Immunological factors, but not clinical features, predict visceral leishmaniasis relapse in patients co-infected with HIV.

Visceral leishmaniasis (VL) has emerged as a clinically important opportunistic infection in HIV patients, as VL/HIV co-infected patients suffer from frequent VL relapse. Here, we follow cohorts of VL patients with or without HIV in Ethiopia. By the end of the study, 78.1% of VL/HIV-but none of the VL patients-experience VL relapse. Despite a clinically defined cure, VL/HIV patients maintain higher parasite loads, lower BMI, hepatosplenomegaly, and pancytopenia. We identify three immunological markers associated with VL relapse in VL/HIV patients: (1) failure to restore antigen-specific production of IFN-γ, (2) persistently lower CD4+ T cell counts, and (3) higher expression of PD1 on CD4+ and CD8+ T cells. We show that these three markers, which can be measured in primary hospital settings in Ethiopia, combine well in predicting VL relapse. The use of our prediction model has the potential to improve disease management and patient care.

Assessment of liver function test and associated factors among visceral leishmaniasis patients attending university of gondar leishmaniasis research and treatment center, Northwest Ethiopia.

BACKGROUND: Visceral leishmaniasis (VL) is one of the major public health burden, mainly distributed throughout tropical and subtropical regions of the world. Among the Sub-Saharan African countries, Ethiopia is the second most affected country with VL. An Alteration of liver function is a typical manifestation of the disease. OBJECTIVE: The purpose of conducting this study was to assess liver function tests and associated risk factors among VL patients at Leishmaniasis Research and Treatment Center of University of Gondar Comprehensive Specialized Hospital, North West Ethiopia. METHOD: Hospital based comparative cross-sectional study design was conducted. A total of 102 study participants were involved in this study. Newly diagnosed VL patients who were attended at Leishmaniasis Research and Treatment Center of University of Gondar Comprehensive Specialized Hospital from 21st February 2020 to 30th September 2020 were included under case group category. On the other hand, age-sex matched apparently healthy study subjects were categorized as control group. Written consent was obtained willingness of patients to participate after ethical clearance was obtained from the Institutional Review Board of School of Medicine, University of Gondar. After overnight fasting, 5ml venous blood was drawn from both VL patients and controls to evaluate liver function tests, including AST, ALT, total bilirubin, albumin, and total protein. Thus, senior health professionals (laboratory technologist) investigate the results using Cobas Integra 400 Plus clinical chemistry analyzer. Data was entered into Epi-data version 4.6 and exported to STATA 14 for analysis of liver function tests and associated risk factors. RESULT: The result of this study showed that significant mean difference was exhibited in aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin, serum albumin, and total protein level among VL patients and controls. It showed that there was a statistically significant elevation in the level of AST, ALT, and total bilirubin among cases as compared to control. The serum AST level was significantly (p<0.001) elevated among cases as compared to controls. Serum ALT was significantly (p<0.001) elevated among cases compared to controls. Additionally, the total serum bilirubin level was significantly increased (P<0.001) among cases as compared to controls. There was a statistically significant (P<0.001) reduction of serum albumin level among VL patients as compared to controls. Similarly, serum total protein was significantly (P<0.001) reduced in VL patients than control groups. CONCLUSION: There were significantly higher mean levels of serum AST, ALT, and total bilirubin among VL patients as compared to controls. On the other hand, VL patients showed significantly lowered level of albumin and total protein as compared to controls.

Association between Hypertriglyceridemia and Disease Severity in Visceral Leishmaniasis.

Visceral leishmaniasis (VL) is a tropical disease endemic to Brazil. The clinical manifestations of the infection range from asymptomatic to severe. In VL, changes in lipid metabolism, such as hypocholesterolemia and hypertriglyceridemia, occur that are believed to be related to its progression and severity. This study investigated the associations between serum levels of cholesterol, triglycerides, and lipoproteins (high-density lipoprotein, low-density lipoprotein, and very low-density lipoprotein) with clinical and hematological parameters that predict severity in a case series of 83 VL patients. Severely ill patients had higher mean serum triglyceride levels than non-severely ill patients. There was a significant positive correlation between disease severity score and serum triglyceride levels, very low-density lipoprotein, international normalized ratio for prothrombin time test, total bilirubin, and age. An inverse correlation was detected between the disease severity score and mean platelet and neutrophil counts. Hypertriglyceridemia can be a prognostic indicator of severity in patients diagnosed with VL.

Acute kidney injury in patients with Visceral Leishmaniasis in Northwest Ethiopia.

BACKGROUND: Visceral Leishmaniasis (VL) is a neglected tropical disease endemic to several countries including Ethiopia. Outside of Africa, kidney involvement in VL is frequent and associated with increased mortality. There is however limited data on acute kidney injury (AKI) in VL patients in East-Africa, particularly in areas with high rates of HIV co-infection. This study aims to determine the prevalence, characteristics and associated factors of AKI in VL patients in Northwest Ethiopia. METHODS: A hospital based retrospective patient record analysis was conducted including patients treated for VL from January 2019 to December 2019 at the Leishmaniasis Research and Treatment Center (LRTC), Gondar, Ethiopia. Patients that were enrolled in ongoing clinical trials at the study site and those with significant incomplete data were excluded. Data was analyzed using SPSS version 20. P values were considered significant if < 0.05. RESULTS: Among 352 VL patients treated at LRTC during the study period, 298 were included in the study. All were male patients except two; the median age was 23 years (IQR: 20-27). The overall prevalence of AKI among VL patients was 17.4% (confidence interval (CI): 13.6%-22.2%). Pre-renal azotemia (57%) and drug-induced AKI (50%) were the main etiologies of AKI at admission and post-admission respectively. Proteinuria and hematuria occurred in 85% and 42% of AKI patients respectively. Multivariate logistic regression revealed HIV co-infection (adjusted odds ratio (AOR): 6.01 95% CI: 1.99-18.27, p = 0.001) and other concomitant infections (AOR: 3.44 95% CI: 1.37-8.65, p = 0.009) to be independently associated with AKI. CONCLUSION: AKI is a frequent complication in Ethiopian VL patients. Other renal manifestations included proteinuria, hematuria, and pyuria. HIV co-infection and other concomitant infections were significantly associated with AKI. Further studies are needed to quantify proteinuria and evaluate the influence of AKI on the treatment course, morbidity and mortality in VL patients.

In-situ proliferation contributes to the accumulation of myeloid cells in the spleen during progressive experimental visceral leishmaniasis.

Visceral leishmaniasis (VL) is characterized by expansion of myeloid cells in the liver and spleen, which leads to a severe splenomegaly associated with higher risk of mortality. This increased cellularity is thought to be a consequence of recruitment of cells to the viscera. We studied whether the local proliferation of splenic myeloid cells contributes to increased splenic cellularity. We found that a monocyte-like population of adherent splenic cells from Leishmania donovani-infected hamsters had enhanced replicative capacity ex vivo and in vivo (BrdU incorporation, p<0.0001). In vitro assays demonstrated that proliferation was more pronounced in the proinflammatory M1 environment and that intracellular infection prevented proliferation. Secondary analysis of the published splenic transcriptome in the hamster model of progressive VL revealed a gene expression signature that included division of tumoral cells (Z = 2.0), cell cycle progression (Z = 2.3), hematopoiesis (Z = 2.8), proliferation of stem cells (Z = 2.5) and overexpression of proto-oncogenes. Regulators of myeloid cell proliferation were predicted in-silico (CSF2, TLR4, IFNG, IL-6, IL-4, RTK signaling, and STAT3). The in-silico prediction was confirmed with chemical inhibitors of PI3K/AKT, MAPK and STAT3 which decreased splenic myeloid cell division ex vivo. Hamsters infected with L. donovani treated with a STAT3 inhibitor had reduced in situ splenic myeloid proliferation (p = 0.03) and parasite burden. We conclude that monocyte-like myeloid cells have increased STAT3-dependent proliferation in the spleen of hamsters with visceral leishmaniasis and that inhibition of STAT3 reduces myeloid cell proliferation and parasite burden.

Clinical features and treatment outcomes of visceral leishmaniasis patients admitted to three centers in Oromia, Ethiopia.

INTRODUCTION: In three health care facilities in the Oromia region, the aim of this study is to report on 1) the number of VL cases registered over time (2013-2018) and 2) the clinical profile, type of treatment used and response to treatment. METHODOLOGY: A retrospective cohort study was conducted among all VL cases admitted with a diagnosis of VL. RESULTS: A total of 434 VL cases were registered at the three health facilities, but patient files were available for only 188. Most (51.6%) were children and only three presented with VL relapse. 78 (41.5%) of the 188 patients presented within one month of symptom onset. Concurrent severe acute malnutrition (27.1%), tuberculosis (6.4%) and malaria (6.4%) were common. There were only two cases with HIV coinfection. Fourty-three percent were treated with antimonials, 34% with antimonials combined with paromomycin and 23% with AmBisome. Amongst the 188 patients with patient files there were no deaths and one treatment failure. Six months outcome data were however missing for all. Aggregated data from the 434 VL cases reported three deaths, two treatment failures and one relapse. CONCLUSIONS: Children were most commonly affected, suggesting long-term endemicity. While short-term outcomes are encouraging, long-term follow-up data are required.

Pulmonary involvement in human visceral leishmaniasis: Clinical and tomographic evaluation.

Visceral leishmaniasis (VL) is a severe, systemic and potentially lethal parasitosis. The lung, like any other organ, can be affected in VL, and interstitial pneumonitis has been described in past decades. This research aimed to bring more recent knowledge about respiratory impairment in VL, characterizing pulmonary involvement through clinical, radiographic and tomographic evaluation. This is an observational, cross-sectional study that underwent clinical evaluation, radiography and high-resolution computed tomography of the chest in patients admitted with the diagnosis of VL in a university service in Northeast Brazil, from January 2015 to July 2018. The sample consisted of 42 patients. Computed tomography was considered abnormal in 59% of patients. Images compatible with pulmonary interstitial involvement were predominant (50%). The most observed respiratory symptom was cough (33.3%), followed by tachypnea (14.1%). Chest radiography was altered in only four patients. VL is a disease characterized by systemic involvement and broad spectrum of clinical manifestations. The respiratory symptoms and tomographic alterations found show that the involvement of respiratory system in VL deserves attention because it is more common than previously thought. Chest X-ray may not reveal this impairment.

Role of IL-17 gene polymorphism in Indian kala-azar.

BACKGROUND & OBJECTIVES: Visceral leishmaniasis or kala-azar is a fatal protozoan disease caused by an obligate intracellular parasite, Leishmania donovani. Susceptibility, establishment of infection and severity of this disease depend upon many factors, but it is the host immune system that plays decisive role in disease progression. Keeping this view into consideration, we investigated the probable relationship between polymorphisms rs2275913 and rs8193036 in IL-17 gene, and its association as a risk factor for kala-azar in an endemic population of Assam, India. METHODS: A total of 209 subjects, 76 kala-azar cases (male: 46, female: 30, mean age ± SD: 34.60 ± 12.61) and 133 controls (male: 66, female: 67, mean age ± SD: 33.35 ± 14.48) were included in this study. We analysed the polymorphisms, rs2275913 and rs8193036 by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. The data were analysed using logistic regression analysis and SPSS software. RESULTS: The results revealed that the mutant rs8193036 TT genotype conferred 4.7-fold higher risk for kala-azar (p = 0.00991, OR = 4.72, 95% CI = 1.330-16.911). A significant difference was found between the allele frequencies of rs8193036 (p = 0.029, OR = 1.64, 95% CI = 1.04-2.57) when comparisons were done using the genetic models of association. When stratification analysis was done on the basis of active and past cases we found that during active infection rs2275913 A allele was significantly associated with increased risk of kala-azar (p = 0.016, OR = 3.95, 95% CI = 1.21-12.87). INTERPRETATION & CONCLUSION: The findings revealed that IL-17 genetic variant, rs8193036 is an independent risk factor for kala-azar infection and may contribute in pathogenesis of the disease. Further, rs2275913 polymorphism of IL-17 gene is associated with kala-azar during active infection.

Serum proteome of dogs at subclinical and clinical onset of canine leishmaniosis.

The objective of this study was to identify changes in serum proteome in dogs that may occur after an experimental infection at subclinical and clinical stages of canine leishmaniosis (CanL). For this purpose, canine pre- and post-infection with Leishmania infantum serum proteomes in the same dogs were analysed by a high-throughput label-based quantitative LC-MS/MS proteomic approach. A total of 169 proteins were identified, and 74 of them including complement C8 alpha chain, adiponectin, transferrin, sphingomyelin phosphodiesterase acid-like 3A and immunoglobulins showed different modulation between the different stages of CanL. These proteins could be considered as potential serum biomarkers of early diagnostic or disease progression in CanL. Additionally, biological pathways modulated during CanL such as blood coagulation or gonadotropin-releasing hormone receptor were revealed, which could help to understand the pathological mechanisms of the disease.

Artemisinin-resistant Leishmania parasite modulates host cell defense mechanism and exhibits altered expression of unfolded protein response genes.

Artemisinin, extracted from a medicinal herb Artemisia annua, is widely used to treat malaria and has shown potent anticancer activity. Artemisinin has been found to be effective against experimental visceral and cutaneous leishmaniasis. Despite extensive research to understand the complex mechanism of resistance to artemisinin, several questions remain unanswered. The artesunate (ART)-resistant line of Leishmania donovani was selected and cellular mechanisms associated with resistance to artemisinin were investigated. ART-resistant (AS-R) parasites showed reduced susceptibility towards ART both at promastigote and amastigote stage compared with ART sensitive (WT) parasites. WT and AS-R parasites were both more susceptible to ART at the early log phase of growth compared with late log phase. AS-R parasites were more infective to the host macrophages (p < 0.05). Evaluation of parasites' tolerance towards host microbicidal mechanisms revealed that AS-R parasites were more tolerant to complement-mediated lysis and nitrosative stress. ROS levels were modulated in presence of ART in AS-R parasites infected macrophages. Interestingly, infection of macrophages by AS-R parasites led to modulated levels of host interleukins, IL-2 and IL-10, in addition to nitric oxide. Additionally, AS-R parasites showed upregulated expression of genes of unfolded protein response pathway including methyltransferase domain-containing protein (HSP40) and flagellar attachment zone protein (prefoldin), that are reported to be associated with ART resistance in Plasmodium falciparum malaria. This study presents in vitro model of artemisinin-resistant Leishmania parasite and cellular mechanisms associated with ART resistance in Leishmania.

Paediatric visceral leishmaniasis: a retrospective study on clinical manifestations, demographic features and laboratory findings of hospitalised cases in Iran between 2006 and 2016.

Visceral leishmaniasis (VL) is a neglected disease. Our retrospective study describes 38 clinical and epidemiological characteristics of VL in patients admitted to a paediatric hospital in Tehran, Iran, who came from different geographical regions, indicating that the disease has spread to most parts of the country. Some 76.3% of the children documented suffered with symptoms of the disease for two months before admission.

Visceral Leishmaniasis in Hospitalized HIV-Infected Patients in Pernambuco, Brazil.

Common in four continents, visceral leishmaniasis (VL) is an important but neglected disease. Human immunodeficiency virus (HIV) infection increases the risk of developing VL in people from leishmaniasis-endemic areas, with worse prognosis when there is coinfection. We conducted a cross-sectional study to determine the prevalence of HIV/VL coinfection in patients admitted in three referral hospitals for HIV/acquired immunodeficiency syndrome (AIDS) in Pernambuco, Brazil, and to compare epidemiological, clinical, and laboratory characteristics among HIV/VL coinfected and HIV mono-infected individuals. The sample consisted of HIV patients aged 18 years or more, in a period of data collection of 6 months. We performed four Leishmania tests-polymerase chain reaction (PCR), direct agglutination test, rK39, and latex agglutination test-and individuals with at least one positive test were considered coinfected. The HIV/VL coinfection prevalence we found was 16.9%. We observed large variation in prevalence according to the Leishmania test used, with low coincidence of positive tests. The most frequent symptoms found were weight loss (75.6%), fever (67.6%), and cough (55.3%). When we compared HIV/VL coinfected and HIV mono-infected groups we did not observe statistically significant differences. Low educational level (P = 0.004) and pallor (P = 0.009) were more frequent in the coinfected group. Serum albumin level was higher in coinfected individuals (P = 0.009). It is important to follow-up these individuals to understand the dynamics of VL in people living with HIV. New tests are necessary, ideally differentiating active from latent infection. Testing for VL in people with HIV is important and should be considered as part of the initial investigation in these individuals.

Biomarkers for Zoonotic Visceral Leishmaniasis in Latin America.

In Latin America, zoonotic visceral leishmaniasis (ZVL) arising from infection by L. infantum is primarily transmitted by Lutzomyia longipalpis sand flies. Dogs, which are chronic reservoirs of L. infantum, are considered a significant risk factor for acquisition of ZVL due to their close proximity to humans. In addition, as a vector-borne disease the intensity of exposure to vector sand flies can also enhance the risk of developing ZVL. Traditionally, IFN-γ and IL-10 are considered as the two main cytokines which determine the outcome of visceral leishmaniasis. However, more recently, the literature has demonstrated that different mediators, such as lipid mediators (PGE-2, PGF-2 alfa, LTB-4, resolvins) and other important inflammatory and anti-inflammatory cytokines are also involved in the pathogenicity of ZVL. Analysis of a greater number of mediators allows for a more complete view of disease immunopathogenesis. Additionally, our knowledge has expanded to encompass different biomarkers associated to disease severity and healing after specific treatments. These parameters can also be used to better define new potential targets for vaccines and chemotherapy for ZVL. Here, we will provide an overview of ZVL biomarkers identified for both humans and dogs and discuss their merits and shortcomings. We will also discuss biomarkers of vector exposure as an additional tool in our arsenal to combat ZVL.

Leishmaniasis.

Leishmaniasis is a poverty-related disease with two main clinical forms: visceral leishmaniasis and cutaneous leishmaniasis. An estimated 0·7-1 million new cases of leishmaniasis per year are reported from nearly 100 endemic countries. The number of reported visceral leishmaniasis cases has decreased substantially in the past decade as a result of better access to diagnosis and treatment and more intense vector control within an elimination initiative in Asia, although natural cycles in transmission intensity might play a role. In east Africa however, the case numbers of this fatal disease continue to be sustained. Increased conflict in endemic areas of cutaneous leishmaniasis and forced displacement has resulted in a surge in these endemic areas as well as clinics across the world. WHO lists leishmaniasis as one of the neglected tropical diseases for which the development of new treatments is a priority. Major evidence gaps remain, and new tools are needed before leishmaniasis can be definitively controlled.

Leishmania donovani induced Unfolded Protein Response delays host cell apoptosis in PERK dependent manner.

BACKGROUND: Endoplasmic reticulum (ER) stress generated unfolded stress response (UPR) is a basic survival mechanism which protects cell under unfavourable conditions. Leishmania parasite modulates host macrophages in various ways to ensure its survival. Modulation of PI3K-Akt pathway in delayed apoptotic induction of host; enables parasite to stabilize the infection for further propagation. METHODOLOGY: Infected RAW macrophages were exposed to campothecin or thagsigargin and phosphorylation status of PERK, Akt, BAD and Cyt-C was determined through western blotting using phospho specific antibody. Expression at transcriptional level for cIAP1 &2, ATF4, CHOP, ATF3, HO-1 and sXBP1 was determined using real time PCR. For inhibition studies, RAW macrophages were pre-treated with PERK inhibitor GSK2606414 before infection. FINDINGS: Our studies in RAW macrophages showed that induction of host UPR against L.donovani infection activates Akt mediated pathway which delays apoptotic induction of the host. Moreover, Leishmania infection results in phosphorylation and activation of host PERK enzyme and increased transcription of genes of inhibitor of apoptosis gene family (cIAP) mRNA. In our inhibition studies, we found that inhibition of infection induced PERK phosphorylation under apoptotic inducers reduces the Akt phosphorylation and fails to activate further downstream molecules involved in protection against apoptosis. Also, inhibition of PERK phosphorylation under oxidative exposure leads to increased Nitric Oxide production. Simultaneously, decreased transcription of cIAP mRNA upon PERK phosphorylation fates the host cell towards apoptosis hence decreased infection rate. CONCLUSION: Overall the findings from the study suggests that Leishmania modulated host UPR and PERK phosphorylation delays apoptotic induction in host macrophage, hence supports parasite invasion at early stages of infection.

Countervailing, time-dependent effects on host autophagy promotes intracellular survival of Leishmania.

Autophagy is essential for cell survival under stress and has also been implicated in host defense. Here, we investigated the interactions between Leishmania donovani, the main etiological agent of visceral leishmaniasis, and the autophagic machinery of human macrophages. Our results revealed that during early infection-and via activation of the Akt pathway-Leishmania actively inhibits the induction of autophagy. However, by 24 h, Leishmania switched from being an inhibitor to an overall inducer of autophagy. These findings of a dynamic, biphasic response were based on the accumulation of lipidated light chain 3 (LC3), an autophagosome marker, by Western blotting and confocal fluorescence microscopy. We also present evidence that Leishmania induces delayed host cell autophagy via a mechanism independent of reduced activity of the mechanistic target of rapamycin (mTOR). Notably, Leishmania actively inhibited mTOR-regulated autophagy even at later stages of infection, whereas there was a clear induction of autophagy via some other mechanism. In this context, we examined host inositol monophosphatase (IMPase), reduced levels of which have been implicated in mTOR-independent autophagy, and we found that IMPase activity is significantly decreased in infected cells. These findings indicate that Leishmania uses an alternative pathway to mTOR to induce autophagy in host macrophages. Finally, RNAi-mediated down-regulation of host autophagy protein 5 (ATG5) or autophagy protein 9A (ATG9A) decreased parasite loads, demonstrating that autophagy is essential for Leishmania survival. We conclude that Leishmania uses an alternative pathway to induce host autophagy while simultaneously inhibiting mTOR-regulated autophagy to fine-tune the timing and magnitude of this process and to optimize parasite survival.

Epidemiological profile of patients co-infected with visceral leishmaniasis and HIV/AIDS in Northeast, Brazil.

INTRODUCTION: Visceral leishmaniasis (VL) and human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) co-infection has been a research topic of interest worldwide. In Brazil, it has been observed that there is a relative underreporting and failure in the understanding and management of this important association. The aim of this study was to analyze epidemiological and clinical aspects of patients with VL with and without HIV/AIDS. METHODS: We conducted an observational and analytical study of patients with VL followed in a Reference Service in the State of Maranhão, Brazil from 2007-2013. RESULTS: In total 126 patients were enrolled, of which 61 (48.4%) were co-infected with HIV/AIDS. There were more males among those with HIV/AIDS (85.2%, P>0.05) or with VL only (81.5%, P>0.05). These findings significantly differed based on age group (P<0.003); the majority of patients were aged 31-40 years (41.0%) and 21-30 years (32.3%) among those with and without HIV/AIDS co-infection, respectively. The incidence of diarrhea and splenomegaly significantly differed between the two groups (P=0.0014 and P=0.019, respectively). The myelogram parasitic examination was used most frequently among those with HIV/AIDS (91.8%), followed by those with VL only (69.2%). VL recurrences and mortality were significantly higher in the HIV/AIDS co-infected patients (P<0.0001 and P=0.012, respectively). CONCLUSIONS: Patients with VL with or without HIV/AIDS co-infection were mostly adult men. Diarrhea was more frequent in HIV/AIDS co-infected patients, whereas splenomegaly was more common in patients with VL only. In the group of HIV/AIDS co-infected patients, there was a higher rate of VL recurrence and mortality.

Visceral leishmaniasis and leishmaniasis-HIV coinfection: comparative study.

INTRODUCTION: This study aimed to draw clinical and epidemiological comparisons between visceral leishmaniasis (VL) and VL associated with human immunodeficiency virus (HIV) infection. METHOD: Retrospective study. RESULTS: Of 473 cases of VL, 5.5% were coinfected with HIV. The highest proportion of cases of both VL and VL/HIV were found among men. A higher proportion of VL cases was seen in children aged 0-10 years, whereas coinfection was more common in those aged 18-50 years. CONCLUSIONS: VL/HIV coinfected patients presented slightly differently to and had a higher mortality rate than those with VL only.