Epidemiological, Diagnostic and Drug Discovery Studies of an Emerging Infection: Human Leishmaniasis

Human leishmaniasis is a vector-borne diseases, caused by the Leishmania parasite and endemic in 100 countries, including southern Europe. The clinical spectrum of Leishmania infection ranges from asymptomatic to visceral leishmaniasis (VL), the latter being fatal if not treated. The real prevalence of asymptomatic infections is unknown, the diagnosis of VL is challenging and lacks standardized methods, and also antileishmanial treatment has critical limitations. In this scenario, this study aimed (1) to determine the prevalence of asymptomatic infections in blood donors of the Bologna province, where increase of VL cases has been recently reported, (2) to compare the performance of nine different serological tests in the diagnosis of VL in northern Italy, (3) to assess the antileishmanial activity of a library of newly synthesized natural like compounds, ie chalcones. (1) The screening on samples of blood donors living in the Bologna province, shows a total prevalence of asymptomatic Leishmania infection of 11.5%, suggesting a high circulation of the parasite in this area. (2) Our findings on serodiagnosis indicate that rK39 immunochromatographic tests are insufficiently sensitive as screening tests for VL, ELISA exhibited suboptimal results in terms of sensitivity, IFAT and WB exhibited excellent sensitivity, but their cost and complexity in execution would not allow their employment as screening tests for VL. These results confirm the complexity of VL serodiagnosis and reveal the variability of diagnostic performance of serological tests. (3) Screening of 33 newly synthesized chalcones on proliferation of Leishmania promastigote and amastigote revealed that 2 compounds showed a remarkable antileishmanial potency against the parasite, a low citotoxicity against mammalian cells and they also were able to efficiently bind trypanothione reductase - a specific parasite enzyme - suggesting that these compounds should be further evaluated as antileishmanial agents

In Vitro Reduced Susceptibility to Pentavalent Antimonials of a Leishmania infantum Isolate from a Human Cutaneous Leishmaniasis Case in Central Italy

Cutaneous leishmaniasis (CL) caused by Leishmania (Leishmania) infantum is endemic in the Mediterranean basin. Here we report an autochthonous case of CL in a patient living in central Italy with an unsatisfactory response to treatment with intralesional Meglumine Antimoniate and in vitro demonstration of reduced susceptibility to SbIII. Parasitological diagnosis was first achieved by histopathology on tissue biopsy and the patient was treated with a local infiltration of Meglumine Antimoniate. Since the clinical response at 12 weeks from the treatment’s onset was deemed unsatisfactory, two further skin biopsies were taken for histopathological examination, DNA extraction and parasite isolation. L. (L.) infantum was identified by molecular typing. The low susceptibility to Meglumine Antimoniate was confirmed in vitro: the promastigotes from the patient strain showed significantly lower susceptibility to SbIII (the active trivalent form of antimonial) compared to the reference strain MHOM/TN/80/IPT1. The patient underwent a new treatment course with intravenous liposomal Amphotericin B, reaching complete healing of the lesion. Additional studies are needed to confirm the epidemiological and clinical relevance of reduced susceptibility to SbIII of human L. (L.) infantum isolate in Italy

A sex and gender perspective for neglected zoonotic diseases

Sex-driven and gender-driven diversities in health and diseases should be considered to promote equitable and more effective health for all. Nevertheless, these diversities are not sufficiently taken into consideration when approaching diseases, including infectious diseases. Neglected infectious diseases (NIDs) are a group of diseases, more prevalent in - but not limited to - tropical areas, that are almost absent from the global health agenda. NIDs are related to poverty, associated with stigma and social exclusion and prone to perpetuate health inequities from different perspectives including sex and gender. There is consensus that women and men are differently affected by NIDs. Many epidemiological studies have shown that being a man is a risk factor for several infectious diseases; a number of evidences indicate that sex-related hormonal and chromosomal factors contribute to distinct host’s response to infections in males and females. Conversely, women and girls experience a greater share of the NID burden due to their disproportionate poverty, illiteracy, lower education and social status particularly in low- and middle-income countries. More generally, different professional and environmental exposure can also influence dissimilar disease burden in men and women, but these aspects are seldom considered in clinical practice and in epidemiological surveillance. This opinion paper has the objective of describing the role of sex and gender-based differences for the management and control of NIDs with the scope of orientating research, clinical management and public health strategies towards a gender- and sex- balanced approach. Various NIDs will be examined as examples (case studies) to describe the biological features as well as organizational structures in healthcare and cultural aspects that should be considered for a gender-neutral management of these diseases as well as for the development of individualized anti-infectious treatments that take sex-specific host factors into account. An increased focus on sex together with gender diversities in health and health care services for NIDs is crucial to develop personalized and targeted therapeutic approaches but also to maximize the impact of interventions and public health strategies aimed at eliminating some of them by 2030

Test combination to detect latent Leishmania infection: A prevalence study in a newly endemic area for L. infantum, northeastern Italy

Dataset disponible en: http://hdl.handle.net/20.500.12105/14600Background: 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/principal 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.This work was supported by funds “Ricerca Corrente 2016 (IZSLER 11/16 – PRC2016011) to SV, and funds “Ricerca Finalizzata 2016” (RF-2016-02361931) from the Italian Ministry of Health to SV. This work was also supported by RFO funds 2019-2020 (to SV) from the University of Bologna. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

Updated diagnosis and graft involvement for visceral leishmaniasis in kidney transplant recipients: a case report and literature review

Visceral leishmaniasis (VL) has become a rising concern to transplantation teams, being associated with graft dysfunction and reduced survival of renal transplant recipients. Here, we describe a case of VL occurring in a kidney transplant (KT) recipient in Italy, a country in which Leishmania infantum is endemic and we reviewed the literature on the clinical course and diagnosis of VL in KT recipients residing or travelling to southern Europe

Identification of asymptomatic Leishmania infection in patients undergoing kidney transplant using multiple tests

Objectives: In immunocompromised patients, asymptomatic Leishmania infection can reactivate, and evolve to severe disease. To date, no test is considered the gold standard for the identification of asymptomatic Leishmania infection. A combination of methods was employed to screen for Leishmania infection in patients undergoing kidney transplant (KT). Methods: We employed polymerase chain reaction for the detection of parasitic DNA in peripheral blood, Western blot to identify serum immunoglobulin G and whole blood assay to detect cytokines/chemokines after stimulation of whole blood with parasitic antigen. Results: One-hundred twenty patients residing in Italy were included in the study at the time of KT. Each patient that tested positive to at least one test was considered as Leishmania positive. Fifty out of 120 patients (42%) tested positive for one or more tests. The detection of specific cell-mediated response (32/111, 29%) was the most common marker of Leishmania infection, followed by a positive serology (24/120, 20%). Four patients (3%) harbored parasitic DNA in the blood. Conclusion: Our findings underline the high prevalence of asymptomatic Leishmania infection in patients undergoing KT in Italy, who are potentially at-risk for parasite reactivation and can benefit from an increased vigilance. Understanding the clinical relevance of these findings deserves further studies.This work was supported by the Italian Ministry of Health (grant number RF-2016-02361931)S

Re-emergence of human leishmaniasis in northern Italy, 2004 to 2022: a retrospective analysis

Background. Human leishmaniasis is a protozoan disease transmitted by sand flies and endemic in the Mediterranean region. In Italy, leishmaniasis is present in the south and the western coastal regions, with an epidemic peak detected in northern Italy in the early 1970s.AimTo examine temporal trends, and demographic, clinical, geographical and environmental features of human leishmaniasis cases recorded by the local health unit (LHU) of Bologna, northern Italy.MethodsIn this retrospective observational study, we analysed human leishmaniasis cases recorded from 2004 to 2022 within the Bologna LHU. We also conducted serological investigations for canine leishmaniasis in owned dogs living near the place of infection of human cases. Results. In total, 173 cases of human leishmaniasis were detected, and 154 cases were considered autochthonous. An increase of human cases was observed since 2004, with incidence peaks above 2 cases/100,000 inhabitants in 2013, 2018 and 2022; epidemic peaks were preceded by dry summers. Most cases lived in the plain and hilly areas less than 400 m above sea level and many resided in isolated housing, in city outskirts, and/or near uncultivated areas, watercourses and railway sections. The incidence of canine leishmaniasis did not increase in the study period.Conclusion. An epidemic of human leishmaniasis with fluctuating annual numbers of cases, probably related to environmental and climatic factors, was identified in the Bologna LHU. Understanding the risk factors and the environmental characteristics related to places of infection is crucial to evaluate the public health implications of leishmaniasis

Serodiagnosis of Visceral Leishmaniasis in Northeastern Italy: Evaluation of Seven Serological Tests

This study compares the performance of seven assays, including two ELISA (Leishmania ELISA IgG + IgM, Vircell Microbiologists; Leishmania infantum IgG ELISA, NovaTec), three rK39-based immunochromatographic tests (rK39-ICTs) (Leishmania Dipstick Rapydtest, Apacor; On Site Leishmania IgG/IgM Combo Rapid Test, CTK Biotech; LEISHMANIA Strip quick Test, Cypress Diagnostic), one indirect immunofluorescent antibody test (IFAT) (Leishmania-Spot IF, BioMérieux), and one western blot (WB) (Leishmania WESTERN BLOT IgG, LDBio Diagnostics) for serodiagnosis of visceral leishmaniasis (VL). Serum samples from 27 VL patients living in northeastern Italy were analyzed, as well as the serum samples from 50 individuals in whom VL diagnosis was excluded. The WB and the IFAT had 96% sensitivity, followed by the ELISA (63% and 74%, respectively). The rK39-ICT exhibited the worst performance among the serological tests, with sensitivities ranging from 52% to 70%. By combining selected ELISA/ICT, the sensitivity of VL detection reached 89%. IFAT and WB outperformed ELISA and rK39-ICT by possessing optimal sensitivity, but their high cost and complexity of execution would not allow their employment as screening tests. In conclusion, the combination of easy-to-perform tests, such as ICT and ELISA, could improve sensitivity in the serodiagnosis of Mediterranean VL

Test combination to detect latent Leishmania infection: a prevalence study in a newly endemic area for L. infantum, northeastern Italy [Dataset]

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.S

Identification of chalcone-based antileishmanial agents targeting trypanothione reductase

All currently used first-line and second-line drugs for the treatment of leishmaniasis exhibit several drawbacks including toxicity, high costs and route of administration. Furthermore, some drugs are associated with the emergence of drug resistance. Thus, the development of new treatments for leishmaniasis is a priority in the field of neglected tropical diseases. The present work highlights the use of natural derived products, i.e. chalcones, as potential source of antileishmanial agents. Thirty-one novel chalcone compounds have been synthesized and their activity has been evaluated against promastigotes of Leishmania donovani; 16 compounds resulted active against L. donovani in a range from 3.0 to 21.5 μM, showing low toxicity against mammalian cells. Among these molecules, 6 and 16 showed good inhibitory activity on both promastigotes and intracellular amastigotes, coupled with an high selectivity index. Furthermore, compounds 6 and 16 inhibited the promastigote growth of other leishmanial species, including L. tropica, L. major and L. infantum. Finally, 6 and 16 interacted with high affinity with trypanothione reductase (TR), an essential enzyme for the leishmanial parasite and compound 6 inhibited TR with sub-micromolar potency. Thus, the effective inhibitory activity against Leishmania, the lack of toxicity on mammalian cells and the ability to block a crucial parasite's enzyme, highlight the potential for compound 6 to be optimized as novel drug candidate against leishmaniasis