produced and tested a panel of 93 proteins identified from the genome of the parasite responsible for this disease. We found that the pattern of host antibody reactivity to these proteins was broadly consistent across naturally acquired infections in both human patients and dogs, as well as experimental rodent infections. We identified a new protein called LdBPK_323600.1 that could accurately diagnose visceral leishmaniasis infections in humans.

Leishmania blood parasite dynamics during and after treatment of visceral leishmaniasis in Eastern Africa: A pharmacokinetic-pharmacodynamic model.

BACKGROUND: With the current treatment options for visceral leishmaniasis (VL), recrudescence of the parasite is seen in a proportion of patients. Understanding parasite dynamics is crucial to improving treatment efficacy and predicting patient relapse in cases of VL. This study aimed to characterize the kinetics of circulating Leishmania parasites in the blood, during and after different antileishmanial therapies, and to find predictors for clinical relapse of disease. METHODS: Data from three clinical trials, in which Eastern African VL patients received various antileishmanial regimens, were combined in this study. Leishmania kinetoplast DNA was quantified in whole blood with real-time quantitative PCR (qPCR) before, during, and up to six months after treatment. An integrated population pharmacokinetic-pharmacodynamic model was developed using non-linear mixed effects modelling. RESULTS: Parasite proliferation was best described by an exponential growth model, with an in vivo parasite doubling time of 7.8 days (RSE 12%). Parasite killing by fexinidazole, liposomal amphotericin B, sodium stibogluconate, and miltefosine was best described by linear models directly relating drug concentrations to the parasite elimination rate. After treatment, parasite growth was assumed to be suppressed by the host immune system, described by an Emax model driven by the time after treatment. No predictors for the high variability in onset and magnitude of the immune response could be identified. Model-based individual predictions of blood parasite load on Day 28 and Day 56 after start of treatment were predictive for clinical relapse of disease. CONCLUSION: This semi-mechanistic pharmacokinetic-pharmacodynamic model adequately captured the blood parasite dynamics during and after treatment, and revealed that high blood parasite loads on Day 28 and Day 56 after start of treatment are an early indication for VL relapse, which could be a useful biomarker to assess treatment efficacy of a treatment regimen in a clinical trial setting.

Challenges of animals shelters in caring for dogs infected with Leishmania and other pathogens.

The incidence of human Visceral Leishmaniasis (VL) has decreased in Brazil; however, the number of areas reporting human and canine cases has increased, with Leishmania infantum usually preceding human infection. This study aimed to analyze the profile of infectious diseases that are endemic for both human and canine VL, in dogs housed in a shelter located in the state of Rio Grande do Norte, Northeast Brazil. Data was obtained between November/2021 to April/2022. All dogs residing at the shelter (98 dogs) were examined and blood was collected for testing for L. infantum, Ehrlichia canis, and Babesia sp. Statistical analyses considered the clinical and laboratory findings. Of the 98 animals, approximately 43% were positive for L. infantum antibodies, 19% were positive for L. infantum kDNA, and 18% were L. infantum positive by culture. Greater levels of anti-leishmania antibodies were observed in dogs with symptoms suggestive of VL. The dogs tested positive for E. canis (19/98) and B. canis (18/98). Lutzomyia longipalpis was captured inside the shelter, representing 74.25% (n = 225) of whole sandflies in the dog shelter. Concomitant infection by L. infantum and E. canis increased the odds of death. Treatment of VL included the use of allopurinol (n = 48) and miltefosine (n = 8). Treated animals showed more signs of Leishmania infection. Tickborn parasites and Leishmania were prevalent in sheltered dogs in a VL-endemic area, which increases the odds of death and poses an additional challenge for caring for abandoned dogs and at the same time setting protocols to manage reservoirs of L. infantum.

FVB/NJ strain as a mouse model for cutaneous leishmaniasis by Leishmania (L.) amazonensis.

BACKGROUND: Leishmaniases encompass a spectrum of neglected diseases caused by parasites of the genus Leishmania, grouped in two forms: tegumentary and visceral leishmaniasis. OBJECTIVES: In this study, we propose Friend Virus B NIH Jackson (FVB/NJ) mouse strain as a new experimental model of infection with Leishmania (Leishmania) amazonensis, the second most prevalent agent of tegumentary leishmaniasis in Brazil. METHODS AND FINDINGS: We performed in vitro infections of FVB/NJ macrophages and compared them with BALB/c macrophages, showing that BALB/c cells have higher infection percentages and a higher number of amastigotes/cell. Phagocytosis assays indicated that BALB/c and FVB/NJ macrophages have similar capacity to uptake parasites after 5 min incubations. We also investigated promastigotes' resistance to sera from FVB/NJ and BALB/c and observed no difference between the two sera, even though FVB/NJ has a deficiency in complement components. Finally, we subcutaneously infected FVB/NJ and BALB/c mice with 2 × 106 parasites expressing luciferase. Analysis of lesion development for 12 weeks showed that FVB/NJ and BALB/c mice have similar lesion profiles and parasite burdens. MAIN CONCLUSIONS: This work characterises for the first time the FVB/NJ mouse as a new model for tegumentary leishmaniasis caused by Leishmania (L.) amazonensis.

Monitoring the Long-Term Effectiveness of Miltefosine in Indian Post-Kala-Azar Dermal Leishmaniasis.

Post-kala-azar dermal leishmaniasis (PKDL), the dermal sequel to visceral leishmaniasis (VL), is characterized by hypopigmented macules (macular) and/or papules and nodules (polymorphic). Post-kala-azar dermal leishmaniasis plays a significant role in disease transmission, emphasizing the need for monitoring chemotherapeutic effectiveness. Accordingly, this study aimed to quantify the parasite burden in PKDL patients after treatment with miltefosine by a quantitative polymerase chain reaction (qPCR). A Leishmania kinetoplastid gene-targeted qPCR was undertaken using DNA from skin biopsy specimens of patients with PKDL at three time points, i.e., at disease presentation (week 0, n = 157, group 1), upon completion of treatment (week 12, n = 39, group 2), and at any time point 6 months after completion of treatment (week ≥36, n = 54, group 3). A cycle threshold (Ct) <30 was considered the cutoff for positivity, and load was quantified as the number of parasites/µg genomic DNA (gDNA); cure was considered when samples had a Ct >30. The parasite load at disease presentation (group 1) was 10,769 (1,339-80,441)/µg gDNA (median [interquartile range]). In groups 2 and 3, qPCR results were negative in 35/39 cases (89.7%) and 48/54 cases (88.8%), respectively. In the 10/93 (10.8%) qPCR-positive cases, the parasite burdens in groups 2 and 3 were 2,420 (1,205-5,661)/µg gDNA and 22,195 (5,524-100,106)/µg gDNA, respectively. Serial monitoring was undertaken in 45 randomly selected cases that had completed treatment; all cases in groups 2 or 3 had a Ct >30, indicating cure. Overall, qPCR confirmed an 89.2% cure (as 83/93 cases showed parasite clearance), and the persistent qPCR positivity was attributed to nonadherence to treatment or unresponsiveness to miltefosine and remains to be investigated.

Peptide-triggered IL-12 and IFN-γ mediated immune response in CD4+ T-cells against Leishmania donovani infection.

Leishmania donovani are intracellular, human blood parasites that cause visceral leishmaniasis or kala-azar. Cell-penetrating peptides (CPPs) have been shown to modulate intracellular processes and cargo delivery, whereas host defense peptides (HDPs) promote proliferation of both naïve and antigen activated CD4+ T-cells. We report newly designed tripeptides that were able to trigger proinflammatory cytokine (IL-12 and IFN-γ) secretion by CD4+CD44+ T-cells in response to Leishmania donovani infection. These peptides can be used to induce antigen specific TH1 responses to combat obstacles of cytotoxicity and drug resistance associated with current anti-leishmanial drugs. Furthermore, these peptides can also be used as adjuvants to develop an effective immunoprophylactic approach for immunity restoration against visceral leishmaniasis.

In vitro growth of Leishmania parasites from biopsy samples of suspected cutaneous and visceral leishmaniasis cases in Sri Lanka: An observational study.

Sri Lanka reports a large focus of Leishmania donovani caused cutaneous leishmaniasis (CL). Subsequent emergence of visceral leishmaniasis (VL) was also reported recently. Expansion of the on-going disease outbreak and many complexities indicate urgent need to enhance early case detection methods. In vitro cultivation (IVC) of parasites causing visceral leishmaniasis (VL) is important for disease confirmation and to obtain sufficient quantities of parasites required in many scientific studies. IVC is carried out as a useful second line investigation for direct microscopy negative patients with CL in this setting. Along with the emergence of VL, current study was carried out to evaluate in vitro growth of local VL parasites and to identify their differences associated with in vitro growth characteristics. Routine parasitological diagnostic methods, i.e., light microscopy (LM), polymerase chain reaction (PCR) were used for confirmation of suspected cases. Lesion samples from 125 suspected CL cases and bone marrow or splenic aspirations from 125 suspected VL patients were used to inoculate IVCs. Media M199 (about 70 µl) supplemented with 15-20% of heat inactivated fetal bovine serum was used for initial culturing procedures in capillaries. Capillary cultures were monitored daily. Total of 44 different compositions/conditions were used for evaluating in vitro growth of VL causing parasite. Daily records on parasite counts, morphological appearance (size, shape, and wriggly movements) were maintained. In vitro transformation of Leishmania promastigotes to amastigotes and outcome of the attempts on recovery of live Leishmania from culture stabilates was also compared between CL and VL parasites. Proportion of cultures showing a transformation of promastigotes were 40/45 (88.9%) and 4/10 (40.0%) for CL and VL respectively. In the transformed cultures, parasites showing typical shape, size and movement patterns were less in VL (1/4, 25.0%) compared to CL (28/40, 70.0%). CL cultures showed a growth up to mass culturing level with mean duration of two weeks while it was about five weeks for VL cultures. Proportion of cultures that reached a parasite density of 1 × 106 cells/ml (proceeded to mass cultures) was significantly low in VL (4/10, 40%) as compared to CL (28/40, 70.0%). None of media compositions/conditions were successful for mass culturing of VL parasites while all of them were shown to be useful for growing CL strains. Also in vitro transformation to amastigote form and recovering of culture stabilates were not successful compared to CL. There were clear differences between in vitro growth of Leishmania parasites causing local CL and VL. Further studies are recommended for optimization of in vitro culturing of VL parasite which will be invaluable to enhance case detection in future.

Myotubularin-related protein 6 is an ion channel-associated pro-leishmanial phosphatase.

Residing obligatorily as amastigotes within the mammalian macrophages, the parasite Leishmania donovani inflicts the potentially fatal, globally re-emerging disease visceral leishmaniasis (VL) by altering intracellular signaling through kinases and phosphatases. Because the phosphatases that modulate the VL outcome in humans remained unknown, we screened a human phosphatase siRNA-library for anti-leishmanial functions in THP-1, a human macrophage-like cell line. Of the 251 phosphatases, the screen identified the Ca++-activated K+-channel-associated phosphatase myotubularin-related protein-6 (MTMR6) as the only phosphatase whose silencing reduced parasite load and IL-10 production in human macrophages. Virulent, but not avirulent, L. donovani infection increased MTMR6 expression in macrophages. As virulent L. donovani parasites expressed higher lipophosphoglycan, a TLR2-ligand, we tested the effect of TLR2 stimulation or blockade on MTMR6 expression. TLR1/TLR2-ligand Pam3CSK4 enhanced, but TLR2 blockade reduced, MTMR6 expression. L. donovani infection of macrophages ex vivo increased, but miltefosine treatment reduced, MTMR6 expression. Corroboratively, compared to endemic controls, untreated VL patients had higher, but miltefosine-treated VL patients had reduced, MTMR6 expression. The phosphatase siRNA-library screening thus identified MTMR6 as the first TLR2-modulated ion channel-associated phosphatase with significant implications in VL patients and anti-leishmanial functions.

Within-host bayesian joint modeling of longitudinal and time-to-event data of Leishmania infection.

The host immune system plays a significant role in managing and clearing pathogen material during an infection, but this complex process presents numerous challenges from a modeling perspective. There are many mathematical and statistical models for these kinds of processes that take into account a wide range of events that happen within the host. In this work, we present a Bayesian joint model of longitudinal and time-to-event data of Leishmania infection that considers the interplay between key drivers of the disease process: pathogen load, antibody level, and disease. The longitudinal model also considers approximate inflammatory and regulatory immune factors. In addition to measuring antibody levels produced by the immune system, we adapt data from CD4+ and CD8+ T cell proliferation, and expression of interleukin 10, interferon-gamma, and programmed cell death 1 as inflammatory or regulatory factors mediating the disease process. The model is developed using data collected from a cohort of dogs naturally exposed to Leishmania infantum. The cohort was chosen to start with healthy infected animals, and this is the majority of the data. The model also characterizes the relationship features of the longitudinal outcomes and time-to-death due to progressive Leishmania infection. In addition to describing the mechanisms causing disease progression and impacting the risk of death, we also present the model's ability to predict individual trajectories of Canine Leishmaniosis (CanL) progression. The within-host model structure we present here provides a way forward to address vital research questions regarding the understanding of the progression of complex chronic diseases such as Visceral Leishmaniasis, a parasitic disease causing significant morbidity worldwide.

Upregulation of ATP6V0D2 benefits intracellular survival of Leishmania donovani in erythrocytes-engulfing macrophages.

Visceral leishmaniasis (VL) is the most severe type of leishmaniasis which is caused by infection of Leishmania donovani complex. In the BALB/c mouse model of VL, multinucleated giant cells (MGCs) with heavy parasite infection consist of the largest population of hemophagocytes in the spleen of L. donovani-infected mice, indicating that MGCs provide the parasites a circumstance beneficial for their survival. Although ATP6V0D2 is a demonstrated factor inducing the formation of hemophagocytic MGCs during L. donovani infection, functions of this protein in shaping the infection outcome in macrophages remain unclear. Here we evaluated the influence of upregulated ATP6V0D2 on intracellular survival of the parasites. L. donovani infection-induced hemophagocytosis of normal erythrocytes by macrophages was suppressed by RNAi-based knockdown of Atp6v0d2. The knockdown of Atp6v0d2 did not improve the survival of amastigotes within macrophages when the cells were cultured in the absence of erythrocytes. On the other hand, reduced intracellular survival of amastigotes in macrophages by the knockdown was observed when macrophages were supplemented with antibody-opsonized erythrocytes before infection. There, increase in cytosolic labile iron pool was observed in the L. donovani-infected knocked-down macrophages. It suggests that ATP6V0D2 plays roles not only in upregulation of hemophagocytosis but also in iron trafficking within L. donovani-infected macrophages. Superior access to iron in macrophages may be how the upregulated expression of the molecule brings benefit to Leishmania for their intracellular survival in the presence of erythrocytes.