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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-1β 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. Author summary: The parasite Leishmania donovani causes the fatal systemic disease visceral leishmaniasis (VL) in endemic nations including India. The parasite is an obligate intracellular protozoan, residing mostly in monocytic cells in the bone marrow, blood and affected organs of infected human or other vertebrate hosts. Cells of the monocytic lineage can either provide a safe intracellular location in which the parasite survives, or they can be activated to kill intracellular parasites. We studied the contribution of monocyte subsets in the blood and bone marrow of patients from India with active visceral leishmaniasis, or control subjects without leishmaniasis. Our studies suggested that circulating monocytes of patients with VL are defective in their hability to kill intracellular parasites and to migrate toward sites of inflammation. These defects are most prominent in expanded subsets of monocytes expressing both the CD16 and CD14 receptors on their surface membranes. The immune defects underlying VL may be greatly influenced by subsets of blood monocytes that expand during disease. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Visceral Leishmaniasis (VL) is endemic in South Sudan, manifesting periodically in major outbreaks. Provision of treatment during endemic periods and as an emergency response is impeded by instability and conflict. Médecins Sans Frontières (MSF) has provided health care in South Sudan since the late 1980's, including treatment for 67,000 VL patients. In recent years, MSF monitoring data have indicated increasing numbers of VL relapse cases. A retrospective analysis of these data was performed in order to provide insight into the possible causes of this increase. Methodology/Principal findings: Programme monitoring data from the MSF hospital in Lankien, Jonglei State, South Sudan, for the period 2001–2018 were analysed to detect trends in VL relapse as a proportion of all VL cases presenting to MSF treatment centres. Routinely collected patient-level data from relapse and primary VL cases treated at all MSF sites in South Sudan over the same period were analysed to describe patient characteristics and treatments received. VL relapse as a proportion of all VL cases increased by 6.5% per annum (95% CI 0.3% to 13.0%, p = 0.04), from 5.2% during 2001–2003 to 14.4% during 2016–2018. Primary VL and VL relapse patients had similar age, sex and anthropometric characteristics, the latter indicating high indices of undernutrition which were relatively constant over time. Clinical factors (Hb, spleen size, and VL severity score) also did not vary substantially over time. SSG/PM was the main treatment regimen from 2001–2018, used in 68.7% of primary and 70.9% of relapse VL cases; AmBisome was introduced in 2013, received by 22.5% of primary VL and 32.6% of VL relapse cases from 2013–2018. Conclusion: Increasing incidence of VL relapse in South Sudan does not appear to be explained by changes in patient characteristics or other factors. Our data are concerning and may indicate an emergence of treatment-resistant parasite strains, decreasing the effectiveness of treatment regimens. This warrants further investigation as a causal factor. New chemical entities that will enable safe and highly effective short-course oral treatments for VL are urgently needed. Author summary: Visceral leishmaniasis is a fatal parasitic disease which affects impoverished populations living in remote areas. It has caused very high mortality in South Sudan where several major outbreaks have occurred in the past 40 years. In East Africa, the disease can be successfully treated with a combination of injectable sodium stibogloconate and paromomycin given for 17 days. South Sudan was the first country where this regimen was introduced. Now, nearly 20 years later, our data show that it may have become less effective. In our clinic in Lankien, situated in the middle of the endemic area, more and more patients present with relapsed disease and need to be retreated. We have analysed our data and found that the relapse rate as a proportion of all VL cases has increased by 6.5% per annum since 2003. It is possible that this is caused by treatment-resistant parasite strains. Since there is currently no alternative first line treatment, this is of concern. We have planned a prospective observational study where we will follow patients for 12 months in order to determine the current relapse rate, and we highlight the need for new chemical entities that will enable safe and effective short-course oral treatments. [ABSTRACT FROM AUTHOR]

Background: Most people infected with Leishmania remain asymptomatic, which is a common element that may promote the resurgence of clinically evident leishmaniasis in individuals with impaired cell-mediated immune responses. Unfortunately, there is no universally accepted assay to identify asymptomatic infection. This cross-sectional study focuses on the employment of three methods targeting different features of the parasitic infection to be used in combination for the screening of latent leishmaniasis in a newly endemic area of northeastern Italy. Methodology/principle findings: The selected methods included highly sensitive Real-Time PCR for detection of parasitic kinetoplast (k)DNA in peripheral blood, Western Blot (WB) for detection of specific IgG, and Whole Blood stimulation Assay (WBA) to evaluate the anti-leishmanial T-cell response by quantifying the production of IL-2 after stimulation of patients' blood with Leishmania specific antigens. Among 145 individuals, living in a municipality of the Bologna province, northeastern Italy, recruited and screened for Leishmania infection, 23 subjects tested positive (15.9%) to one or more tests. Positive serology was the most common marker of latent leishmaniasis (15/145, 10%), followed by the detection of specific cell-mediated response (12/145, 8%), while only few individuals (6/145, 4%) harbored parasitic DNA in the blood. Conclusions/significance: Combining different tests substantially increased the yield of positivity in detecting latent Leishmania infection. The test combination that we employed in this study appears to be effective to accurately identify latent leishmaniasis in an endemic area. Author summary: After infection with Leishmania infantum cutaneous or visceral leishmaniasis can develop, but in most cases the host becomes an asymptomatic carrier of the parasite. This key population of individuals with latent leishmaniasis can undergo reactivation of the infection with severe outcome in case of immunosuppression and can contribute to disease transmission. The public health impact of leishmaniasis is currently underestimated in Europe, despite Leishmania infection is endemic in the Mediterranean countries and outbreaks of human leishmaniasis have been reported in the last decades, including northeastern Italy. One of the causes contributing to this neglect is that the available diagnostic tests exhibit scarce sensitivity in identification of latent leishmaniasis. In this study, we identified latent Leishmania infection in a newly endemic area of northeastern Italy by using three different diagnostic methods; serological, molecular and immunological tests. Sixteen percent of individuals residing in the selected area were shown to be positive to at least one of the three tests, which indicates that they have been in contact with the parasite. This study highlights the importance of test combination to enhance the ability to identify latent Leishmania infection. [ABSTRACT FROM AUTHOR]

Leishmaniasis is an infectious disease caused by protozoan parasites belonging to the genus Leishmania for which there are no approved human vaccines. Infections localise to different tissues in a species-specific manner with the visceral form of the disease caused by Leishmania donovani and L. infantum being the most deadly in humans. Although Leishmania spp. parasites are predominantly intracellular, the visceral disease can be prevented in dogs by vaccinating with a complex mixture of secreted products from cultures of L. infantum promastigotes. With the logic that extracellular parasite proteins make good subunit vaccine candidates because they are directly accessible to vaccine-elicited host antibodies, here we attempt to discover proteins that are essential for in vitro growth and host infection with the goal of identifying subunit vaccine candidates. Using an in silico analysis of the Leishmania donovani genome, we identified 92 genes encoding proteins that are predicted to be secreted or externally anchored to the parasite membrane by a single transmembrane region or a GPI anchor. By selecting a transgenic L. donovani parasite that expresses both luciferase and the Cas9 nuclease, we systematically attempted to target all 92 genes by CRISPR genome editing and identified four that were required for in vitro growth. For fifty-five genes, we infected cohorts of mice with each mutant parasite and by longitudinally quantifying parasitaemia with bioluminescent imaging, showed that nine genes had evidence of an attenuated infection although all ultimately established an infection. Finally, we expressed two genes as full-length soluble recombinant proteins and tested them as subunit vaccine candidates in a murine preclinical infection model. Both proteins elicited significant levels of protection against the uncontrolled development of a splenic infection warranting further investigation as subunit vaccine candidates against this deadly infectious tropical disease. Author summary: Leishmaniasis is a parasitic infectious disease that is responsible for many tens of thousands of human deaths per year, primarily in impoverished parts of the world. Although there are drugs to treat this parasite infection, resistance is emerging and there are no approved human vaccines. Extracellular parasite proteins can make good vaccine targets because they are directly accessible to host antibodies; however, not all parasite surface proteins can elicit protective immune responses. With the goal of identifying new vaccine targets, we selected over 90 genes that encode parasite cell surface and secreted proteins and used the latest CRISPR gene editing technology to individually target them. Using these mutant parasites, we identified four genes required for parasite growth in the laboratory. We expressed two of the proteins as subunit vaccines and a preclinical infection model was used to determine if they could elicit protective immune responses. We found that two of our candidates were able to confer significant levels of protection in a murine model of visceral leishmaniasis. Our study will contribute to the search for a highly effective vaccine against visceral leishmaniasis to improve the lives of people living in some of the poorest regions on the planet. [ABSTRACT FROM AUTHOR]

Background: Visceral leishmaniasis (VL) is a life-threatening parasitic disease next to malaria, which is responsible for the death of 50,000 patients annually. It has three major clinical stages, including visceral, cutaneous, and mucocutaneous leishmaniasis. Ethiopia is one of the east African countries commonly affected with leishmanisis disease. There are many drugs for leishmaniasis, including sodium stibogluconate and paromomycin combined therapy. However, the adverse effect of those combined drugs is not well-defined. Therefore, the purpose of this study was to assess serum amylase, lipase, and associated factors among patients with VL treatment with those combined drugs. Methods: Hospital-based cross-sectional study was conducted at the University of Gondar Comprehensive Specialized Hospital Leishmaniasis Research and Treatment Center from February to September 2020 G.C. Simple random sampling technique was utilized to select study participants. The study participants who fulfill the inclusion criteria were included in the study with written informed consent. 5 ml of blood was withdrawn by an experienced health professional to analyze serum amylase and lipase level. Descriptive data was presented by tables, charts and graphs. Data was cleared, entered by Epi-data version 3.1 then transfer to STATA 14.1 SE version and for analysis paired t-test was used, for factors correlation and regression was used. Those factor variable who have p-value <0.25 was filtered and goes to multivariate regression and p-value <0.05 was considered as significant variables. Results: The result of this study showed that there was a significant mean difference between serum pancreatic amylase and lipase before and after treatment. The mean ± SD level of serum amylase after treatment showed a statistically significant elevation (P<0.001) as compared to its level before treatment. Similarly, the mean ± SD level of serum lipase after treatment showed a statistically significant elevation (P<0.001) as compared to its level before treatment. There was also significant association between age and baseline serum amylase as compared to serum amylase after treatment. Similarly, there was also significant relation of age and serum lipase with serum lipase after treatment. Conclusion: In this study, the level of serum amylase and lipase at treatment of cure was higher and there was an increase in mean serum amylase and lipase after a patient taking sodium stibogluconate and paromomycin combined drugs. Consequently, the elevated result of these biochemical profiles mainly associated with drug induced adverse effect and associated risk factors in VL patients. [ABSTRACT FROM AUTHOR]

Background: Visceral leishmaniasis (VL) is a zoonotic protozoal vector-borne disease that is a major public health challenge. In Argentina, canine (CVL) and human visceral leishmaniasis (HVL) have recently emerged. There is a lack of standardised diagnostic tests for CVL, which hinders control of CVL and HVL. Methodology/Principal findings: Sampling was carried out in Puerto Iguazú, Argentina, comprising 190 asymptomatic, oligosymptomatic and polysymptomatic dogs. The following diagnostics were applied: microscopy of lymph node aspirate (LNA); three immunochromatographic rapid diagnostic tests (RDTs), prototype rK28-ICT, rK39-ICT (both Coris BioConcept), commercial rK39 (InBios); ELISA for IgG, IgG1 and IgG2, against rK28, rK39 or crude lysate antigen. DNA detection and analysis, with 30 dogs, was of the ITS1 region using skin samples, and loop-mediated isothermal amplification (LAMP; Eiken Loopamp) of buffy coat, skin scrape or LNA. 15.4% of dogs were positive by LNA microscopy. The rK28 RDT had higher seropositivity rate (61%) than either a prototype rK39 RDT (31.4%) or commercial rK39 RDT (18.8%), without cross-reactivity with six other pathogens. IgG anti-rK39 ELISA antibody titres, but not IgG2, were positively correlated with number of clinical signs. LAMP with LNA had a higher positivity rate than PCR; buffy coat sampling was more sensitive than skin scrape. ITS1 confirmed Leishmania (Leishmania) infantum as the agent of CVL. Leishmania (Viannia) spp. was detected in skin samples from two dogs, compatible with Leishmania (Viannia) braziliensis. Conclusions/Significance: Seroprevalence confirmed rapid increase in CVL in Puerto Iguazú. The rK28 RDT test potentially has great value for improved point-of-care diagnosis. Given cost reduction and accessibility, commercial LAMP may be applicable to buffy coat. RDT biomarkers of CVL clinical status are required to combat spread of CVL and HVL. The presence of Viannia, perhaps as an agent of human mucocutaneous leishmaniasis (MCL), highlights the need for vigilance and surveillance. Author summary: Visceral leishmaniasis (VL) is a widespread parasitic disease caused by sand fly-borne parasites of the Leishmania donovani complex (L. donovani and L. infantum). Without early diagnosis and successful chemotherapy symptomatic human VL is fatal. Dogs are reservoir hosts of L. infantum, and canine visceral leishmaniasis (CVL) often precedes outbreaks in local human populations. With dogs sampled within a region of emergent CVL in northern Argentina, we compared a range of diagnostic techniques, including microscopy, serology by enzyme linked immunosorbent assays (ELISAs) and rapid diagnostic tests (RDTs), and detection and identification of Leishmania DNA. Novel serological assays based on the rK28 antigen were more sensitive than those based on rK39, and without evidence of cross reaction with six other canine pathogens. A commercial DNA detection kit (LAMP; Eiken Loopamp), used for the first time with CVL, was more sensitive than PCR on lymph node samples. Unexpectedly, we also found canine infection with the Viannia sub-genus of Leishmania. Our results reinforce the need for improved diagnosis, vigilance, surveillance and control of CVL. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis is a vector-borne protozoan infection that is fatal if untreated. There is no vaccination against the disease, and the current chemotherapeutic agents are ineffective due to increased resistance and severe side effects. Buparvaquone is a potential drug against the leishmaniases, but it is highly hydrophobic resulting in poor bioavailability and low therapeutic efficacy. Herein, we loaded the drug into silicon nanoparticles produced from barley husk, which is an agricultural residue and widely available. The buparvaquone-loaded nanoparticles were several times more selective to kill the intracellular parasites being non-toxic to macrophages compared to the pure buparvaquone and other conventionally used anti-leishmanial agents. Furthermore, the in vivo results revealed that the intraperitoneally injected buparvaquone-loaded nanoparticles suppressed the parasite burden close to 100%. By contrast, pure buparvaquone suppressed the burden only by 50% with corresponding doses. As the conclusion, the biogenic silicon nanoparticles are promising carriers to significantly improve the therapeutic efficacy and selectivity of buparvaquone against resistant visceral leishmaniasis opening a new avenue for low-cost treatment against this neglected tropical disease threatening especially the poor people in developing nations. Author summary: Visceral leishmaniasis is a neglected tropical disease caused by a protozoan parasite. It infects macrophage associated organs and invades the immune system. Currently, there is no vaccination against the disease and the conventional chemotherapeutic agents are ineffective due to the increased resistance and toxicity. In the present work, we utilized porous silicon nanoparticles as carriers for an anti-leishmanial drug (buparvaquone) to improve its therapeutic efficacy against visceral leishmaniasis (S1 Scheme). The nanoparticles were synthesized from an agricultural residue (barley husk), which is cheap and widely available. Due to the nano size of the nanoparticles, they internalized into the infected cells successfully. The efficacies of buparvaquone loaded in the nanoparticles were greatly improved in comparison with pure buparvaquone to evacuate the parasitic infection both in vitro and in vivo studies. Therefore, the biogenic silicon nanocarriers are promising candidates to be utilized as low-cost treatment regimen in leishmaniases therapy. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis (VL) is one of the most prevalent parasitic diseases worldwide. In 2019, 97% of the total numbers of cases in Latin America were reported in Brazil. In São Paulo state, currently 17.6% of infected individuals live in the western region. To study this neglected disease on a regional scale, we describe the spread of VL in 45 municipalities of the Regional Network for Health Assistance11(RNHA11). Environmental, human VL (HVL), and canine VL (CVL) cases, Human Development Index, and Lutzomyia longipalpis databases were obtained from public agencies. Global Moran's I index and local indicators of spatial association (LISA) statistics were used to identify spatial autocorrelation and to generate maps for the identification of VL clusters. On a local scale, we determined the spread of VL in the city of Teodoro Sampaio, part of the Pontal of Paranapanema. In Teodoro Sampaio, monthly peri-domicile sand fly collection; ELISA, IFAT and Rapid Test serological CVL; and ELISA HVL serum surveys were carried out. In RNHA11 from 2000 to 2018, Lu. longipalpis was found in 77.8%, CVL in 69%, and HVL in 42.2% of the 45 municipalities, and 537 individuals were notified with HVL. Dispersion occurred from the epicenter in the north to Teodoro Sampaio, in the south, where Lu. longipalpis and CVL were found in 2010, HVL in 2018, and critical hotspots of CVL were found in the periphery. Moran's Global Index showed a weak but statistically significant spatial autocorrelation related to cases of CVL (I = 0.2572), and 11 municipalities were identified as priority areas for implementing surveillance and control actions. In RNHA11, a complex array of socioeconomic and environmental factors may be fueling the epidemic and sustaining endemic transmission of VL, adding to the study of a neglected disease in a region of São Paulo, Brazil. Author summary: Visceral leishmaniasis (VL) is considered the second most important disease caused by a protozoan worldwide. Currently, 97% of the cases in Latin America are in Brazil. In São Paulo state, since 1997, the disease has been found in an increasing number of municipalities, mainly in the western region. The reasons why VL is spreading in a crescent shape in this region is not well understood, however, socioeconomic and environmental risk factors that increase vulnerability to the disease may be involved. From 2000 to 2018, vectors of Lutzomyia longipalpis were found in 77.8%, canine VL in 69%, and human VL in 44.4% of the 45 municipalities of the western region. Dispersion occurred from the epicenter in the north and followed central and radial highways, reaching Teodoro Sampaio in the south. Among the 45 municipalities, 11 were identified as priority areas for implementing surveillance and control actions. Considered the last frontier and one of the poorest regions of São Paulo, our findings are relevant for planning interventions aimed at reducing the cases of VL in the region. [ABSTRACT FROM AUTHOR]

The microbial consortium associated with sandflies has gained relevance, with its composition shifting throughout distinct developmental stages, being strongly influenced by the surroundings and food sources. The bacterial components of the microbiota can interfere with Leishmania development inside the sandfly vector. Microbiota diversity and host-microbiota-pathogen interactions regarding New World sandfly species have yet to be thoroughly studied, particularly in Lutzomyia longipalpis, the primary vector of visceral leishmaniasis in Brazil.The native microbiota of different developmental stages and physiological conditions of Lu. longipalpis (Lapinha Cave), was described by culturing and 16s rRNA gene sequencing. The 16s rRNA sequencing of culture-dependent revealed 13 distinct bacterial genera (Bacillus, Enterococcus, Erwinia, Enterobacter, Escherichia, Klebsiella, Lysinibacillus, Pseudocitrobacter, Providencia, Pseudomonas, Serratia, Staphylococcus and Solibacillus). The in vitro and in vivo effects of each one of the 13 native bacteria from the Lu. longipalpis were analyzed by co-cultivation with promastigotes of L.i. chagasi, L. major, L. amazonensis, and L. braziliensis. After 24 h of co-cultivation, a growth reduction observed in all parasite species. When the parasites were co-cultivated with Lysinibacillus, all parasites of L. infantum chagasi and L. amazonensis died within 24 hours. In the in vivo co-infection of L.chagasi, L. major and L. amazonensis with the genera Lysinibacillus, Pseudocitrobacter and Serratia it was possible to observe a significant difference between the groups co-infected with the bacterial genera and the control group.These findings suggest that symbiont bacteria (Lysinibacillus, Serratia, and Pseudocitrobacter) are potential candidates for paratransgenic or biological control. Further studies are needed to identify the nature of the effector molecules involved in reducing the vector competence for Leishmania. Author summary: According to the World Health Organization Leishmaniasis is the second parasitic disease that kills the most in the world; the first is malaria. Despite this, knowledge about the Leishmania parasite and its interaction with vertebrate hosts concerning the transmitting insect is still relatively fewer and fragmented. Studies on insects microbiota have great importance to obtain basic information. How a vector responds to the presence of different microorganisms and how they interact with various pathogens and may lead to the development of new strategies or tools that can be used to prevent or hinder the transmission of the protozoan by the vector insect. Considering the knowledge about the intestinal microbiota of sandflies, we aim to study the effect of bacterial isolates on Lu. longipalpis infection by different species of Leishmania, and it believed that these bacteria might influence the development of Leishmania, preventing, and hindering transmission, contributing to Leishmaniasis control strategies. [ABSTRACT FROM AUTHOR]

Genetic polymorphisms in natural Leishmania populations have been reported in endemic areas. Microsatellite typing is a useful tool to elucidate the genetic variability of parasite strains, due to its capability for high-resolution mapping of genomic targets. The present study employed multilocus microsatellite typing (MLMT) to explore the genotypic composition of Leishmania infantum in naturally infected dogs by genotyping parasites infecting different tissues with or without in vitro expansion. Eighty-six samples were collected from 46 animals in an endemic region of visceral leishmaniasis (VL). MLMT was performed for 38 spleen samples and 48 L. infantum cultures isolated from different tissues. Of the 86 samples, 23 were effectively genotyped by MLMT, identifying nine multilocus genotypes (MLG; referred to as MLG A–I). MLGs A, B and C were detected in more than one type of tissue and in more than one sample. Conversely, MLG D-I were uniquely detected in one sample each. The results showed that multiple genotype infections occur within a single host and tissue. Paired sample analysis revealed the presence of different MLMT alleles in 14 dogs, while the same MLG allele was present in 15 animals. STRUCTURE analysis demonstrated the presence of two populations; 13 samples displayed a similar admixture of both ancestral populations, and these were not assigned to any population. Only samples for which Q ≥ 0.70 after CLUMPP alignment were considered to be part of Population 1 (POP1) or Population 2 (POP2). POP2 comprised the majority of samples (n = 54) compared to POP1 (n = 19). This study presents evidence of multiple genotype infections (caused by L. infantum) in dogs in an area with high VL transmission. Further investigations must be undertaken to determine the multiple effects of infection on the host immune response and disease dynamics and treatment. Author summary: American visceral leishmaniasis (VL) is a parasitic disease caused by the protozoan Leishmania infantum. This parasite can infect humans and animals and is transmitted by sand flies. Domestic dogs are considered an important host, and like humans, they can manifest the disease or present asymptomatic infections. Studies have identified genetic variations among L. infantum parasites from different endemic regions in the American continent. For other parasitic diseases (e.g., malaria), studies have suggested that multigenetic infection predicts the development of symptoms and can lead to a high level of transmission. However, the effects of the genetic composition of Leishmania parasites on VL need to be ascertained. This study used highly variable microsatellite markers to investigate multigenotype L. infantum populations among naturally infected dogs living in an area in which VL is highly prevalent. Samples obtained from different tissues were examined to identify the occurrence of multiple genotypes in the same animal and even within the same tissue. [ABSTRACT FROM AUTHOR]

Background: Bangladesh, India, and Nepal aim for the elimination of Visceral Leishmaniasis (VL), a systemic parasitic infectious disease, as a public health problem by 2020. For decades, male patients have comprised the majority of reported VL cases in this region. By comparing this reported VL sex ratio to the one observed in population-based studies conducted in the Indian subcontinent, we tested the working hypothesis that mainly socio-cultural gender differences in healthcare-seeking behavior explain this gender imbalance. Methodology/Principal findings: We compared the observed sex ratio of male versus female among all VL cases reported by the health system in Nepal and in the two most endemic states in India with that observed in population-based cohort studies in India and Nepal. Also, we assessed male sex as a potential risk factor for seroprevalence at baseline, seroconversion, and VL incidence in the same population-based data. The male/female ratio among VL cases reported by the health systems was 1.40 (95% CI 1.37–1.43). In the population cohort data, the age- and study site-adjusted male to female risk ratio was 1.27 (95% CI 1.08–1.51). Also, males had a 19% higher chance of being seropositive at baseline in the population surveys (RR 1.19; 95% CI 1.11–1.27), while we observed no significant difference in seroconversion rate between both sexes at the DAT cut-off titer defined as the primary endpoint. Conclusions/Significance: Our population-based data show that male sex is a risk factor for VL, and not only as a socio-cultural determinant. Biological sex-related differences likely play an important role in the pathogenesis of this disease. Author summary: Visceral Leishmaniasis (VL) is a parasitic disease that is lethal if not treated timely and mainly affects impoverished populations. Bangladesh, India, and Nepal have targeted the elimination of this disease as a public health problem by 2020. The majority of VL patients attending the health services are male, and this is usually attributed to unequal access to health care for men and women in this sociocultural context. We analyzed two large datasets obtained in population surveillance projects, including regular door-to-door screening for VL, in India and Nepal. Thereby we minimized any potential differences in access to health care between both sexes, as every suspect VL case occurring in the community received a full diagnostic work-up, and the research project facilitated transport to treatment centers if needed. By comparing the observed sex ratio in the health services records with those of the population surveillance records, we aimed to reach meaningful conclusions about the pathway through which male gender exerts its leverage for increasing VL risk: socio-cultural determinants blocking women from accessing care (nurture) or biological factors (nature) making men more vulnerable to VL. Because in the population-based age adjusted-data, male VL cases were significantly more frequent than female, our findings strongly suggest that in the Indian subcontinent, biological differences between men and women play a more critical role in the pathogenesis of VL than previously assumed, and the observed male predominance in VL cases in health services cannot be explained by socio-cultural factors only. Moreover, data show that above the age of 14 years, males are seropositive more often than females and are at higher risk to develop VL disease. [ABSTRACT FROM AUTHOR]

Background: The aim of this study was to compare cases of Visceral Leishmaniasis (VL) with and without HIV in a state in northeastern Brazil. Methodology: We performed a comparative study in the state's referral hospital for infectious/parasitic diseases located in Northeast Brazil between January 2007 and July 2017. The data obtained using this protocol were analyzed with SPSS. Principal findings: In total, 252 patients were evaluated, including 126 with coincident VL/HIV and 126 with VL alone. Both groups primarily consisted of male patients. The most commonly affected ages were 30–39 years in the coinfected group and 19–29 years in the VL group (p < 0.001). Fever and anorexia (p = 0.001), which were more common in those with VL alone, were frequently observed, while diarrhea, vomiting, bleeding and dyspnea were more common in patients with VL/HIV coinfection (p<0.005). According to the hemogram results, leukocyte levels were lower in the VL group (p < 0.0001). Additionally, AST (aspartate aminotransferase) and ALT (alanine aminotransferase) levels differed between the groups, with higher levels in patients with VL (p < 0.001). On average, HIV was diagnosed 2.6 years before VL (p < 0.001), and VL relapse was observed only in the coinfection group (36.5% of cases). Fever (β = +0.17; p = 0.032) in the first VL/HIV episode was identified as a risk factor for relapse (R2 = 0.18). More deaths occurred in the VL/HIV group (11.1%) than in the VL group (2.4%). Conclusion/Significance: VL/HIV was found to be prevalent among young adults, although the median patient age was higher in the VL/HIV group. The classic symptomatology of VL was more common in patients not coinfected with HIV; therefore, attention is needed in patients with HIV who present with any symptoms that suggest the presence of VL, especially in endemic areas. No cases of VL relapse occurred in patients without HIV, and death was more common in the VL/HIV co-infected group. [ABSTRACT FROM AUTHOR]

Background: In endemic regions, asymptomatic Leishmania infection is common. In HIV patients, detection of asymptomatic Leishmania infection could potentially identify those at risk of visceral leishmaniasis (VL). However, data on the prevalence, incidence, and determinants of asymptomatic infection, and the risk of VL are lacking. Methods: We conducted a cross-sectional survey at a single ART centre, followed by a prospective cohort study amongst HIV-infected adults in HIV care in a district hospital in a VL-endemic area in North-West Ethiopia (9/2015-8/2016). Asymptomatic Leishmania infection was detected using the direct agglutination test (DAT), rK39-rapid diagnostic test (RDT)), PCR on peripheral blood and the KAtex urine antigen test, and defined as positivity on any Leishmania marker. All individuals were followed longitudinally (irrespective of the Leishmania test results). Risk factors for asymptomatic Leishmania infection were determined using logistic regression. Results: A total of 534 HIV-infected individuals enrolled in HIV care were included in the study. After excluding 13 patients with a history of VL and an 10 patients with incomplete baseline Leishmania tests, 511 were included in analysis. The median age was 38 years (interquartile range (IQR) 30–45), 62.6% were male. The median follow-up time was 12 months (IQR 9–12). No deaths were reported during the study period. Most (95.5%) were on antiretroviral treatment at enrolment, for a median of 52 months (IQR 27–79). The median CD4 count at enrolment was 377 cells/mm3 (IQR 250–518). The baseline prevalence of Leishmania infection was 12.8% in males and 4.2% in females. Overall, 7.4% tested positive for rK39, 4.3% for DAT, 0.2% for PCR and 0.2% for KAtex. Independent risk factors for a prevalent infection were male sex (odds ratio (OR) 3.2; 95% confidence intervals (CI) 14–7.0) and concurrent malaria infection (OR 6.1; 95% CI 1.9–18.9). Amongst the 49 prevalent (baseline) infections with further follow-up, the cumulative incidence of losing the Leishmania markers by one year was 40.1%. There were 36 incident infections during the course of the study, with a cumulative one-year risk of 9.5%. Only one case of VL was detected during follow-up. Conclusions: We found a high prevalence of asymptomatic Leishmania infection, persisting in most cases. The incidence was more modest and overt VL was rare. Larger and longer studies with more complete follow-up may help to decide whether a test and treat strategy would be justified in this context. Trial registration: ClinicalTrials.gov [ABSTRACT FROM AUTHOR]

Background: An earlier open label, prospective, non-randomized, non-comparative, multi-centric study conducted within public health facilities in Bihar, India (CTRI/2012/08/002891) measured the field effectiveness of three new treatment regimens for visceral leishmaniasis (VL): single dose AmBisome (SDA), and combination therapies of AmBisome and miltefosine (AmB+Milt) and miltefosine and paromomycin (Milt+PM) up to 6 months follow-up. The National Vector Borne Disease Control Program (NVBDCP) recommended an extended follow up at 12 months post-treatment of the original study cohort to quantify late relapses. Methods: The 1,761 patients enrolled in the original study with the three new regimens were contacted and traced between 10 and 36 months following completion of treatment to determine their health status and any occurrence of VL relapse. Results: Of 1,761 patients enrolled in the original study, 1,368 were traced at the extended follow-up visit: 711 (80.5%), 295 (83.2%) and 362 (71.5%) patients treated with SDA, AmB+Milt and Milt+PM respectively. Of those traced, a total of 75 patients were reported to have relapsed by the extended follow-up; 45 (6.3%) in the SDA, 25 (8.5%) in the AmB+Milt and 5 (1.4%) in the Milt+PM arms. Of the 75 relapse cases, 55 had already been identified in the 6-months follow-up and 20 were identified as new cases of relapse at extended follow-up; 7 in the SDA, 10 in the AmB+Milt and 3 in the Milt+PM arms. Conclusion: Extending follow-up beyond the standard 6 months identified additional relapses, suggesting that 12-month sentinel follow-up may be useful as a programmatic tool to better identify and quantify relapses. With limited drug options, there remains an urgent need to develop effective new chemical entities (NCEs) for VL. [ABSTRACT FROM AUTHOR]

Keywords: Editorial; Medicine and health sciences; Tropical diseases; Neglected tropical diseases; Leishmaniasis; Parasitic diseases; Protozoan infections; Infectious diseases; Zoonoses; Disease vectors; Insect vectors; Sand flies; Biology and life sciences; Species interactions; Infectious disease control; Vaccines; Kala-azar; Immunology; Vaccination and immunization; Vaccine development; Public and occupational health; Preventive medicine; Organisms; Eukaryota; Protozoans; Parasitic protozoans; Leishmania; Leishmania donovani The gaps in vector bionomics knowledge about leishmaniasis elimination include estimates of sand fly biting rates, parasite infection rates in the vector, and the spatial and temporal variations of these in response to indoor residual spraying (IRS) [[31]]. 4 Molina R, Ghosh D, Carrillo E, Monnerat S, Bern C, et al. (2017) Infectivity of Post-Kala-azar Dermal Leishmaniasis Patients to Sand Flies: Revisiting a Proof of Concept in the Context of the Kala-azar Elimination Program in the Indian Subcontinent. 24 Osman M, Mistry A, Keding A, Gabe R, Cook E, et al. (2017) A third generation vaccine for human visceral leishmaniasis and post kala azar dermal leishmaniasis: First-in-human trial of ChAd63-KH. [Extracted from the article]

Background: Visceral leishmaniasis (VL) is a parasitic disease, transmitted by the sand fly species Phlebotomus argentipes in the Indian sub-continent. Effective vector control is highly desirable to reduce vector density and human and vector contact in the endemic communities with the aim to curtail disease transmission. We evaluated the effect of long lasting insecticide treated bed nets (LLIN) and bed nets impregnated with slow-release insecticide tablet K-O TAB 1-2-3 (jointly insecticide-treated nets or ITN) on VL incidence in a highly endemic sub-district (upazila) in Bangladesh. Methods: Several distributions of LLIN or K-O TAB 1-2-3 for self-impregnation of bed nets at home took place in Fulbaria upazila, Mymensigh district from 2004 to 2008 under three research projects, respectively funded by CDC, Atlanta, USA (2004) and WHO-TDR, Geneva, Switzerland (2006 & 2008). We included all households (n = 8142) in the 20 villages that had benefited in the past from one of these interventions (1295 donated LLIN and 11,918 local bed nets impregnated with K-O TAB 1-2-3) in the “exposed cohort”. We recruited a “non-exposed cohort” in villages with contemporaneously similar incidence rates who had not received such vector control interventions (7729 HHs from nine villages). In both cohorts, we visited all families house to house and ascertained any VL cases for the 3 year period before and after the intervention. We evaluated the incidence rate (IR) of VL in both cohorts as primary endpoint, applying the difference-in-differences method. Results: The study identified 1011 VL cases (IR 140.47/10,000 per year [py]) before the intervention, of which 534 and 477 cases in the intervention and control areas respectively. The IR was 144.13/10,000 py (534/37050) and 136.59/10,000 py (477/34923) in the intervention and control areas respectively, with no significant difference (p = 0.3901) before the intervention. After the intervention, a total of 555 cases (IR 77.11/10,000 py) were identified of which 178 (IR 48.04/10,000 py) in the intervention and 377 (107.95/10,000 py) in the control area. The intervention area had a significant lower IR than the control area during follow up, rate difference = –59.91, p<0.0001. The IR during follow up was significantly reduced by 96.09/10,000 py in the intervention area (p<0.0001) and 28.63/10,000 py in control area (p<0.0001) compared to baseline. There was a strong and significant overall effect of the ITN intervention, δ = –67.45, p <0.0001. Sex (OR = 1.36, p<0.0001) and age (OR = 0.99, p<0.0001) also had a significant effect on VL incidence. Male had a higher risk of VL than female and one year increase in age decreased the likelihood of VL by about 0.92%. Two third of the VL incidence occurred in the age range 2 to 30 years (median age of VL patients was 17 years). Conclusion: VL incidence rate was significantly lower in the ITN intervention cohort compared to control in Bangladesh. Some bias due to more intense screen-and-treat activities or other interventions in the intervention area cannot be ruled out. Nonetheless, given their feasibility and sustainability, ITNs should be considered for integrated vector control during the maintenance phase of the VL elimination programme. [ABSTRACT FROM AUTHOR]

Lipophosphoglycan (LPG) is the major surface glycoconjugate of Leishmania protozoan and has an important biological role in host-parasite interactions both in the midgut epithelium of the sand fly vector and in the vertebrate macrophages. Canine leishmaniasis (CanL) is a chronic infectious disease predominantly caused by Leishmania infantum. An early and accurate immunodiagnosis of the disease is crucial for veterinary clinical practice and for disease control. In this work, we evaluated L. infantum LPG as an antigen in an indirect enzyme-linked immunosorbent assay (ELISA) for CanL immunodiagnosis (LPG-ELISA) by testing serum samples from 97 naturally infected dogs with diverse clinical presentations ranging from subclinical infection to severe disease, as evaluated by veterinarian infectologists. Serum samples from healthy dogs from non-endemic areas (n = 68) and from dogs with other infectious diseases (n = 64) were used as controls for assay validation. The performance of the LPG-ELISA was compared with that of an ELISA using the soluble fraction of L. infantum total lysate antigen (TLA). LPG-ELISA presented a superior performance in comparison to TLA-ELISA, with 91.5% sensitivity, 98.5% specificity and 99.7% accuracy. A distinguishing feature of the LPG-ELISA compared to the TLA-ELISA was its higher ability to identify subclinical infection in clinically healthy dogs, in addition to the absence of cross-reactivity with other canine infectious diseases. Finally, LPG-ELISA was compared to TR DPP visceral canine leishmaniasis test, the immunochromatographic test recommended by the Brazilian Ministry of Agriculture. LPG-ELISA exhibited higher values of specificity (98.5% versus 93.1%) and sensitivity (91.5% versus 90.6%) compared to TR DPP. In conclusion, L. infantum-derived LPG was recognized by antibodies elicited during CanL in different infection stages and was shown to be a suitable antigen for specific clinical settings of veterinary diagnosis and for public health usage. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis (VL) caused by Leishmania donovani remains of public health concern in rural India. Those at risk of VL are also at risk of other neglected tropical diseases (NTDs) including soil transmitted helminths. Intestinal helminths are potent regulators of host immune responses sometimes mediated through cross-talk with gut microbiota. We evaluate a meta-taxonomic approach to determine the composition of prokaryotic and eukaryotic gut microflora using amplicon-based sequencing of 16S ribosomal RNA (16S rRNA) and 18S rRNA gene regions. The most abundant bacterial taxa identified in faecal samples from Bihar State India were Prevotella (37.1%), Faecalibacterium (11.3%), Escherichia-Shigella (9.1%), Alloprevotella (4.5%), Bacteroides (4.1%), Ruminococcaceae UCG-002 (1.6%), and Bifidobacterium (1.5%). Eukaryotic taxa identified (excluding plant genera) included Blastocystis (57.9%; Order: Stramenopiles), Dientamoeba (12.1%; Family: Tritrichomonadea), Pentatrichomonas (10.1%; Family: Trichomonodea), Entamoeba (3.5%; Family: Entamoebida), Ascaridida (0.8%; Family: Chromodorea; concordant with Ascaris by microscopy), Rhabditida (0.8%; Family: Chromodorea; concordant with Strongyloides), and Cyclophyllidea (0.2%; Order: Eucestoda; concordant with Hymenolepis). Overall alpha (Shannon’s, Faith’s and Pielou’s indices) and beta (Bray-Curtis dissimilarity statistic; weighted UniFrac distances) diversity of taxa did not differ significantly by age, sex, geographic subdistrict, or VL case (N = 23) versus endemic control (EC; N = 23) status. However, taxon-specific associations occurred: (i) Ruminococcaceae UCG- 014 and Gastranaerophilales_uncultured bacterium were enriched in EC compared to VL cases; (ii) Pentatrichomonas was more abundant in VL cases than in EC, whereas the reverse occurred for Entamoeba. Across the cohort, high Escherichia-Shigella was associated with reduced bacterial diversity, while high Blastocystis was associated with high bacterial diversity and low Escherichia-Shigella. Individuals with high Blastocystis had low Bacteroidaceae and high Clostridiales vadin BB60 whereas the reverse held true for low Blastocystis. This scoping study provides useful baseline data upon which to develop a broader analysis of pathogenic enteric microflora and their influence on gut microbial health and NTDs generally. [ABSTRACT FROM AUTHOR]

Leishmania (Leishmania) infantum is the zoonotic agent of visceral leishmaniasis (VL), a disease with a global distribution. The transmission scenario of VL has been undergoing changes worldwide, with the biologic cycle invading urbanized areas and dispersing the parasites into other previously free areas. The epidemiological cycle in Brazil has dispersed from the Northeast to other regions of the country. In this study, an integrative approach, including genotyping Brazilian strains of L. (L.) infantum for 14 microsatellite markers and reviewing historical records of the disease, was used to assess dispersion routes throughout central-southern Brazil. Our results support three L. (L.) infantum dispersion routes: A) dispersion from Bolivia to the states of Mato Grosso, Mato Grosso do Sul and São Paulo via the Bolivia-Brazil gas pipeline from 1998 to 2005; B) VL dispersion from Paraguay to the Brazilian side of the triple border (Foz do Iguaçu and Santa Terezinha de Itaipu) during after 2012; and C) emergence of a new L. (L.) infantum cluster in western Santa Catarina State and its dispersion to southern Paraná State (municipality of Pato Branco), after 2013. Hypotheses regarding possible entries of Leishmania (L.) infantum into the area of the triple border are presented and discussed. Understanding how VL has dispersed is vital to the development of control measures for this disease and to avoid future dispersion events. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis (VL) is a zoonosis caused by the protozoan Leishmania infantum and in Brazil is transmitted mainly by the bite of Lutzomuyia longipalpis sand flies. Data about the presence, distribution, natural infection rate, seasonal and monthly dynamics of the vector population are important for optimizing the measures to control VL in endemic areas. This study aimed to identify sand fly fauna in an endemic area for VL to detect the prevalence of L. infantum infection in the Lu. longipalpis population and to elucidate the influence of bioclimatic factors on the monthly fluctuations of this vector. HP light traps were monthly set in the intradomicile and peridomicile of residences located in the central and beachfront areas of Camaçari, a VL endemic area. The sand fly collection was conducted in two periods: i) period 1—between December 2011 and November 2012 and ii) period 2—August 2014 and July 2015. Sand fly species were identified and detection of L. infantum infection by qPCR was performed in pools of female Lu. longipalpis. For the first time, the parasite load of positive pools was correlated with the number of Lu. longipalpis captured per month in both periods. Correlation analyses between the monthly fluctuation of the sand fly population and bioclimatic indices of the municipality in both collection periods were also performed. In both evaluated periods, more than 98% of the collected sand flies were Lu. longipalpis, confirming the predominance of this species in the region. It was captured mostly in the beachfront area in all months evaluated (99%). For the period 1, Leishmania DNA was detected in 81% of tested pools representing a minimal infection rate of 9.6%. In the period 2, 40% of the pools were positive with a minimal infection rate of 10.2%. Infected sand flies were only detected in the beachfront area in both periods. The parasite load was low and did not vary in the evaluated months despite the number of collected sand flies. No correlation was observed for climatic factors in both areas of Camaçari. These findings emphasize the high risk of Leishmania transmission in Camaçari regardless of the season and that other factors, aside from bioclimatic elements, are influencing the sand fly population monthly fluctuation. [ABSTRACT FROM AUTHOR]

Amphotericin B provides improved therapy for visceral leishmaniasis (VL) caused by Leishmania donovani, with single dose liposomal-encapsulated Ambisome providing the best cure rates. The VL elimination program aims to reduce the incidence rate in the Indian subcontinent to <1/10,000 population/year. Ability to predict which asymptomatic individuals (e.g. anti-leishmanial IgG and/or Leishmania-specific modified Quantiferon positive) will progress to clinical VL would help in monitoring disease outbreaks. Here we examined whole blood transcriptional profiles associated with asymptomatic infection, active disease, and in treated cases. Two independent microarray experiments were performed, with analysis focussed primarily on differentially expressed genes (DEGs) concordant across both experiments. No DEGs were identified for IgG or Quantiferon positive asymptomatic groups compared to negative healthy endemic controls. We therefore concentrated on comparing concordant DEGs from active cases with all healthy controls, and in examining differences in the transcriptome following different regimens of drug treatment. In these comparisons 6 major themes emerged: (i) expression of genes and enrichment of gene sets associated with erythrocyte function in active cases; (ii) strong evidence for enrichment of gene sets involved in cell cycle in comparing active cases with healthy controls; (iii) identification of IFNG encoding interferon-γ as the major hub gene in concordant gene expression patterns across experiments comparing active cases with healthy controls or with treated cases; (iv) enrichment for interleukin signalling (IL-1/3/4/6/7/8) and a prominent role for CXCL10/9/11 and chemokine signalling pathways in comparing active cases with treated cases; (v) the novel identification of Aryl Hydrocarbon Receptor signalling as a significant canonical pathway when comparing active cases with healthy controls or with treated cases; and (vi) global expression profiling support for more effective cure at day 30 post-treatment with a single dose of liposomal encapsulated amphotericin B compared to multi-dose non-liposomal amphotericin B treatment over 30 days. (296 words; 300 words allowed). [ABSTRACT FROM AUTHOR]

Background: We investigated the relationship of treatment regimens for visceral leishmaniasis (VL) with post-kala-azar dermal leishmaniasis (PKDL) and visceral leishmaniasis relapse (VLR) development. Methods: Study subjects included cohorts of patients cured of VL since treatment with monotherapy by sodium stibogluconate (SSG), miltefosine (MF), paromomycin intramuscular injection (PMIM), liposomal amphotericin B [AmBisome (AMB)] in a single dose (SDAMB) and in multidose (MDAMB), and combination therapies by AMB+PMIM, AMB+MF, and PMIM+MF. Follow up period was 4 years after treatment. Cohorts were prospective except SSG (retrospective) and MF (partially retrospective). We compared incidence proportion and rate in 100-person-4year of PKDL and VLR by treatment regimens using univariate and multivariate analysis. Findings: 974 of 984 enrolled participants completed the study. Overall incidence proportion for PKDL and VLR was 12.3% (95% CI, 10.4%–14.5%) and 7.0% (95% CI, 5.6%–8.8%) respectively. The incidence rate (95% CI) of PKDL and VLR was 14.0 (8.6–22.7) and 7.6 (4.1–14.7) accordingly. SSG cohort had the lowest incidence rate of PKDL at 3.0 (1.3–7.3) and VLR at 1.8 (0.6–5.6), followed by MDAMB at 8.2 (4.3–15.7) for PKDL and at 2.7 (0.9–8.4) for VLR. Interpretation: Development of PKDL and VLR is related with treatment regimens for VL. SSG and MDAMB resulted in less incidence of PKDL and VLR compared to other treatment regimens. MDAMB should be considered for VL as a first step for prevention of PKDL and VLR since SSG is highly toxic and not recommended for VL. [ABSTRACT FROM AUTHOR]

Background: Accurate and accessible diagnosis is key for the control of visceral leishmaniasis (VL). Yet, current diagnostic tests for VL have severe limitations: they are invasive or not suitable as point of care (POC) test or their performance is suboptimal in East Africa. We analysed the antigens in the VL serodiagnostics development pipeline to identify shortcomings and to propose strategies in the development of an alternative POC test for VL in East Africa. Objectives: The objective of this study was to identify and to analyse all antigens for VL serodiagnosis that have been published before 2018 in order to identify candidates and gaps in the pipeline for a new POC test in East Africa. Methods: A systematic literature search was performed on PubMed for original research articles on Leishmania-specific antigens for antibody detection of VL in humans. From each article, the following information was extracted: the antigen name, test format and characteristics, its reported sensitivity and specificity and study cohort specifications. Results: One hundred and seven articles containing information about 96 tests based on 89 different antigens were included in this study. Eighty six of these tests, comprising 80 antigens, were evaluated in phase I and II studies only. Only 20 antigens, all of which are native, contain a carbohydrate and/or lipid moiety. Twenty-four antigens, of which 7 are non-native, are composed of antigen mixtures. Nineteen tests, comprising 18 antigens, have been evaluated on East African specimens, of which only 2 (rK28 based immunochromatographic test and intact promastigote based indirect fluorescent antibody technique) consistently showed sensitivities above 94 and specificities above 97% in a phase III study and one in a phase II study (dot blot with SLA). Only rK28 is a non-native mixture of antigens which we consider suitable for further evaluation and implementation. Conclusions: The development pipeline for an alternative serodiagnostic test for VL is almost empty. Most antigens are not sufficiently evaluated. Non-protein antigens and antigen mixtures are being neglected. We propose to expand the evaluation of existing antigen candidates and to investigate the diagnostic potential of defined non-native carbohydrate and lipid antigens for VL serodiagnosis in East Africa. [ABSTRACT FROM AUTHOR]

Kala-azar, a worldwide disease, is caused by the parasitic protozoan species of genus Leishmania and transmitted by species of sandflies. Awareness of the population about the disease is essential to run a successful control-strategies program. This cross-sectional study was conducted to assess the awareness of residents about it and related practices in two highly-endemic areas of Bangladesh. In total, 511 household respondents were selected conveniently from two unions (Kushmail and Kanihari) under two sub-districts (Trishal and Fulbaria) of Mymensingh district. Interviewer-administered questionnaires were used for assessing the awareness of the respondents about kala-azar and their practices. Knowledge scores were categorized as poor (mean +1SD). Statistical tests were considered significant at p value of ≤5% (≤0.05). Chi-squared and Student’s t-tests were performed for statistical analysis. The mean knowledge score of the respondents of Kanihari union regarding kala-azar was significantly higher than that of the respondents of Kushmail union (mean ±SD, 4.30 ±0.86 versus 4.12 ±0.75, p = 0.002). Of the respondents, 11% and 20% had good (>5.04), 77% and 72% had average (3.43–5.04), 12% and 8% had poor (<3.43) (GAP) knowledge on kala-azar in Kushmail union and Kanihari union respectively. However, the mean knowledge score of the respondents of Kanihari union relating sandfly was significantly lower compared to that of the respondents of Kushmail union (mean ±SD, 2.49 ±0.79 versus 2.65 ±0.85, p = 0.03). Of them, 22% and 19% had good (>3.39), 67% and 64% had average (3.39–1.75), and 11% and 17% had poor (<1.75) knowledge on sandfly in Kushmail union and Kanihari union respectively. More than 70% of the respondents from Kushmail and Kanihari mentioned only injection as the preferred treatment of kala-azar. Eighty-seven percent and 88% of the respondents in the two unions had chosen upazilla health complex for the treatment of their kala-azar. About 50% of the respondents in Kushmail and Kanihari liked to use coil or mat as a preventive measure to avoid sandfly bites. The findings of this study indicate the importance of awareness and related practices of the community people in two endemic areas that will help implement the kala-azar-elimination program successfully in Bangladesh. [ABSTRACT FROM AUTHOR]

Canine Visceral Leishmaniasis (CVL) prevalence, spatial distribution and associated factors were assessed in four locations in Iguazú department in 2014 and in Puerto Iguazú city again in 2018. The city areas were divided into a grid of 400x400m cells. All cells were sampled in 2014 and a random subsampling was developed in 2018. In each cell, five dogs clustered in a ‘critical scenario’ (prone to have vectors) were sampled. A rapid immunochromatographic dipstick was used to detect antibodies against Leishmania infantum, confirming by lymph node smears observation and PCR. For Puerto Iguazú, Generalized Linear Models (GLMs) were constructed considering environmental, dog and clinical variables. Pearson's Chi square and Fisher's exact tests were employed to evaluate the association between CVL, dog clinical signs and infestation with other parasites. Cartographic outputs were made and Moran's I indices were calculated as spatial autocorrelation indicators. CVL prevalence rates were 26.18% in 2014 and 17.50% in 2018. No associations were established in environmental models, but dog age and repellent use were significant when running 2014 dog models. Clinical models showed significant associations between seropositive dogs and ophthalmological, dermal signs and onychogryphosis in 2014. In 2018, only adenomegaly was associated. The results of global Moran´s I were not significant but regarding local analysis, six sites in 2014 and one in 2018 presented autocorrelation with neighboring sites. The decrease in CVL prevalence may be associated to transmission stabilization, which could explain the lack of associations with dog-related variables. Further, spatial distribution of CVL is a poor evidence for design of transmission control measures but could be important in case of intensive parasite circulation or when the first autochthonous cases appear. For control success, sensitivity of diagnostic methods, political will and adequate material resources remain critical. Modeling of multiple variables will be required to identify factors that drive disease stabilization/destabilization. [ABSTRACT FROM AUTHOR]

Background: Visceral Leishmaniasis in humans presents with fever, anemia, and splenomegaly and can be lethal if not treated. Nevertheless, the majority of Leishmania infantum-infected individuals does not manifest symptoms and remain so provided they are not immunosuppressed. In this work, the performance of different tests was evaluated to detect asymptomatic individuals who were living in Teresina, Piauí state, Brazil, an endemic area for VL. Methodology: L. infantum-specific antibodies were detected by ELISA and two different rapid immunochromatographic (IC) diagnostic tests, Kalazar Detect and OnSite, and parasitic loads were detected by real time PCR [qPCR]. Additionally, we measured levels of the biomarkers monokine induced by IFN-γ (MIG) and IFN-γ-induced protein 10 (IP-10) before and after stimulation of whole blood with soluble Leishmania antigen [SLA]. Principal findings: Kalazar Detect and OnSite detected, respectively, 76% and 64% of patients presenting with active Visceral Leishmaniasis; 50% and 57% of patients remained positive in these tests, respectively, after treatment. Of the healthy participants in the study who were living in the endemic area, only 1.7% were positive with both of the IC tests. On the other hand, reactivity in ELISA tests revealed that 13% of these individuals presented asymptomatic infections; among VL patients, 84% presenting with active disease were reactive in ELISA, and after treatment, 55.5% were seropositive. L. infantum DNA was present in the blood of 37.9% of infected individuals living in the endemic area, while IP-10 and MIG biomarkers were detected in 26.7% of them. The greatest concordance of positivity occurred between ELISA and qPCR. Conclusion: The association of different techniques can detect asymptomatic infections, however, more research is necessary to develop ideal biomarkers that are simple to use in the clinic and in field studies in areas endemic for Visceral Leishmaniasis. [ABSTRACT FROM AUTHOR]

Introduction: Diagnosis and treatment for visceral leishmaniasis (VL) is considered to be delayed amongst poor, rural women in highly endemic districts of Bihar and Jharkhand. The objective of this study was to assess and understand barriers to VL diagnosis and treatment for women in endemic districts with a high burden of VL. Methods: The study used a stratified and purposive sample of 33 female patients with VL, 11 health staff, 11 local (unqualified) health providers and 12 groups of community elders drawn from ten districts in Bihar and four in Jharkhand with high burdens of VL. The study was conducted within an exploratory and inductive framework, using semi-structured in-depth interviews and discussions. Results: Women accessing treatment more quickly tended to move faster from treating their symptoms on their own to seeking care from local providers. Perception among female patients of the illness being not serious (owing to initially non-specific and mild symptoms), lack of money, prioritisation of household chores over their need to seek care and the absence of a male guardian to accompany them in seeking care at facilities worked together to drive these choices. Most patients and their families did not suspect VL as the cause for their non-specific symptoms, but when VL was suspected, treatment shopping ended. Lack of prioritization of women’s health issues appears to be a pervasive underlying factor. Public health facilities were not an early treatment choice for the majority, but where it was, the diagnosis of VL was often not considered when presenting with under 2 weeks of symptoms, nor were appropriate follow-up plans instituted. Conclusion: The insidious presentation of VL and the low prioritisation of women’s health need to be jointly addressed through messages that emphasise the importance of early diagnosis and treatment of disease, which is low-cost in time and money when managed in public health facilities. Clear messages that project prioritising women’s care-seeking over household work as a smart choice and the need for rallying male support are needed. Additionally, efforts to reduce missed opportunities through early case suspicion and engaging private providers to better counsel women with suspected VL could close critical gaps in the continuum of care. [ABSTRACT FROM AUTHOR]

In the present study, we assessed the annual screening coverage (i.e., the percentage of dogs that are screened for anti-Leishmania antibodies annually) in the municipality of Sobral, Ceará state, Brazil. Data on the number of dogs screened during 2008−2017 (except 2010) were obtained from the Centre for Zoonoses Control of Sobral. The annual screening coverage during 2012−2017 was calculated. Data on human visceral leishmaniasis (VL) cases during 2008−2017 were compiled from the National Disease Notification System. Correlation analyses were performed to assess the correlation between canine and human data. During 2008−2017, 73,964 dogs (range, 0 to 13,980 dogs/year) were serologically screened and 2,833 (3.8%) were positive. The annual screening coverage during 2012−2017 ranged from 11.1% to 45.7%. There were no significant correlations between the number of dogs culled and the number of human VL cases, canine positivity and human VL incidence, number of dogs culled and human VL incidence, or between canine positivity and number of human VL cases. An inconsistent and relatively low annual screening coverage was found in the study area, with no dog being screened in 2010 due to the lack of serological tests. Our results highlight that many dogs potentially infected with Leishmania infantum have been virtually overlooked by public health workers in the study area, perhaps with a negative, yet underestimated, impact on the control of canine and human VL. Hence, the failure of the dog culling strategy in controlling human VL in Brazil may be due to the low screening coverage and low percentage of culled dogs, rather than the absence of associations between canine and human infections. [ABSTRACT FROM AUTHOR]

In French Guiana, cutaneous leishmaniasis is highly endemic, whereas no autochthonous case of visceral leishmaniasis have been reported so far. However, due to its proximity to Brazil which is highly endemic for visceral leishmaniasis, and the high transboundary population flow, an epidemiological challenge could arise at any time. As an overseas department and region and the largest outermost region of the European Union, epidemiological surveillance of visceral leishmaniasis is of great importance. Our study aimed to investigate the presence of Leishmania spp. in domestic (dogs) and sylvatic (bats) animals from French Guiana. Over the 2008–2018 period, samples from 349 animals were collected. They included blood from 179 autochthonous dogs and 59 bats, spleen samples from 33 bats and, blood from 78 military working dogs (MWD) collected before their departure from continental France and at the end of their four-month stay in French Guiana. Samples were screened using real-time polymerase chain reaction (qPCR) assays targeting Leishmania DNA followed by sequencing of 18S rRNA, kDNA and ITS2 genes. L. infantum was detected in 2.3% (8/349) of animals with 1.7% (3/179) of autochthonous dogs, 5.1% (4/78) of MWD returning from French Guiana, whereas they were negative before their departure. One of them dates back to 2012. All these dogs were positive for serological tests. In addition, L. infantum DNA was detectable in one bat spleen sample, belonging to Carollia perspicillata species. We report here for the first time an infection with L. infantum in dogs and bat from French Guiana. Our results suggest the existence of potential reservoir and transmission cycle for visceral leishmaniasis, at least since 2012, which was unknown in this territory until now. Further studies are needed to determine how these animals were infected and which vectors are involved in the transmission in this area. [ABSTRACT FROM AUTHOR]

Background: Leishmania parasites are transmitted to vertebrate hosts by phlebotomine sandflies and, in humans, may cause tegumentary or visceral leishmaniasis. The role of PKR (dsRNA activated kinase) and Toll-like receptor 3 (TLR3) activation in the control of Leishmania infection highlights the importance of the engagement of RNA sensors, which are usually involved in the antiviral cell response, in the fate of parasitism by Leishmania. We tested the hypothesis that Phlebovirus, a subgroup of the Bunyaviridae, transmitted by sandflies, would interfere with Leishmania infection. Methodology/Principal findings: We tested two Phlebovirus isolates, Icoaraci and Pacui, from the rodents Nectomys sp. and Oryzomys sp., respectively, both natural sylvatic reservoir of Leishmania (Leishmania) amazonensis from the Amazon region. Phlebovirus coinfection with L. (L.) amazonensis in murine macrophages led to increased intracellular growth of L. (L.) amazonensis. Further studies with Icoaraci coinfection revealed the requirement of the PKR/IFN1 axis on the exacerbation of the parasite infection. L. (L.) amazonensis and Phlebovirus coinfection potentiated PKR activation and synergistically induced the expression of IFNβ and IL-10. Importantly, in vivo coinfection of C57BL/6 mice corroborated the in vitro data. The exacerbation effect of RNA virus on parasite infection may be specific because coinfection with dengue virus (DENV2) exerted the opposite effect on parasite load. Conclusions: Altogether, our data suggest that coinfections with specific RNA viruses shared by vectors or reservoirs of Leishmania may enhance and sustain the activation of host cellular RNA sensors, resulting in aggravation of the parasite infection. The present work highlights new perspectives for the investigation of antiviral pathways as important modulators of protozoan infections. [ABSTRACT FROM AUTHOR]

Phlebotomines have been recorded from a wide variety of habitats, and some of these vector species have shown preference for human environments, with high levels of adaptation. This study evaluated the degree of preference of these vectors for urban, rural, and forested environments (synanthropic behavior), as well as the diversity of these species, in three areas (forested, rural, and urban, exhibiting different degrees of anthropogenic changes) in a region of intense transmission of visceral leishmaniasis in Corumbá county, Mato Grosso do Sul, Brazil. Using light traps, sand fly specimens were collected from the three environments simultaneously, from May 2015 to April 2017, totaling 7 213 sand flies of 14 species in eight genera. Nuorteva’s synanthropy index was determined for the species Lutzomyia cruzi, Brumptomyia brumpti, Micropygomyia peresi, Lu. forattinii, Martinsmyia oliveirai and Evandromyia corumbaensis. Lutzomyia cruzi, the vector of Leishmania infantum in Corumbá, was the most abundant vector species, recorded from all three areas and sampling plots, on all 24 months investigated. This species exhibited the highest synanthropic index (+75.09), indicating a strong preference for urban environments. Brumptomyia brumpti, Micropygomyia peresi, Lu. forattinii, and Martinsmyia oliveirai showed preference, albeit not strong, for urban environments. Overall, males were more abundant than females (W = 490; p < 0.0001). High density, high synanthropic index, and sustained indoor presence were found for Mi. peresi in the rural area. Monitoring changes in the ecological behavior of sand flies is of vital importance, as these changes may indicate an increased likelihood of leishmaniasis emergence or reemergence. [ABSTRACT FROM AUTHOR]

Background: There is a pressing need for drug discovery against visceral leishmaniasis, a life-threatening protozoal infection, as the available chemotherapy is antiquated and not bereft of side effects. Plants as alternate drug resources has rewarded mankind in the past and aimed in this direction, we investigated the antileishmanial potential of Cinnamomum cassia. Methodology: Dichloromethane, ethanolic and aqueous fractions of C. cassia bark, prepared by sequential extraction, were appraised for their anti-promastigote activity along with apoptosis-inducing potential. The most potent, C. cassia dichloromethane fraction (CBD) was evaluated for anti-amastigote efficacy in infected macrophages and nitric oxide (NO) production studied. The in vivo antileishmanial efficacy was assessed in L. donovani infected BALB/c mice and hamsters and various correlates of host protective immunity ascertained. Toxicity profile of CBD was investigated in vitro against peritoneal macrophages and in vivo via alterations in liver and kidney functions. The plant secondary metabolites present in CBD were identified by gas chromatography-mass spectroscopy (GC-MS). Principal findings: CBD displayed significant anti-promastigote activity with 50% inhibitory concentration (IC50) of 33.6 μg ml-1 that was mediated via apoptosis. This was evidenced by mitochondrial membrane depolarization, increased proportion of cells in sub-G0-G1 phase, ROS production, PS externalization and DNA fragmentation (TUNEL assay). CBD also inhibited intracellular amastigote proliferation (IC50 14.06 μg ml-1) independent of NO production. The in vivo protection achieved was 80.91% (liver) and 82.92% (spleen) in mice and 75.61% (liver) and 78.93% (spleen) in hamsters indicating its profound therapeutic efficacy. CBD exhibited direct antileishmanial activity, as it did not specifically induce a T helper type (Th)-1-polarized mileu in cured hosts. This was evidenced by insignificant modulation of NO production, lymphoproliferation, DTH (delayed type hypersensitivity), serum IgG2a and IgG1 levels and production of Th2 cytokines (IL-4 and IL-10) along with restoration of pro-inflammatory Th1 cytokines (INF-γ, IL-12p70) to the normal range. CBD was devoid of any toxicity in vitro as well as in vivo. The chemical constituents, cinnamaldehyde and its derivatives present in CBD may have imparted the observed antileishmanial effect. Conclusions: Our study highlights the profound antileishmanial efficacy of C. cassia bark DCM fraction and merits its further exploration as a source of safe and effective antieishmanial. [ABSTRACT FROM AUTHOR]

Background: The search for diagnostic biomarkers has been profiting from a growing number of high quality sequenced genomes and freely available bioinformatic tools. These can be combined with wet lab experiments for a rational search. Improved, point-of-care diagnostic tests for visceral leishmaniasis (VL), early case detection and surveillance are required. Previous investigations demonstrated the potential of IgG1 as a biomarker for monitoring clinical status in rapid diagnostic tests (RDTs), although using a CLA as capturing antigen. Replacing the CLA by specific antigens would lead to more robust RDTs. Methodology: Immunoblots revealed L. donovani protein bands detected by IgG1 from VL patients. Upon confident identification of these antigens by mass spectrometry (MS), we searched for evidence of constitutive protein expression and presence of antigenic domains or high accessibility to B-cells. Selected candidates had their linear epitopes mapped with in silico algorithms. Multiple high-scoring predicted epitopes from the shortlisted proteins were screened in peptide arrays. The most promising candidate was tested in RDT prototypes using VL and nonendemic healthy control (NEHC) patient sera. Results: Over 90% of the proteins identified from the immunoblots did not satisfy the selection criteria and were excluded from the downstream epitope mapping. Screening of predicted epitope peptides from the shortlisted proteins identified the most reactive, for which the sensitivity for IgG1 was 84% (95% CI 60—97%) with Sudanese VL sera on RDT prototypes. None of the sera from NEHCs were positive. Conclusion: We employed in silico searches to reduce drastically the output of wet lab experiments, focusing on promising candidates containing selected protein features. By predicting epitopes in silico we screened a large number of peptides using arrays, identifying the most promising one, for which IgG1 sensitivity and specificity, with limited sample size, supported this proof of concept strategy for diagnostics discovery, which can be applied to the development of more robust IgG1 RDTs for monitoring clinical status in VL. [ABSTRACT FROM AUTHOR]

Background: Asymptomatic Leishmania donovani infections outnumber clinical presentations, however the predictors for development of active disease are not well known. We aimed to identify serological, immunological and genetic markers for progression from L. donovani infection to clinical Visceral Leishmaniasis (VL). Methods: We enrolled all residents >2 years of age in 27 VL endemic villages in Bihar (India). Blood samples collected on filter paper on two occasions 6–12 months apart, were tested for antibodies against L. donovani with rK39-ELISA and DAT. Sero converters, (negative for both tests in the first round but positive on either of the two during the second round) and controls (negative on both tests on both occasions) were followed for three years. At the start of follow-up venous blood was collected for the following tests: DAT, rK39- ELISA, Quantiferon assay, SNP/HLA genotyping and L.donovani specific quantitative PCR. Results: Among 1,606 subjects enrolled,17 (8/476 seroconverters and 9/1,130 controls) developed VL (OR 3.1; 95% CI 1.1–8.3). High DAT and rK39 ELISA antibody titers as well as positive qPCR were strongly and significantly associated with progression from seroconversion to VL with odds ratios of 19.1, 30.3 and 20.9 respectively. Most VL cases arose early (median 5 months) during follow-up. Conclusion: We confirmed the strong association between high DAT and/or rK39 titers and progression to disease among asymptomatic subjects and identified qPCR as an additional predictor. Low predictive values do not warrant prophylactic treatment but as most progressed to VL early during follow-up, careful oberservation of these subjects for at least 6 months is indicated. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis (VL) is the most severe clinical form of leishmaniasis, and if untreated may be fatal. It affects important organs of the immune system and is characterized by a specific immunosuppression, along with intense cellular activation and cytokine storm. Moreover, VL is now recognized as a systemic inflammatory response syndrome (SIRS), in which multiple cytokines and other pro-inflammatory molecules are released. The action of these inflammatory mediators may be considered risk factors for poor prognosis and death. Leptin, a hormone derived from adipose tissue, has been described with several immunoregulatory functions in vitro and in vivo Leishmania infection models, particularly for enhancing the macrophage microbicidal mechanisms. Considering that evaluation of immunologic parameters that may be associated with this clinical scenario may help to decrease VL lethality, we evaluated whether leptin is associated with VL pathogenesis. Thirty-one patients were recruited in the active phase of VL, of which 22 were followed up until one month after therapy (1mpt). Except for creatinine levels, all clinical parameters were altered in active VL patients, especially leucocyte counts and albumin and hemoglobin levels. Also, elevated levels of lipopolysaccharide (LPS), immunoglobulins (Ig)G1 and G3 anti-Leishmania and interleukins (IL)-6 and -10 were higher than in healthy individuals. In contrast, active VL patients presented diminished serum leptin levels and positive correlation with leukocytes counts and hemoglobin and albumin levels. After 1mpt, VL patients showed a significant increase in leptin levels, reaching values similar to healthy volunteers. As expected, only LPS levels remained elevated after 1mpt. These findings suggest that leptin levels are affected in Leishmania infection and the correlation with important parameters associated with the prognosis of VL points to the involvement of this molecule in VL immunopathogenesis. Additional studies are needed to evaluate the possibility of leptin as a prognostic marker of VL. [ABSTRACT FROM AUTHOR]

Background: Post Kala-azar Dermal Leishmaniasis (PKDL) develops in patients apparently cured of Visceral Leishmaniasis (VL), and is the strongest contender for being the disease reservoir. Therefore, existence of a few cases is sufficient to trigger an epidemic of VL in a given community, emphasizing the need for its active detection and in turn ensuring success of the current elimination program. This study explored the impact of active surveillance on the demographic profile of PKDL patients in West Bengal. Methodology/Principal findings: Patients with PKDL were recruited through passive (2003-date, n = 100) and active surveillance (2015-date, n = 202), the former from outpatient departments of dermatology in medical colleges in West Bengal and the latter through an active door-to-door survey in four VL hyper-endemic districts of West Bengal. Passive surveillance indicated a male preponderance and a predominance of polymorphic lesions, whereas active surveillance indicated absence of any gender bias and more importantly, macular PKDL constituted almost 50% of the population burden. In terms of polymorphic vs. macular PKDL, the former appeared at a later age, their disease duration was longer and had a higher parasite burden. In the polymorphic variant, the lesional distribution was asymmetrical, comprised of papules/nodules/macules that were present mainly in sun-exposed areas whereas in macular cases, the hypopigmented patches were diffusely present all over the body. Conclusions/Significance: Active surveillance unraveled a disease component whose demographic profile showed important differences with PKDL cases who sought treatment in government hospitals. Detection of a higher proportion of macular cases indicates that this variant is not an uncommon presentation as conventionally stated in text books, and should be studied in greater detail to ensure success of the ongoing Leishmaniasis elimination programme. [ABSTRACT FROM AUTHOR]

Objective: To assess the effectiveness of community-wide deployment of insecticide–impregnated collars for dogs- the reservoir of Leishmania infantum–to reduce infantile clinical visceral leishmaniasis (VL). Methods: A pair matched–cluster randomised controlled trial involving 40 collared and 40 uncollared control villages (161 [95% C.L.s: 136, 187] children per cluster), was designed to detect a 55% reduction in 48 month confirmed VL case incidence. The intervention study was designed by the authors, but implemented by the Leishmaniasis Control Program in NW Iran, from 2002 to 2006. Results: The collars provided 50% (95% C.I. 17·8%–70·0%) protection against infantile VL incidence (0·95/1000/yr compared to 1·75/1000/yr). Reductions in incidence were observed across 76% (22/29) of collared villages compared to pair–matched control villages, with 31 fewer cases by the end of the trial period. In 11 paired villages, no further cases were recorded post–intervention, whereas in 7 collared villages there were 9 new clinical cases relative to controls. Over the trial period, 6,835 collars were fitted at the beginning of the 4 month sand fly season, of which 6.9% (95% C.I. 6.25%, 7.56%) were lost but rapidly replaced. Collar coverage (percent dogs collared) per village varied between 66% and 100%, with a mean annual coverage of 87% (95% C.I. 84·2, 89·0%). The variation in post-intervention clinical VL incidence was not associated with collar coverage, dog population size, implementation logistics, dog owner compliance, or other demographic variables tested. Larger reductions and greater persistence in incident case numbers (indicative of transmission) were observed in villages with higher pre-existing VL case incidence. Conclusion: Community–wide deployment of collars can provide a significant level of protection against infantile clinical VL, achieved in this study by the local VL Control Program, demonstrating attributes desirable of a sustainable public health program. The effectiveness is not dissimilar to the community-level protection provided against human and canine infection with L. infantum. [ABSTRACT FROM AUTHOR]

Visceral Leishmaniasis is a very dangerous form of leishmaniasis and, shorn of appropriate diagnosis and handling, it leads to death and physical disability. Depicting the spatiotemporal pattern of disease is important for disease regulator and deterrence strategies. Spatiotemporal modeling has distended broad veneration in recent years. Spatial and spatiotemporal disease modeling is extensively used for the analysis of registry data and usually articulated in a hierarchical Bayesian framework. In this study, we have developed the hierarchical spatiotemporal Bayesian modeling of the infected rate of Visceral leishmaniasis in Human (VLH). We applied the Stochastics Partial Differential Equation (SPDE) approach for a spatiotemporal hierarchical model for Visceral leishmaniasis in human (VLH) that involves a GF and a state process is associated with an autoregressive order one temporal dynamics and the spatially correlated error term, along with the effect of land shield, metrological, demographic, socio-demographic and geographical covariates in an endemic area of Amhara regional state, Ethiopia. The model encompasses a Gaussian Field (GF), affected by an error term, and a state process described by a first-order autoregressive dynamic model and spatially correlated innovations. A hierarchical model including spatially and temporally correlated errors was fit to the infected rate of Visceral leishmaniasis in human (VLH) weekly data from January 2015 to December 2017 using the R package R-INLA, which allows for Bayesian modeling using the stochastic partial differential equation (SPDE) approach. We found that the mean weekly temperature had a significant positive association with infected rate of VLH. Moreover, net migration rate, clean water coverage, average number of households, population density per square kilometer, average number of persons per household unit, education coverage, health facility coverage, mortality rate, and sex ratio had a significant association with the infected rate of visceral leishmaniasis (VLH) in the region. In this study, we investigated the dynamic spatiotemporal modeling of Visceral leishmaniasis in Human (VLH) through a stochastic partial differential equation approach (SPDE) using integrated nested Laplace approximation (INLA). Our study had confirmed both metrological, demographic, sociodemographic and geographic covariates had a significant association with the infected rate of visceral leishmaniasis (VLH) in the region. [ABSTRACT FROM AUTHOR]

Background: The long-term treatment outcome of visceral leishmaniasis (VL) patients with HIV co-infection is complicated by a high rate of relapse, especially when the CD4 count is low. Although use of secondary prophylaxis is recommended, it is not routinely practiced and data on its effectiveness and safety are limited. Methods: A prospective cohort study was conducted in Northwest Ethiopia from August 2014 to August 2017 (NCT02011958). HIV-VL patients were followed for up to 12 months. Patients with CD4 cell counts below 200/μL at the end of VL treatment received pentamidine prophylaxis starting one month after parasitological cure, while those with CD4 count ≥200 cells/μL were followed without secondary prophylaxis. Compliance, safety and relapse-free survival, using Kaplan-Meier analysis methods to account for variable time at risk, were summarised. Risk factors for relapse or death were analysed. Results: Fifty-four HIV patients were followed. The probability of relapse-free survival at one year was 50% (95% confidence interval [CI]: 35–63%): 53% (30–71%) in 22 patients with CD4 ≥200 cells/μL without pentamidine prophylaxis and 46% (26–63%) in 29 with CD4 <200 cells/μL who started pentamidine. Three patients with CD4 <200 cells/μL did not start pentamidine. Amongst those with CD4 ≥200 cells/μL, VL relapse was an independent risk factor for subsequent relapse or death (adjusted rate ratio: 5.42, 95% CI: 1.1–25.8). Except for one case of renal failure which was considered possibly related to pentamidine, there were no drug-related safety concerns. Conclusion: The relapse-free survival rate for VL patients with HIV was low. Relapse-free survival of patients with CD4 count <200cells/μL given pentamidine secondary prophylaxis appeared to be comparable to patients with a CD4 count ≥200 cells/μL not given prophylaxis. Patients with relapsed VL are at higher risk for subsequent relapse and should be considered a priority for secondary prophylaxis, irrespective of their CD4 count. [ABSTRACT FROM AUTHOR]

Background: Miltefosine (MF) is the only oral drug available for treatment of visceral leishmaniasis (VL) and post-kala-azar dermal leishmaniasis (PKDL). Although the drug is effective and well tolerated in treatment of VL, the efficacy and safety of MF for longer treatment durations (>28 days) in PKDL remains unclear. This study provides an overview of the current knowledge about safety and efficacy of long treatment courses with MF in PKDL, as a strategy in the VL elimination in South Asia. Methodology/Principal findings: Literature was searched systematically for articles investigating MF treatment in PKDL. A meta-analysis included eight studies (total 324 PKDL patients) to estimate the efficacy of MF in longer treatment regimens (range 6–16 weeks). We found a per-protocol (PP) initial cure rate of 95.2% (95%CI 89.6–100.8) and a PP definite cure rate of 90% (95%CI 81.6–96.3). Descriptive analysis showed that 20% of patients experienced adverse events, which mostly had an onset in the first week of treatment and were likely to get more severe after four weeks of treatment. Gastrointestinal (GI) side effects such as vomiting, nausea, diarrhoea, and abdominal pain were most common. Conclusions/Significance: Longer treatment regimens with MF are effective in PKDL patients in India, however with the caveat that the efficacy has recently been observed to decline. GI side effects are frequent, although mostly mild or moderate. However, on the basis of limited data, we cannot conclude that longer MF treatment regimens are safe. Moreover, VL and PKDL pharmacovigilance studies indicate a risk for serious adverse events, questioning the safety of MF. The provision of safer treatment regimens for PKDL patients are therefore recommended. Until these regimens are identified, it should be considered to halt the use of MF monotherapy for PKDL in order to preserve the drug’s efficacy. [ABSTRACT FROM AUTHOR]

The arsenic contamination of ground water in visceral leishmaniasis (VL) endemic areas in Bihar, India leads to human exposure through drinking water. Possibly, the consumed arsenic (As) accumulates in the tissues of VL patients, who subsequently internalize intracellular amastigotes to confer resistance against chemotherapy to the parasite, leading to modulation in the host’s immune response. This hypothesis appears to be consistent with the in vitro findings that in arsenic-exposed parasites, the mitochondrial membrane potential became depolarized, whereas the reduced thiol and lactate production was overexpressed with enhanced glucose consumption; therefore, the reduced thiol possibly supports an immunosuppressive state in the host cells. This observation was well supported by the down-regulated expression of pro-inflammatory cytokines (IL-2, IL-12, IFN-γ, and TNF-α) with a suppressed anti-leishmanial function of macrophage (NO, ROS). In contrast, the pathophysiological mechanism of VL has received ample support by the promotion of Th2 cytokines (IL-4 and IL-10) in the presence of arsenic-exposed Leishmania parasites (LdAS). Dysfunction of mitochondria and the overexpression of lactate production raise the possibility of the Warburg effect being operative through the up-regulation of glucose consumption by parasites to enhance the energy production, possibly augmenting virulence. Therefore, we surmise from our data that arsenic exposure to Leishmania donovani modulates the immune response and infection pattern by impairing parasite function, which may affect the anti-leishmanial effect in VL. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis (VL) or kala-azar, the most severe form of leishmaniasis, can lead to death if not properly diagnosed and treated. Correct identification of infected patients and reservoirs is vital for controlling the spread of leishmaniasis. Current diagnostic kits for leishmaniasis show high sensitivity and specificity, but can also result in false negatives and cross reactions with related parasitic infections. New diagnostic methods with greater accuracy are urgently needed for diagnosis of leishmaniasis. In this study, we aimed to uncover a new highly effective antigen for the diagnosis of visceral leishmaniasis in dogs and humans, aiming to improve the accuracy compared with those of current methods of diagnosis. Initially, in-silico epitope prediction analyses identified several potential B-cell epitopes in the repetitive region of Leishmania infantum kinesin, which co-localized with predicted structural disordered regions, suggesting high potential for antigenicity. Based on this analysis, 8.5 genomic motifs, which encode the repetitive sequence of 39 degenerate amino acids, were selected for recombinant expression. BLASTn analysis of this repetitive region indicated that it is absent in the T. cruzi parasite, which is closely related to Leishmania, indicating the specificity of this region. This potentially antigenic protein, named recombinant kinesin degenerated derived repeat (rKDDR), was recombinantly expressed in Escherichia coli BL21-Star using the pET28a-TEV expression vector. We then evaluated the performance of rKDDR in correctly diagnosing Leishmania infection and compared this new assay with currently used diagnostic tests for leishmaniasis. rKDDR showed greater sensitivity and specificity in correctly diagnosing leishmaniasis both in human (sensitivity 92.86% and specificity 100%) and canine (sensitivity 88.54% and specificity 97.30%) sera compared with those of rK39 (human: sensitivity 90.48% and specificity 97.92%; canine: sensitivity 78.13% and specificity 90.09%). In addition, the rKDDR-ELISA outperformed the EIE-LVC kit, which is the serologic kit recommended by the Brazilian Ministry of Health for the diagnosis of canine visceral leishmaniasis. These results indicate that rKDDR is a highly promising candidate for diagnosis of visceral leishmaniasis, and is more accurate than the currently used gold-standard antigens. [ABSTRACT FROM AUTHOR]

Background: Visceral leishmaniasis (VL) in human immunodeficiency virus (HIV) co-infected patients requires special case management. AmBisome monotherapy at 40 mg/kg is recommended by the World Health Organization. The objective of the study was to assess if a combination of a lower dose of AmBisome with miltefosine would show acceptable efficacy at the end of treatment. Methodology/Principal findings: An open-label, non-comparative randomized trial of AmBisome (30 mg/kg) with miltefosine (100 mg/day for 28 days), and AmBisome monotherapy (40 mg/kg) was conducted in Ethiopian VL patients co-infected with HIV (NCT02011958). A sequential design was used with a triangular continuation region. The primary outcome was parasite clearance at day 29, after the first round of treatment. Patients with clinical improvement but without parasite clearance at day 29 received a second round of the allocated treatment. Efficacy was evaluated again at day 58, after completion of treatment. Recruitment was stopped after inclusion of 19 and 39 patients in monotherapy and combination arms respectively, as per pre-specified stopping rules. At D29, intention-to-treat efficacy in the AmBisome arm was 70% (95% CI 45–87%) in the unadjusted analysis, and 50% (95% CI 27–73%) in the adjusted analysis, while in the combination arm, it was 81% (95% CI 67–90%) and 67% (95% CI 48–82%) respectively. At D58, the adjusted efficacy was 55% (95% CI 32–78%) in the monotherapy arm, and 88% (95% CI 79–98%) in the combination arm. No major safety concerns related to the study medication were identified. Ten SAEs were observed within the treatment period, and 4 deaths unrelated to the study medication. Conclusions/Significance: The extended treatment strategy with the combination regimen showed the highest documented efficacy in HIV-VL patients; these results support a recommendation of this regimen as first-line treatment strategy for HIV-VL patients in eastern Africa. Trial registration number: . [ABSTRACT FROM AUTHOR]

In South America, the Protist parasite that causes visceral leishmaniasis, a potentially fatal human disease, is transmitted by blood-feeding female Lutzomyia longipalpis sand flies. A synthetic copy of the male produced sex-aggregation pheromone offers new opportunities for vector control applications. We have previously shown that the pheromone placed in plastic sachets (lures) can attract both females and males to insecticide treated sites for up to 3 months. To use the pheromone lure in a control program we need to understand how the application of lures in the field can be optimised. In this study we investigated the effect of increasing the number of lures and their proximity to each other on their ability to attract Lu. longipalpis. Also for the first time we applied a Bayesian log-linear model rather than a classic simple (deterministic) log-linear model to fully exploit the field-collected data. We found that sand fly response to pheromone is significantly related to the quantity of pheromone and is not influenced by the proximity of other pheromone sources. Thus sand flies are attracted to the pheromone source at a non-linear rate determined by the amount of pheromone being released. This rate is independent of the proximity of other pheromone releasing traps and indicates the role of the pheromone in aggregation formation. These results have important implications for optimisation of the pheromone as a vector control tool and indicate that multiple lures placed in relatively close proximity to each other (5 m apart) are unlikely to interfere with one another. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis (VL) still represents a serious public health problem in Brazil due to the inefficiency of the control measures currently employed, that included early diagnosis and treatment of human cases, vector control, euthanasia of infected dogs and, recently approved in Brazil, treatment with Milteforam drug. Effective clinical management depend largely on early and unequivocal diagnosis, however, cross-reactivity have also been described in serological tests, especially when it refers to individuals from areas where Chagas’ disease is also present. Thus, to discover new antigens to improve the current serological tests for VL diagnosis is urgently needed. Here, we performed an immunogenomic screen strategy to identify conserved linear B-cell epitopes in the predicted L. infantum proteome using the following criteria: i) proteins expressed in the stages found in the vertebrate host, amastigote stage, and secreted/excreted, to guarantee greater exposure to the immune system; ii) divergent from proteins present in other infectious disease pathogens with incidence in endemic areas for VL, as T. cruzi; iii) highly antigenic to humans with different genetic backgrounds, independently of the clinical stage of the disease; iv) stable and adaptable to quality-control tests to guarantee reproducibility; v) using statistical analysis to determine a suitable sample size to evaluate accuracy of diagnostic tests established by receiver operating characteristic strategy. We selected six predicted linear B-cell epitopes from three proteins of L. infantum parasite. The results demonstrated that a mixture of peptides (Mix IV: peptides 3+6) were able to identify VL cases and simultaneously able to discriminate infections caused by T. cruzi parasite with high accuracy (100.00%) and perfect agreement (Kappa index = 1.000) with direct methods performed by laboratories in Brazil. The results also demonstrated that peptide-6, Mix III (peptides 2+6) and I (peptides 2+3+6) are potential antigens able to used in VL diagnosis, represented by high accuracy (Ac = 99.52%, 99.52% and 98.56%, respectively). This study represents an interesting strategy for discovery new antigens applied to serologic diagnosis which will contribute to the improvement of the diagnosis of VL and, consequently, may help in the prevention, control and treatment of the disease in endemic areas of Brazil. [ABSTRACT FROM AUTHOR]

To reduce morbidity and mortality caused by visceral leishmaniasis (VL), the Brazilian Visceral Leishmaniasis Control and Surveillance Program promotes the diagnosis and treatment of cases, vector control, euthanasia of seropositive dogs, and health education. Nevertheless, the effectiveness of these measures is questionable as they lead to little reduction in the transmission of the disease. Thus, the effectiveness of strategies such as insecticide-impregnated collars, spot-on insecticides, and immunization of dogs should be assessed. Herein, we evaluated the effectiveness of deltamethrin-impregnated collars on reducing the incidence of Leishmania infantum infection in dogs living in an endemic area of VL. An intervention study was conducted and a total 5,850 dogs were analyzed in baseline. Of these 3,742 seronegative dogs were divided into two groups: collared and uncollared (control). Dogs were followed for 12 months and three interventions were performed. The Cox regression model was used to evaluate the effectiveness of the collar. All analyzes were performed by Intention-to-treat and per-protocol. By intention-to-treat, the incidence rates of L. infantum infection were 7.5 and 7.9 in the collar group, and 6.5 and 13.2 per 1,000 dogs-months in the control group after 6 and 12 months, respectively. In the per-protocol analysis, the incidence rates in the control group were similar to those observed in the intention-to-treat analysis. In the collar group, the incidence rate was 5.1/1,000 dogs-months after 6 and 12 months. The effectiveness by intention-to-treat after adjustment by the multivariate Cox model was 48%. In the analysis per-protocol, the effectiveness increased to 63%. Although collar use was effective when it was evaluated by intention-to-treat, higher effectiveness was found in the per-protocol analysis after one year of follow-up. The data emphasize the importance of the uninterrupted use of deltamethrin-impregnated collars to increase protection against canine VL. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis (VL) in humans is a chronic and often fatal disease if left untreated. Dogs appear to be the main reservoir host for L. infantum infection, however, in many regions other canids such as jackals, foxes, wolves and other mammals, such as hares or black rats, have been implicated as wild reservoirs. Most dogs cannot form an effective immune response against this infection, and this could be modulated by small non-coding RNAs, called microRNAs, responsible for post-transcriptional control of gene expression. Here, we evaluated the expression of miRNAs in peripheral blood mononuclear cells (PBMC) of symptomatic dogs naturally infected with Leishmania (L.) infantum (n = 10) and compared to those of healthy dogs (n = 5). Microarray analysis revealed that miR-21, miR-424, miR-194 and miR-451 had a 3-fold increase in expression, miR-192, miR-503, and miR-371 had a 2-fold increase in expression, whereas a 2-fold reduction in expression was observed for miR-150 and miR-574. Real-time PCR validated the differential expression of miR-21, miR-150, miR-451, miR-192, miR-194, and miR-371. Parasite load of PBMC was measured by real-time PCR and correlated to the differentially expressed miRNAs, showing a strong positive correlation with expression of miR-194, a regular positive correlation with miR-371 expression, and a moderate negative correlation with miR-150 expression in PBMC. These findings suggest that Leishmania infection interferes with miRNAs expression in PBMC, and their correlation with parasite load may help in the identification of therapeutic targets in Canine Visceral Leishmaniasis (CVL). [ABSTRACT FROM AUTHOR]

Background: The World Health Organization has targeted elimination of visceral leishmaniasis (VL) in the Indian subcontinent (ISC) by 2020. Despite distinctive decline seen in the number of VL cases in ISC, there is still a quest for development of a diagnostic test which has the utility for detection of active infection and relapse cases and as a test of cure. The present study validated the sensitivity and specificity of SYBR Green I based closed tube LAMP assay reported by us for diagnosis of VL. Methodology: The validation study was carried out at two endemic sites in India, located at Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Patna and Institute of Medical Sciences (IMS), Banaras Hindu University (BHU), Varanasi. Standard operating protocols were provided at the two sites for applying LAMP assay on confirmed VL cases. The diagnostic accuracy of LAMP assay was evaluated by Receiver operator curve (ROC) analysis. Furthermore, a simplified LAMP assay based on direct blood lysis, DBL-LAMP, was developed and verified for its diagnostic accuracy. Principal findings: A total of 267 eligible participants were included in the study which comprised of 179 VL cases and 88 controls. Sensitivity and specificity of the LAMP assay were 98.32% (95% C.I– 95.2–99.7%) and 96.59% (95% C.I.-90.4–99.3%), respectively. ROC curve analysis depicted no significant difference between area under curve (AUCROC) for LAMP assay and rK39 RDT, indicative of LAMP as an excellent diagnostic test. DBL-LAMP assay, performed on 67 VL and 100 control samples, yielded a sensitivity of 93.05% (95% C.I- 84.75–97%) and specificity of 100% (95% C.I.- 96.30–100%). Conclusions/Significance: The validated closed tube LAMP for diagnosis of VL will provide impetus to the ongoing VL elimination programme in ISC. The assay based on direct blood lysis promotes its scope for application in field settings by further reducing time and cost. [ABSTRACT FROM AUTHOR]

Background: Ethiopia bears a high burden of visceral leishmaniasis (VL). Early access to VL diagnosis and care improves clinical prognosis and reduces transmission from infected humans; however, significant obstacles exist. The approximate 250,000 seasonal mobile workers (MW) employed annually in northwestern Ethiopia may be particularly disadvantaged and at risk of VL acquisition and death. Our study aimed to assess barriers, and recommend interventions to increase access, to VL diagnosis and care among MWs. Methodology/Principal findings: In 2017, 50 interviews and 11 focus group discussions were conducted with MWs, mobile residents, VL patients and caretakers, community leaders and healthcare workers in Kafta Humera District, Tigray. Participants reported high vulnerability to VL among MWs and residents engaged in transitory work. Multiple visits to health facilities were consistently needed to access VL diagnosis. Inadequate healthcare worker training, diagnostic test kit unavailability at the primary healthcare level, lack of VL awareness, insufficient finances for care-seeking and prioritization of income-generating activities were significant barriers to diagnosis and care. Social (decision-making and financial) support strongly and positively influenced care-seeking; workers unable to receive salary advances, compensation for partial work, or peer assistance for contract completion were particularly disadvantaged. Participants recommended the government/stakeholders intervene to ensure: MWs access to bed-nets, food, shelter, water, and healthcare at farms or sick leave; decentralization of diagnostic tests to primary healthcare facilities; surplus medications/staff during the peak season; improved referral/feedback/reporting/training within the health system; free comprehensive healthcare for all VL-related services; and community health education. Conclusions/Significance: Contrary to what health policy for VL dictates in this endemic setting, study participants reported very poor access to diagnosis and, consequently, significantly delayed access to treatment. Interventions tailored to the socio-economic and health needs of MWs (and other persons suffering from VL) are urgently needed to reduce health disparities and the VL burden. [ABSTRACT FROM AUTHOR]