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Background: Visceral Leishmaniasis (VL) is a neglected tropical disease (NTD) with the highest regional burden in East Africa. Relapse and Post Kala-azar Dermal Leishmaniasis (PKDL) contribute to the spread of VL in endemic areas, making their surveillance imperative for control and elimination. Little is known about long-term patient outcomes in Kenya through follow-up after VL treatment, despite its requirement for control and elimination by the World Health Organization (WHO) and the Kenya Ministry of Health (KMOH). Methodology/Principal findings: 36 follow-up patients in Tiaty East and West, sub-counties, Kenya, and records from 248 patients at the regional Chemolingot Sub-county Hospital (CSCH) were analyzed separately using Fisher's Exact Tests, two-sample t-tests, and Welch's t-tests in R (Version 4.3.0). The study found a prevalence rate of 88.89% (n = 32) final cure, 5.56% (n = 2) relapse, and 5.56% (n = 2) PKDL in follow-up patients and 92.74% (n = 230) initial cure, 6.86% (n = 17) relapse, and 0.80% (n = 2) PKDL in overall CSCH patients. The mean lengths of time at which follow-up patients relapsed and developed PKDL were 4.5 and 17 months, respectively. Young age (p = 0.04, 95% CI 0.63–24.31), shorter length of time from initial treatment to follow-up (p = 0.002, 95% CI 1.03-∞), lower Hb level at primary treatment (p = 0.0002, 95% CI 1.23–3.24), and living in Tiaty East sub-county (p = 0.04, 95% CI 0.00–1.43) were significantly associated (p<0.05) with VL relapse in follow-up study patients. Female sex (p = 0.04, 95% CI 0.84-∞) and living in Tiaty East sub-county (p = 0.03, 95% CI 0.00–1.43) were significantly associated with PKDL in follow-up study patients. Conclusions/Significance: More research should be done on PKDL in Kenya with active follow-up to understand its true burden. These results on prevalence and risk factors for PKDL and relapse in Kenya should inform knowledge of patient outcomes and interventions in the region. [ABSTRACT FROM AUTHOR]

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. Author summary: Visceral leishmaniasis (VL) is a severe infectious disease caused by unicellular parasites of the Leishmania donovani complex. As VL is fatal when left untreated, early and confirmatory laboratory diagnosis is crucial for adequate patient management. However, currently available serological assays, such as the rK39 rapid diagnostic test, fall short in terms of sensitivity and cannot be used for detection of relapsing infections. Therefore, we evaluated an innovative and user-friendly diagnostic assay called mini-dbPCR-NALFIA, which is based on the direct detection of Leishmania kinetoplast DNA in human blood samples. We found that mini-dbPCR-NALFIA can detect down to 500 parasites per millilitre of blood, which is lower than the parasite loads generally seen in untreated VL patients. Furthermore, we assessed the accuracy of mini-dbPCR-NALFIA using a set of Spanish and East-African samples from VL patients and control groups. In the Spanish samples, sensitivity of mini-dbPCR-NALFIA was very high and somewhat better than in the East-African samples. The specificity of mini-dbPCR-NALFIA was excellent in both sample sets. In conclusion, considering its robust performance and simplicity, the mini-dbPCR-NALFIA is a promising diagnostic assay that has the potential to improve VL diagnosis and follow-up of treated patients, especially in resource-limited situations. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis (VL, kala azar), caused by Leishmania donovani, transmitted by Phlebotomus orientalis, is a serious systemic disease that causes high morbidity and mortality rates in Sudan and other parts of East Africa and the world. Despite progress in understanding the epidemiology of the disease in East Africa, little is known about the host preference of P. orientalis in kala azar endemic villages of Sudan, which have some of the highest VL incidence rates in the world. The present study used host choice experiments and blood‐meal identification approaches to determine the host preference of P. orientalis in kala azar endemic villages in Gedarif state, eastern Sudan. In the host choice experiment, tent traps were used to compare the attractiveness of cows, donkeys, sheep and goats for host‐seeking P. orientalis. In the blood‐meal identification study, blood‐fed P. orientalis females, captured inside houses and peri‐domestic habitats, were subjected to molecular typing using cytochrome b gene (cyt b) amplification and sequence analysis. Cows and donkeys were the most attractive to blood‐seeking P. orientalis, followed by goats. Similarly, the blood‐meal analysis of P. orientalis showed that the vector preferentially feeds on cows, followed by donkeys, humans and goats. The human blood index of P. orientalis was 19.4% (42/216), indicating a high zoophilic habit of the vector, both inside and outside the houses. Although the order of host preference varied by location, it was clear that cows are the most preferred host of P. orientalis in the area. Results are discussed in relation to the role of domestic/livestock animals in VL zoopotentiation and zooprophylaxis. Inference is made on the potential impact of insecticide treatment of cows in control of the vector and the transmission of VL in Sudan and other parts of East Africa. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis (VL) is caused by Leishmania donovani (Ld), and most cases occur in Brazil, East Africa, and India. The treatment for VL is limited and has many adverse effects. The development of safer and more efficacious drugs is urgently needed. Drug repurposing is one of the best processes to repurpose existing drugs. Ornithine decarboxylase (ODC) is an important target against L. donovani in the polyamine biosynthesis pathway. In this study, we have modeled the 3D structure of ODC and performed high‐throughput virtual screening of 8630 ZINC database ligands against Leishmania donovani ornithine decarboxylase (Ld ODC), selecting 45 ligands based on their high binding score. It is further validated through molecular docking simulation and the selection of the top two lead molecules (ceftaroline fosamil and rimegepant) for Molecular Dynamics (MD) simulation, Density functional theory (DFT), and molecular mechanics generalized born surface area (MMGBSA) analysis. The results showed that the binding affinities of ceftaroline fosamil, and rimegepant are, respectively, −10.719 and 10.159 kcal/mol. The docking complexes of the two lead compounds, ceftaroline fosamil, and rimegepant, with the target ODC, were found stable during molecular dynamics simulations. Furthermore, the analysis of MMGBSA revealed that these compounds had a high binding free energy. The DFT analysis showed that the top lead molecules were more reactive than the standard drug (pentamidine). In‐silico findings demonstrated that ceftaroline fosamil, and rimegepant might be recognized as potent antagonists against ODC for the treatment of VL. [ABSTRACT FROM AUTHOR]

Background: In visceral leishmaniasis (VL) patients coinfected with human immunodeficiency virus (HIV), combination therapy (liposomal amphotericin B infusion and oral miltefosine) is being considered as an alternative to liposomal amphotericin B monotherapy. We aimed to assess the views of stakeholders in relation to these treatment options. Methodology: In a mixed methods study, we surveyed and interviewed patients, government functionaries, programme managers, health service providers, nongovernmental organizations, researchers, and World Health Organization (WHO) personnel. We used the Evidence to Decision (EtD) framework for data collection planning and analysis. Constructs of interest included valuation of outcomes, impact on equity, feasibility and acceptability of the treatment options, implementation considerations, monitoring and evaluation, and research priorities. Principal findings/Conclusion: Mortality and non-serious adverse events were rated as "critical" by respectively the highest (61%) and lowest percentages (47%) of survey participants. Participants viewed clinical cure as essential for patients to regain productivity. Non-patient stakeholders emphasized the importance of "sustained" clinical cure. For most survey participants, combination therapy, compared with monotherapy, would increase health equity (40%), and be more acceptable (79%) and feasible (57%). Interviews revealed that combination therapy was more feasible and acceptable than monotherapy when associated with a shorter duration of hospitalization. The findings of the interviews provided insight into those of the survey. When choosing between alternative options, providers should consider the outcomes that matter to patients as well as the impact on equity, feasibility, and acceptability of the options. Author summary: In East Africa and South Asia, the number of patients with visceral leishmaniasis (VL) co-infected with human immunodeficiency virus (HIV) has been increasing over the years. In addition to independently posing major health challenges, the two conditions have detrimental effects on each other. In light of new evidence on treatment regimens for this patient population, the World Health Organization (WHO) recently updated its evidence-based region-specific treatment recommendations. To inform this process, we assessed the views of stakeholders on the outcomes of interest and on medication treatment options. The mixed methods study consisted of an online survey and semi-structured interviews. Outcomes of mortality, complications, clinical cure, relapse and serious adverse events were viewed as important because of the burden associated with their experience, their consequences, and the co-infection status of the patients. Outcomes such as non-serious adverse events were viewed as less important for reasons relating to patient tolerability and ease of treatment. As compared to monotherapy, combination therapy was found to be more equitable, acceptable, and feasible. While our findings are important to consider by clinicians treating patients with VL patients coinfected with HIV, they also inform decisions made by other stakeholders such as guideline developers and program managers. [ABSTRACT FROM AUTHOR]

Background: Neutrophils are involved in the initial host responses to pathogens. Neutrophils can activate T cell responses either independently or through indirect involvement of Dendritic cells (DCs). Recently we have demonstrated direct neutrophil-T cell interactions that initiate adaptive immune responses following immunization with live attenuated Leishmania donovani centrin deleted parasite vaccine (LdCen-/-). However, neutrophil-DC interactions in T cell priming in vaccine immunity in general are not known. In this study we evaluated the interaction between neutrophils and DCs during LdCen-/- infection and compared with wild type parasite (LdWT) both in vitro and in vivo. Methodology/findings: LdCen-/- parasite induced increased expression of CCL3 in neutrophils caused higher recruitment of DCs capable of inducing a strong proinflammatory response and elevated co-stimulatory molecule expression compared to LdWT infection. To further illustrate neutrophil-DCs interactions in vivo, we infected LYS-eGFP mice with red fluorescent LdWT/LdCen-/- parasites and sort selected DCs that engulfed the neutrophil containing parasites or DCs that acquired the parasites directly in the ear draining lymph nodes (dLN) 5d post infection. The DCs predominantly acquired the parasites by phagocytosing infected neutrophils. Specifically, DCs containing LdCen-/- parasitized neutrophils exhibited a proinflammatory phenotype, increased expression of costimulatory molecules and initiated higher CD4+T cell priming ex-vivo. Notably, potent DC activation occurred when LdCen-/- parasites were acquired indirectly via engulfment of parasitized neutrophils compared to direct engulfment of LdCen-/- parasites by DCs. Neutrophil depletion in LdCen-/- infected mice significantly abrogated expression of CCL3 resulting in decreased DC recruitment in ear dLN. This event led to poor CD4+Th1 cell priming ex vivo that correlated with attenuated Tbet expression in ear dLN derived CD4+ T cells in vivo. Conclusions: Collectively, LdCen-/- containing neutrophils phagocytized by DC markedly influence the phenotype and antigen presenting capacity of DCs early on and thus play an immune-regulatory role in shaping vaccine induced host protective response. Author summary: Visceral Leishmaniasis (VL), caused by the protozoan parasites of the genus Leishmania is a neglected tropical disease. Leishmania donovani is the principal causative agent of VL in East Africa and the Indian subcontinent whereas in Europe, North Africa, and Latin America VL is mainly caused by Leishmania infantum. No licensed vaccine exists against VL. We have reported previously that live attenuated centrin gene-deleted L. donovani (LdCen-/-) parasite vaccine induced strong innate immunity which leads to a protective Th1 response in animal models. We recently demonstrated that neutrophils play an indispensable role following immunization with LdCen-/- parasites in inducing protective Th1 immune response. However, neutrophils also secrete chemokines that attract other innate cells such as dendritic cells and regulate their activities. In the current study we analyzed the interplay between neutrophils and DCs, and its effects on T cell activation during LdCen-/- infection and compared with wild type parasite (LdWT) infection. We observed that higher recruitment of DCs occurred in LdCen-/- infected mice ear draining lymph nodes compared to LdWT. This recruitment is facilitated by increased secretion of the chemokine CCL3 by neutrophils. A markedly decreased DC recruitment was observed in LdCen-/- infected mice following CCL3 neutralization indicating the key role of neutrophils in DC recruitment. Further, we demonstrated that DCs that ingest LdCen-/- infected neutrophils are better activated than those that acquire the parasites independent of neutrophils. Notably neutrophil depletion in LdCen-/- infected mice also attenuated activation of DCs in the ear dLN that resulted in poor CD4+T cell priming. Our results reveal that interaction between neutrophils and DCs play an important role in shaping proinflammatory immune response induced by a live attenuated Leishmania vaccine. [ABSTRACT FROM AUTHOR]

Background: The assessment of chemotherapeutic responses in Post Kala-azar Dermal Leishmaniasis (PKDL), especially its macular form is challenging, emphasizing the necessity for 'test of cure' tools. This study explored the diagnostic and prognostic potential of IgG subclasses and associated cytokines for monitoring the effectiveness of chemotherapy in PKDL. Methods: Participants included PKDL cases at (a) disease presentation, (b) immediately at the end of treatment (12 weeks for Miltefosine or 3 weeks for Liposomal Amphotericin B, LAmB and (c) at any time point 6 months later, for estimating anti-leishmanial immunoglobulin (Ig, IgG, IgM, IgG1, IgG2 and IgG3) and cytokines (IL-10, IL-6). Results: In PKDL, Ig levels were elevated, with IgG3 and IL-10 being the major contributors. Miltefosine decreased both markers substantially and this decrease was sustained for at least six months. In contrast, LAmB failed to decrease IgG3 and IL-10, as even after six months, their levels remained unchanged or even increased. Conclusions: In PKDL, IgG3 and IL-10 proved to be effective predictors of responsiveness to chemotherapy and may be considered as a non invasive alternative for longitudinal monitoring. Author summary: Post Kala-azar Dermal Leishmaniasis (PKDL) is a dermal condition that occurs in East Africa and South Asia, the latter in 5–10% of patients after apparent cure from Visceral Leishmaniasis (VL). Till date, conventional knowledge in South Asia was that the polymorphic form of PKDL comprising of macules, papules and nodules was the predominant disease form, constituting 85–90%. However, since 2014, implementation of active-case surveillance led to unearthing of a large number of macular, hypopigmented cases, and was reported to contribute to nearly 50% of the disease burden. In particular, the macular form poses a diagnostic dilemma as microscopically parasites are difficult to identify in their lesions, and repigmentation occurs months after parasite clearance, emphasizing the need for developing non-invasive approaches for measurement of parasite burden. Till date, no formal clinical trial for treatment of PKDL has been undertaken where the parasite load was quantified and treatment remains empirical. This is primarily due to PKDL cases being unwilling to provide a repeat skin biopsy once their lesions have declined. Therefore, in cases where treatment failure occurs, it cannot be precisely identified, and could potentially lead to these cases becoming mobile disease reservoirs, thereby adversely impacting on the ongoing VL elimination programme. This study addressed this critical lacuna, where it was established that in both clinical types of PKDL, circulating levels of IgG3 and IL-10 can be considered as effective markers for monitoring treatment outcome. At disease presentation, the raised levels of IgG subclasses and associated cytokines (IL-10 and IL-6) declined following therapy with Miltefosine, the maximum decrease being with IgG3 along with IL-10; importantly, this decrease was sustained for at least six months. In contrast, LAmB failed to decrease the levels of immunoglobulins and associated cytokines even six months after completion of treatment; in fact the antibody levels either increased or remained unchanged. Taken together, this study has established the potential of IgG3 and IL10 as a non-invasive alternative for monitoring of chemotherapeutic responses in PKDL. [ABSTRACT FROM AUTHOR]