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

BackgroundHuman 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.ResultsIn 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.ConclusionAn 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.

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.

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

PURPOSE: 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. RESULTS: The VL case was diagnosed 18 months after transplant and 28 days after the onset of symptoms by quantitative PCR (qPCR) on peripheral blood. A graft biopsy showed renal involvement, and PCR performed on graft tissue displayed the presence of Leishmania DNA. The retrospective confirmation of Leishmania-positive serology in a serum sample collected before transplantation, as well as the absence of anti-Leishmania IgG in the graft donor strongly suggest that reactivation of a latent parasitic infection caused VL in the current case. CONCLUSION: VL is often underdiagnosed in transplant recipients, despite the presence of latent Leishmania infection being reported in endemic countries. This case report, as well as the literature review on leishmaniasis in KT recipients, underline the importance of rapid VL diagnosis to promptly undergo treatment. Serology is scarcely sensitive in immunocompromised patients, thus molecular tests in peripheral blood should be implemented and standardized for both VL identification and follow-up.

Molecular Typing of Leishmania spp. Causing Tegumentary Leishmaniasis in Northeastern Italy, 2014-2020.

Tegumentary leishmaniasis (TL) is endemic but neglected in southern Europe. Therefore, this study aimed to analyze the Leishmania strains causing TL cases in northeastern Italy, where an upsurge of TL cases has been observed in the last decade. Sections from 109 formalin-fixed and paraffin-embedded (FFPE) biopsies of skin and mucosal tissues were collected from TL cases in the selected area. Two DNA targets were amplified and sequenced: the ribosomal internal transcribed spacer 1 (ITS1) and the heat-shock protein 70 gene (hsp70). An in silico analysis was also performed on 149 genomes belonging to the Leishmania donovani complex. A total of 88 out of 109 (80.7%) samples from 83 TL cases were successfully typed by ITS1 and/or hsp70. ITS1 analysis identified L. infantum in 67 cases (91.8%), while L. major (n = 4, 5.5%) and L. tropica (n = 2, 2.7%) were detected in the remaining cases that were categorized as imported. Further, the hsp70 typing of 75 autochthonous cases showed the presence of eight distinct sequence variants belonging to the Leishmania donovani complex, with high genetic variability when compared to known L. infantum populations. In conclusion, our findings show that peculiar L. infantum variants are emerging in the novel focus on TL in northeastern Italy.

Tegumentary Leishmaniasis in Northeastern Italy from 2017 to 2020: A Neglected Public Health Issue.

Tegumentary leishmaniasis (TL) includes cutaneous (CL) and mucosal (ML) leishmaniasis; despite being endemic in southern Europe, it is often underdiagnosed and underreported. This study aimed to retrospectively examine data collected from patients with TL in a selected area of northeastern Italy (Emilia-Romagna region, RER). A network of 10 diagnostic units within RER was established, and TL cases diagnosed in RER from 2017 to 2020 were evaluated. A total of 135 TL cases were collected (62% male, 38% female); patients ranged from 1 to 84 years, with a median age of 57. Among these cases, 113 (84%) were notified to the public health authorities. The average annual incidence of TL was 0.76 cases per 100,000 inhabitants. Infections were acquired within the RER in 84% of cases; the 113 autochthonous cases were distributed in the foothills areas of the region. We provide evidence of a defined spatial distribution of TL cases in a selected area of northeastern Italy, as well as a relevant number of ML cases. Our observations suggest the need to raise awareness about TL among clinicians and pathologists, promote the molecular confirmation of cases by reference laboratories, and encourage the establishment of surveillance networks for this neglected disease.

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

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/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.

A large outbreak of giardiasis in a municipality of the Bologna province, north-eastern Italy, November 2018 to April 2019.

Giardiasis, the disease caused by the flagellate Giardia duodenalis (syn. G.lamblia, G. intestinalis), is the most commonly reported among the five food- and waterborne parasitic diseases under mandatory surveillance in 24 EU countries. From November 2018 to April 2019, an outbreak of giardiasis occurred in a municipality of the Bologna province, in north-eastern Italy. Microscopy and immunochromatography identified cysts and antigens, respectively, of the parasite in stool samples of 228 individuals. Molecular typing of 136 stool samples revealed a vast predominance (95%) of G. duodenalis assemblage B. Investigations into potential sources indicated tap water as the most likely vehicle of infection, although cysts were not detected in water samples. Control measures mostly aimed at preventing secondary transmission by informing citizens about the outbreak, and by treatment of patients with anti-parasitic drugs. This is the first documented human outbreak of giardiasis in Italy; its investigation has highlighted the difficulties in the timely detection and management of this parasite, which is often overlooked as a cause of human gastroenteritis. The long and variable incubation time, absence of specific symptoms and a general lack of awareness about this pathogen contributed to delay in diagnosis.

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.

Sickle Cell Trait Modulates the Proteome and Phosphoproteome of Plasmodium falciparum-Infected Erythrocytes.

The high prevalence of sickle cell disease in some human populations likely results from the protection afforded against severe Plasmodium falciparum malaria and death by heterozygous carriage of HbS. P. falciparum remodels the erythrocyte membrane and skeleton, displaying parasite proteins at the erythrocyte surface that interact with key human proteins in the Ankyrin R and 4.1R complexes. Oxidative stress generated by HbS, as well as by parasite invasion, disrupts the kinase/phosphatase balance, potentially interfering with the molecular interactions between human and parasite proteins. HbS is known to be associated with abnormal membrane display of parasite antigens. Studying the proteome and the phosphoproteome of red cell membrane extracts from P. falciparum infected and non-infected erythrocytes, we show here that HbS heterozygous carriage, combined with infection, modulates the phosphorylation of erythrocyte membrane transporters and skeletal proteins as well as of parasite proteins. Our results highlight modifications of Ser-/Thr- and/or Tyr- phosphorylation in key human proteins, such as ankyrin, ß-adducin, ß-spectrin and Band 3, and key parasite proteins, such as RESA or MESA. Altered phosphorylation patterns could disturb the interactions within membrane protein complexes, affect nutrient uptake and the infected erythrocyte cytoadherence phenomenon, thus lessening the severity of malaria symptoms.

Screening strategies for the diagnosis of asymptomatic Leishmania infection in dialysis patients as a model for kidney transplant candidates.

Despite being considered a tropical disease, visceral leishmaniasis (VL) caused by L. infantum is also endemic in the Mediterranean Europe and represents an increasing cause of morbidity and mortality in solid organ transplant (SOT) recipients. VL occurring in kidney transplant recipients is a severe event, often worsening the renal damage and leading to poor outcome. It is believed that most of VL cases in transplant recipients are caused by reactivation of a pre-existent, dormant leishmanial infection induced by the immunosuppressive drugs. Nevertheless, the prevalence of asymptomatic Leishmania infection in candidates to kidney transplant residing in or visiting endemic areas is unknown. As L. infantum is highly circulating in northeastern Italy, we aimed to examine the occurrence of this parasitic infection in 119 dialysis patients living in the mentioned area, 71 of whom were potential candidates to kidney transplant. By employing a combination of sensitive serological and molecular methods, we observed a prevalence of 15.9% asymptomatic Leishmania infection in the study cohort. This finding emphasizes the need of further evaluating potential screening strategies for Leishmania infection in solid organ transplant candidates residing in or visiting endemic areas.

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.

Evaluation of the Pharmacophoric Role of the O-O Bond in Synthetic Antileishmanial Compounds: Comparison between 1,2-Dioxanes and Tetrahydropyrans.

Leishmaniases are neglected diseases that can be treated with a limited drug arsenal; the development of new molecules is therefore a priority. Recent evidence indicates that endoperoxides, including artemisinin and its derivatives, possess antileishmanial activity. Here, 1,2-dioxanes were synthesized with their corresponding tetrahydropyrans lacking the peroxide bridge, to ascertain if this group is a key pharmacophoric requirement for the antileishmanial bioactivity. Newly synthesized compounds were examined in vitro, and their mechanism of action was preliminarily investigated. Three endoperoxides and their corresponding tetrahydropyrans effectively inhibited the growth of Leishmania donovani promastigotes and amastigotes, and iron did not play a significant role in their activation. Further, reactive oxygen species were produced in both endoperoxide- and tetrahydropyran-treated promastigotes. In conclusion, the peroxide group proved not to be crucial for the antileishmanial bioactivity of endoperoxides, under the tested conditions. Our findings reveal the potential of both 1,2-dioxanes and tetrahydropyrans as lead compounds for novel therapies against Leishmania.

Asymptomatic Leishmania infantum infection in blood donors living in an endemic area, northeastern Italy.

OBJECTIVES: Human leishmaniasis can be severe and fatal, yet in the Mediterranean region only a small percentage of infections progress to clinical disease. We evaluated the percentage of asymptomatic Leishmania infection in the Bologna province, northeastern Italy. METHODS: We examined the presence of specific antibodies by Western Blot (WB) and parasitic DNA by real time PCR in peripheral blood of 240 blood donors residing in the Bologna province. RESULTS: Anti-Leishmania IgG were detected by WB in 27 subjects (11.2%, 95% CI 7%-15%), while Leishmania kinetoplast DNA was detected in peripheral blood specimens of 4 out of 240 donors (1.7%, 95% CI 0.2%-3.2%). Overall, the prevalence of Leishmania infection in the blood donor cohort was 12.5%, thus indicating an elevated cumulative exposure to the Leishmania parasite in the examined municipality. CONCLUSIONS: Our results suggest that a surveillance system for monitoring Leishmania infection in blood donors and/or strategies of protozoan inactivation in whole blood should be taken into consideration in areas with circulation of the Leishmania parasite.

18F-FDG PET/CT in visceral leishmaniasis: uptake patterns in the context of a multiannual outbreak in Northern Italy.

OBJECTIVES: Visceral leishmaniasis (VL) is the most severe manifestation of the infection caused by the protozoan Leishmania, recently on increase in Italy and Spain. The aim of the study was to describe FDG uptake patterns in VL patients (pts) who underwent 18F-FDG PET/CT. METHODS: A retrospective monocentric study of pts who underwent FDG PET/CT between 2008 and 2017 and later diagnosed with VL was performed. Semi-quantitative parameters were calculated in FDG-positive lesions: SUVmax, SUVmax spleen/SUVmax liver ratio (SLR), SUVmax focal/diffuse spleen ratio (FDR). RESULTS: Overall, 23 pts were included. PET/CT was negative in 2 immunocompromised pts, positive in 21/23 (91%) [6 spleen only, 2 spleen + nodes, 7 spleen + bone marrow (BM), 4 spleen + BM + nodes, 1 spleen + BM + lung, 1 BM only + nodes, 2 nodes only]. Splenic involvement was demonstrated in 20/23 (87%) pts. Two different splenic patterns were observed: diffuse (13/20 pts, mean spleen SUVmax = 7.3 ± 4.2 [4.0-14.1], mean SLR = 2.2 ± 1.6 [1.3-6.7]) and focal over diffuse (7/20 pts, mean SUVmax = 12.6 ± 4.5 [9.5-20.5], mean SLR = 2.8 ± 0.8 [2.1-4.4], mean FDR = 2.1 ± 0.8 [1.2-3.6]). Extra-splenic FDG-avid findings were detected in 15/21 pts (65%): bone marrow in 13/15 (mean SUVmax = 4.0 ± 1.3 [2.8-6.0]), nodes in 67/15 and lung in 1/15. CONCLUSIONS: PET/CT demonstrated splenic FDG uptake in all immunocompetent VL pts; two splenic patterns (diffuse/focal over diffuse) were observed and indistinguishable from splenic involvement by other disorders. The most frequent extra-splenic FDG-positive sites were BM and lymph nodes. Considering the potential disease aggressiveness and recent outbreaks in north-eastern Italy, VL should be considered in the differential diagnosis of FDG-positive splenic findings in pts from endemic areas or reporting travels to endemic countries.

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.

Serological and molecular tools to diagnose visceral leishmaniasis: 2-years' experience of a single center in Northern Italy.

The diagnosis of visceral leishmaniasis (VL) remains challenging, due to the limited sensitivity of microscopy, the poor performance of serological methods in immunocompromised patients and the lack of standardization of molecular tests. The aim of this study was to implement a combined diagnostic workflow by integrating serological and molecular tests with standardized clinical criteria. Between July 2013 and June 2015, the proposed workflow was applied to specimens obtained from 94 in-patients with clinical suspicion of VL in the Emilia-Romagna region, Northern Italy. Serological tests and molecular techniques were employed. Twenty-one adult patients (22%) had a confirmed diagnosis of VL by clinical criteria, serology and/or real-time polymerase chain reaction; 4 of these patients were HIV-positive. Molecular tests exhibited higher sensitivity than serological tests for the diagnosis of VL. In our experience, the rK39 immunochromatographic test was insufficiently sensitive for use as a screening test for the diagnosis of VL caused by L. infantum in Italy. However, as molecular tests are yet not standardized, further studies are required to identify an optimal screening test for Mediterranean VL.