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The World Health Organization (WHO) defines neglected tropical diseases (NTDs) as a diverse group of primarily infectious diseases, which disproportionately affect poor and marginalized populations worldwide. In this context, NTDs are responsible for important morbidity and mortality and justify a global response. Moreover, NTDs are relatively neglected by research and development as well as by funding, if compared with the magnitude of the public health problem they represent. This happens even though, unlike other infectious diseases, they can be prevented, controlled and eliminated by targeted public health interventions. NTDs are mainly prevalent in communities from low-income countries in tropical and sub-tropical areas but are also present in upper–middle-income countries, including several in Europe. Here, we provide an update on the most relevant parasitic endemic or imported NTDs in Italy and illustrate the rationale for the establishment of the Italian network on NTDs, an alliance of scientific societies, institutes, foundations, universities and non-profit organizations united to fight NTDs.

BackgroundHuman cystic echinococcosis (CE) is a zoonotic parasitic infection caused by the larval stage of the species belonging to the Echinococcus granulosus sensu lato (s.l.) complex. Parasitic cysts causing human CE are mainly localized in the liver and in the lungs. In a smaller number of cases, larvae may establish in any organ or tissue, including the central nervous system (CNS). Cerebral CE (CCE) is rare but poses serious clinical challenges.MethodsThis study presents a case of CCE in a child living in the countryside near Rome (Italy), along with a comparative molecular analysis of the isolated cyst specimens from the patient and sheep of local farms. We also systematically searched the literature to summarize the most relevant epidemiological and clinical aspects of this uncommon localization.FindingsThe comparative molecular analysis confirmed that the infection was caused by E. granulosus sensu stricto (s.s.) (G3 genotype), and most likely acquired in the family farm. The literature search identified 2,238 cases of CCE. In 80.51% of cases, brain was the only localization and single CCE cysts were present in 84.07% of cases. Mean patients' age was 20 years and 70.46% were children. Cyst rupture was reported in 12.96% and recurrence of CCE after treatment in 9.61% of cases. Permanent disability was reported in 7.86% of cases, while death occurred in 6.21%. In case series reporting all CE localization, CCE represented 1.5% of all CE cases. In the few reports that identified at molecular level the CCE cyst, E. granulosus s.s. was found in 40% and E. canadensis in 60% of cases.ConclusionsWe report a rare case of CCE and evidenced the probable local origin of infection. The proportions of CE cases with uncommon localizations and with high impact on patients' lives have been globally neglected and should be included in the computation of the global burden of CE.

Abstract Background There are close similarities between the life-cycles of Echinococcus granulosus sensu lato (E. granulosus s.l.) that causes cystic echinococcosis (CE) in humans and Taenia multiceps/Coenurus cerebralis that causes cerebral coenurosis in small ruminants. Recent evidence highlights that livestock in Maasai communities of northern Tanzania are suffering from increases in the prevalence of cerebral coenurosis, leading to concerns about a possible concurrent increased risk of human CE. The aim of this study was to estimate the prevalence of human abdominal CE and the prevalence and species/genotypes of E. granulosus s.l. in livestock in Maasai communities. Methods Human CE was diagnosed by abdominal ultrasound on volunteers aged ≥ 7 years in five villages in the Longido and Ngorongoro Districts in northern Tanzania. Infection in ruminants was evaluated through inspection in local abattoirs, followed by molecular identification of one cyst per animal, with a priority for hepatic cysts, using PCR targeting of the cytochrome c oxidase I gene (COX1), followed by restriction fragment length polymorphism and multiplex PCR, and sequencing of non-E. granulosus s.l. samples. Results Ultrasound was performed on 823 volunteers (n = 352 in two villages in Longido District, and n = 471 in three villages of Ngorongoro). Hepatic CE cases were diagnosed only in Ngorongoro (n = 6; 1.3%), of which three had active cysts. Village-level prevalence of CE ranged between 0 and 2.4%. Of the 697 ruminants inspected, 34.4% had parasitic cysts. Molecular identification was achieved for 140 of the 219 (63.9%) cysts sampled. E. granulosus s.l. and T. hydatigena/Cysticercus tenuicollis were identified in 51.4% and 48.6%, respectively, of livestock cysts. E. granulosus s.l. was identified in livestock from both Longido (35.3% of 116 genotyped cysts) and Ngorongoro (91.2% of 34 genotyped cysts). Of the total of 72 E. granuslosus s.l. cysts identified in livestock, 87.5% were E. granulosus sensu strict o (G1–G3 genotypes), 9.7% were E. ortleppi (G5) and one cyst was E. canadensis (G6–10). The three active human cysts, which were removed surgically, were G1–G3 genotypes. Conclusions Multiple species/genotypes of E. granulosus s.l. are circulating in Maasai communities of northern Tanzania. Human CE was detected in villages of Ngorongoro District and a high prevalence of echinococcal cysts was observed in livestock in both districts. More precise estimation of the prevalence in this area and a better understanding of the specific risk factors for CE among Maasai communities in northern Tanzania is needed. Interventions targeting transmission routes common to both E. granulosus s.l. and T. multiceps would have dual benefits for preventing both human and livestock disease. Graphical Abstract

A high-quality genome for the parasitic tapeworm, Echinococcus granulosus, provides further insight into the EG95 vaccine target for cystic echinococcosis.

Abstract Background This study aimed to fill a gap of knowledge by providing a quantitative measure of molecularly identified species and genotypes belonging to Echinococcus granulosus sensu lato (s.l.) causing human cystic echinococcosis (CE) in Europe during the period 2000–2021. As these species and genotypes are characterized by genetic, animal host and geographical differences, studying the E. granulosus s.l. complex is epidemiologically relevant. Methods A systematic review (SR) was conducted on the basis of both scientific and grey literature considering primary studies between 2000 and 2021 in four databases. From a total of 1643 scientific papers, 51 records were included in the SR. The main inclusion criterion for this study was the molecular confirmation of E. granulosus s.l. at the genotype/species level as a causative agent of human CE cases in selected European countries. Results Relevant data were obtained from 29 out of 39 eligible European countries. This SR identified 599 human molecularly confirmed echinococcal cysts: 460 (76.8%) identified as E. granulosus sensu stricto (s.s.), 130 (21.7%) as E. canadensis cluster (G6/7 and G10), 7 (1.2%) as E. ortleppi (G5), and 2 as E. vogeli (0.3%). Three geographical hotspots of human CE caused by different species of the E. granulosus s.l. complex were identified: (1) E. granulosus s.s. in Southern and South-eastern Europe (European-Mediterranean and Balkan countries); (2) E. canadensis (G6/7) in Central and Eastern Europe; (3) E. ortleppi in Central and Western Europe. This SR also identified data gaps that prevented a better definition of the geographical distribution of the Echinococcus granulosus s.l. species complex in Europe: western Balkan countries, part of Central Europe, and Baltic countries. Conclusions These results mandate longitudinal, multi-centre, intersectoral and transdisciplinary studies which consider both molecular and clinical epidemiology in animals and humans. Such studies would be valuable for a better understanding of the transmission of the E. granulosus s.l. species complex and their potential clinical impact on humans. Graphical Abstract

Abstract Background Cystic echinococcosis (CE) is a parasitic zoonosis caused by infection with the larval stage of Echinococcus granulosus (s.l.). This study investigated the prevalence and potential risk factors associated with human CE in the towns and rural areas of Ñorquinco and Ramos Mexia, Rio Negro province, Argentina. Methods To detect abdominal CE cysts, we screened 892 volunteers by ultrasound and investigated potential risk factors for CE using a standardized questionnaire. Prevalence ratio (PR) with 95% confidence intervals (CI) was used to measure the association between CE and the factors investigated, applying bivariate and multivariate analyses. Results Abdominal CE was detected in 42/892 screened volunteers (4.7%, 95% CI 3.2–6.1), only two of whom were under 15 years of age. Thirteen (30.9%) CE cases had 25 cysts in active stages (CE1, CE2, CE3a, according to the WHO Informal Working Group on Echinococcosis [WHO-IWGE] classification). The most relevant risk factors identified in the bivariate analysis included: living in rural areas (P = 0.003), age > 40 years (P = 0.000), always drinking water from natural sources (P = 0.007), residing in rural areas during the first 5 years of life (P = 0.000) and having lived more than 20 years at the current address (P = 0.013). In the multivariate final model, the statistically significant risk factors were: frequently touching dogs (P = 0.012), residing in rural areas during the first 5 years of life (P = 0.004), smoking (P = 0.000), age > 60 years (P = 0.002) and living in rural areas (P = 0.017). Conclusions Our results point toward infection with CE being acquired since childhood and with constant exposure throughout life, especially in rural areas with a general environmental contamination. Graphic Abstract

Abstract Background Human alveolar echinococcosis (AE) caused by Echinococcus multilocularis is an underreported, often misdiagnosed and mistreated parasitic disease mainly due to its low incidence. The aim of this study was to describe the epidemiological and clinical characteristics of human AE patients in Hungary for the first time. Method Between 2003 and 2018, epidemiological and clinical data of suspected AE patients were collected retrospectively from health database management systems. Results This case series included a total of 16 AE patients. The mean age of patients was 53 years (range: 24–78 years). The sex ratio was 1:1. Four patients (25%) revealed no recurrence after radical surgery and adjuvant albendazole (ABZ) therapy. For five patients (31.3%) with unresectable lesions, a stabilization of lesions with ABZ treatment was achieved. In seven patients (43.8%), progression of AE was documented. The mean diagnostic delay was 33 months (range: 1–122 months). Three AE related deaths (fatality rate 18.8%) were recorded. Conclusions AE is an emerging infectious disease in Hungary with a high fatality rate since based on our results, almost every fifth AE patient died in the study period. Differential diagnosis and appropriate surgical and medical therapy for AE is an urging challenge for clinicians in Hungary, as well as in some other European countries where E. multilocularis is prevalent.

A direct enantio-, diastereo-, and chemo-selective high-performance liquid chromatographic method was developed for determining the content, enantiomeric purity, and related substances of the chiral antidepressant drug sertraline HCl in a single chromatographic run. The separation was achieved on a chiral stationary phase based on amylose tris(3-chloro-5-methylphenylcarbamate) under reversed-phase conditions. The method was optimized by evaluating the influence of the temperature and mobile phase composition on the retention and selectivity. The application of the single-run approach allowed to baseline resolve all investigated species in less than 15 min, without using buffers or tandem-coupled columns. The chromatographic method was validated according to the guidelines of the Official Medicines Control Laboratory and applied to control the content of sertraline HCl and related chiral substances in a generic antidepressant formulation.

BackgroundCystic echinococcosis (CE) caused by Echinococcus granulosus sensu lato (s.l.) is a neglected and underdiagnosed parasitic zoonosis that has a significant socioeconomic impact on rural communities relying on livestock farming. CE is endemic across Latin America, including Chile, where the Coquimbo region exhibits a relatively high record of hospital-based human cases and infected animals. However, the incidence of hospitalized CE cases may underestimate the real burden of infection in a population, since the majority of cases never reach medical attention or official disease records.Methodology/principal findingsIn 2019, a cross-sectional, community-based study was conducted with the objectives of estimating for the first time the prevalence of human abdominal CE using abdominal ultrasound (US) screening in volunteers residing in urban and rural localities of the Monte Patria municipality located in Limarí province, Coquimbo region, Chile, and identifying the risk factors associated with human infection. Pre-screening activities included a 16-h lecture/hands-on training aimed at rural physicians that focused on the diagnosis of CE by US, based on current WHO recommendations. A total of 2,439 (~8% of municipality inhabitants) people from thirteen target localities were screened by abdominal US in June-July 2019. We found an overall CE prevalence of 1.6% (95% CI 1.1-2.2) with a significantly higher likelihood of infection in rural localities, older age classes and people drinking non-potable water; 84.6% of infected volunteers were newly diagnosed with CE. Cysts were either in active or inactive stages in equal proportions; active cysts were detected in all age classes, while 95.7% of inactive cysts occurred in >40 years-old subjects.Conclusions/significanceThis is the first US survey aimed at detecting human infection caused by Echinococcus granulosus s.l. in Chile. Our findings indicate a high CE prevalence in the area, and contribute to define the demographic and behavioral risk factors promoting the transmission of the parasitic infection within target communities. Our results support the implementation of cost-effective strategies for the diagnosis, treatment and control of CE, and the need to improve the epidemiological surveillance system in Chile.

Abstract Background The real burden of human cystic echinococcosis (CE) remains elusive, due to the peculiar characteristics of the disease and the heterogeneous and incomplete data recording of clinical cases. Furthermore, official notification systems do not collect pivotal clinical information, which would allow the comparison of different treatment outcomes, and thus circumvent the difficulty of implementing clinical trials for CE. The Italian Register of CE (RIEC) was launched in 2012 and expanded in 2014 into the European Register of CE (ERCE). The primary aim of the ERCE was to highlight the magnitude of CE underreporting, through the recording of cases that were not captured by official records. We present an overview of data collated in the ERCE and discuss its future, five years after its inception. Methods The ERCE database was explored on March 31st 2019; data concerning participating centres and registered cases were descriptively analysed. Results Forty-four centres from 15 countries (7 non-European) were affiliated to the ERCE. Thirty-four centres (77%) registered at least one patient; of these, 18 (53%) recorded at least one visit within the past 18 months. A total of 2097 patients were registered, 19.9% of whom were immigrants. Cyst characteristics were reported for at least one cyst at least in one visit in 1643 (78.3%) patients, and cyst staging was used by 27 centres. In total, 3386 cysts were recorded at first registration; mostly located in the liver (75.5%). Data concerning clinical management could be analysed for 920 “cyst stage-location-management” observations, showing great heterogeneity in the implementation of the stage-specific management approach recommended by the WHO. Conclusions The ERCE achieved its goal in showing that CE is a relevant but neglected public health problem in Europe and beyond, since a proportion of patients reaching medical attention are not captured by official notification systems. The ERCE may provide a valuable starting platform to complement hospital-derived data, to obtain a better picture of the epidemiology of clinical CE, and to collect clinical data for the issue of evidence-based recommendations. The ERCE will be expanded into the International Register of CE (IRCE) and restructured aiming to overcome its current criticalities and fulfil these aims.

An integrated model, based on a One Health approach, was implemented to estimate the epidemiological and economic outcomes of cystic echinococcosis (CE) in Veneto region, an hypo-endemic area of Northern Italy, and the costs for its prevention. Data and information needed to populate the model were retrieved from published literature, official statistics, expert opinions, or actively searched through data mining (i.e., Hospital and slaughterhouse data), when fundamental data were not available. Human-health and animal-health costs, both public and private, were considered. The overall impact of CE in the study area was estimated in an yearly cost of about 0.5 million €, due to an average of 19.5 human hospitalized cases and about 200 infected animals among cattle and sheep, per year. The human:animal costs ratio was about 8:1. Most of the infected animals were autochthonous, while the identification of an autochthonous source of the infection for the human cases was extremely difficult, and unlikely in most cases. No specific action resulted to be in place for human surveillance, while veterinary surveillance accounted for a yearly cost of about 22,000 €. Sheepherders were found to pay privately an overall amount of around 2000 € for the preventive treatment of their dogs every year, but the applied protocol proved to be sub-optimal. The source of most of the human cases was likely external to the study area, and their economic impact accounts for a cost that is far exceeding that of surveillance and preventive actions in place in the veterinary sector. Although autochthonous human cases appeared to be very rare, the strengthening of preventive actions and surveillance systems can reduce the risk of their increment.

Carvedilol is a chiral drug with potent antihypertensive and antianginal activities. Although it is clinically used as a racemic mixture, its enantiomers show different pharmacokinetic and pharmacodynamic profiles. Here, the direct chiral separation of racemic drug by high performance liquid chromatography using two immobilized-type amylose-based chiral stationary phases is presented. Some chromatographic parameters, such as retention and selectivity, were determined under multimodal eluent conditions and different temperatures. A temperature-dependent inversion of the elution order of enantiomers was observed in the operative temperature range of chiral chromatographic support. Finally, an effective direct enantioselective method was successfully applied to the separation of the enantiomers of carvedilol on a semipreparative scale. Keywords: Chiralpak® IG-3, Enantioselective HPLC, Carvedilol, Column temperature, Isoenantioselective temperature, Semipreparative enantioseparation

Abstract Background Cystic echinococcosis (CE) is a neglected parasitic zoonosis prioritized by the WHO for control. Several studies have investigated potential risk factors for CE through questionnaires, mostly carried out on small samples, providing contrasting results. We present the analysis of risk factor questionnaires administered to participants to a large CE prevalence study conducted in Bulgaria, Romania and Turkey. Methods A semi-structured questionnaire was administered to 24,687 people from rural Bulgaria, Romania and Turkey. CE cases were defined as individuals with abdominal CE cysts detected by ultrasound. Variables associated with CE at P

The European distribution of Echinococcus multilocularis has been reported to be expanding east and north, whereas its southern limits are deemed stable. During a study on Echinococcus granulosus s.l. infections in wolves and dogs in the Italian Maritime Alps, we unexpectedly detected the presence of E. multilocularis eggs in four fecal samples from at least two shepherd dogs, and in five wolf fecal samples. This finding, in an area about 130 km south of the southernmost E. multilocularis report in the Alps, may suggest a rapid expansion southward. While infections in foxes are currently being investigated, these data seem to indicate the potential for a new E. multilocularis endemic area. If this will be confirmed, the implementation of surveillance programs in wild and domestic canids and preventative measures will become a priority. Keywords: Echinococcus multilocularis, Italy, Parasite range expansion, Canis lupus, Domestic dog, Alveolar echinococcosis

The second World Neglected Tropical Diseases (NTDs) Day was celebrated on 30 January 2021. To mark the occasion, the World Health Organization (WHO) launched its roadmap for NTDs for the period 2021 to 2030, which is aimed at increasing prevention and control of these too-long neglected diseases. Described here is a global overview on past achievements, current challenges, and future prospects for the WHO NTDs roadmap 2021-2030.

Cystic echinococcosis (CE) is a neglected worldwide distributed parasitic disease caused by the Echinococcusgranulosus sensu lato (s.l.) species complex. For a better understanding of the pathways of transmission of this parasite, clinical and molecular epidemiological studies are particularly needed from endemic areas where data are scant, such as in the Middle East. The study aimed to identify the characteristics, location, cyst stage and species/genotypes of E. granulosus s.l. complex in humans from the Kurdistan region, Iraq. To this aim, from June 2019 to February 2021, 64 echinococcal cysts were surgically removed from 62 patients in Azadi and Vajeen reference Hospitals at Duhok city, Duhok governorate (Kurdistan region, Iraq). The results confirmed the liver as the most common anatomical site of CE with 72.58% of the cases, followed by the lungs in 19.35%, while 66.13% of CE cases were females. The highest rate of infections occurred in the age class 21–30 (27.42%). High rates of CE were reported among patients living in rural areas and housewives, which were 54.84% and 43.55% of the CE patients, respectively. The fertility of echinococcal cysts was 82.81%, and the viability of fertile protoscoleces was 70.53%. Cysts were staged with ultrasound according to the WHO-IWGE classification as 32.8% CE1, 32.8% CE2, 7.8% CE3a, 9.4% CE3b, 15.6% CE4 and 1.6% CE5. Molecular analyses using mitochondrial NAD5 gene showed that all analyzed samples (n = 59) belonged to the genotypes G1 or G3 of E. granulosussensu stricto (s.s.), thus, confirming sheep–dog–human transmission in the Kurdistan region, Iraq. No statistically significant correlation was found between the genotypes G1–G3 of E. granulosus s.s. and variables, such as the fertility, location and cyst stage classification. Based on the present findings, it is necessary to implement monitoring and control programs in sheep and dog populations to decrease the odds of human infections. Public health education campaigns are required to be implemented at the community level to reduce the risk of acquiring CE in humans in the Kurdistan region, Iraq.

Cystic echinococcosis (CE) is a neglected zoonotic disease caused by Echinococcus granulosus sensu lato. Diagnosis and monitoring of CE rely primarily on imaging while serology is used as a confirmatory test. However, imaging is not always conclusive and currently available serological assays have suboptimal sensitivity and specificity, lack standardization, and are not useful for patients´ follow-up. Seroassays for CE are usually based on hydatid fluid (HF), a complex, variable antigenic mixture, and cross-reactivity exists especially with alveolar echinococcosis. Recombinant proteins based on immunogenic antigens most abundant in HF, such as AgB1, AgB2 and Ag5, have been used to overcome these limitations. None of them so far showed potential to replace HF; however, their performance have been largely tested on a limited number of samples, and comparison of different antigens using the same cohort has been rarely performed. The combination of several immunogenic epitopes in a single recombinant protein could enhance test sensitivity. For the diagnosis and follow-up of patients with CE, we compared the performance of the crude HF, previously described recombinant 2B2t antigen, and GST-tagged version of 2B2t, and novel designed recombinants (GST-Ag5t and the GST-DIPOL chimera containing AgB1, AgBB2 and Ag5 epitopes) by IgG-ELISA format. Samples belong to a retrospective cohort of 253 well-characterized patients with CE, previously described for the evaluation of the 2B2t antigen, 92 patients with alveolar echinococcosis, and 82 healthy donors. The reference standard for CE diagnosis was the presence of a CE lesion as diagnosed by ultrasonography. The highest sensitivity was obtained with HF [86.7%, 95% confidence interval (CI): 81.2-91.0], followed by GST-2B2t (70.0%, 95% CI: 63.1-76.2), 2B2t (65.5%, 95% CI: 58.5-72.0), GST-Ag5t (64.5%, 95% CI: 57.5-71.1) and GST-DIPOL (63.1%, 95% CI: 56.0-69.7). The GST-2B2t had the best specificity (95.8%, 95% CI: 88.3-99.1) and the lowest cross-reactivity (38.7%, 95% CI: 27.6-50.6). Good response to treatment also correlated to negative test results in the GST-2B2t ELISA. While none of the tested recombinant antigen appears suitable to replace HF for the diagnosis of CE, GST-2B2t should be further explored as a confirmation test, based on its high specificity and low cross-reactivity, and for the follow-up after treatment in those patients with positive serology for this antigen.

The reference diagnostic method of human abdominal Cystic Echinococcosis (CE) is imaging, particularly ultrasound, supported by serology when imaging is inconclusive. However, current diagnostic tools are neither optimal nor widely available. The availability of a test detecting circulating biomarkers would considerably improve CE diagnosis and cyst staging (active vs inactive), as well as treatments and follow-up of patients. Exosomes are extracellular vesicles involved in intercellular communication, including immune system responses, and are a recognized source of biomarkers. With the aim of identifying potential biomarkers, plasma pools from patients infected by active or inactive CE, as well as from control subjects, were processed to isolate exosomes for proteomic label-free quantitative analysis. Results were statistically processed and subjected to bioinformatics analysis to define distinct features associated with parasite viability. First, a few parasite proteins were identified that were specifically associated with either active or inactive CE, which represent potential biomarkers to be validated in further studies. Second, numerous identified proteins of human origin were common to active and inactive CE, confirming an overlap of several immune response pathways. However, a subset of human proteins specific to either active or inactive CE, and central in the respective protein-protein interaction networks, were identified. These include the Src family kinases Src and Lyn, and the immune-suppressive cytokine TGF-β in active CE, and Cdc42 in inactive CE. The Src and Lyn Kinases were confirmed as potential markers of active CE in totally independent plasma pools. In addition, insights were obtained on immune response profiles: largely consistent with previous evidence, our observations hint to a Th1/Th2/regulatory immune environment in patients with active CE and a Th1/inflammatory environment with a component of the wound healing response in the presence of inactive CE. Of note, our results were obtained for the first time from the analysis of samples obtained in vivo from a well-characterized, large cohort of human subjects.

Human cystic echinococcosis (CE) is a worldwide-distributed parasitic zoonotic disease, which represents a threat for both human and animals. The current study aimed at estimating the prevalence of human CE in Khyber Pakhtunkhwa (KPK) province of Pakistan. Clinical records from four major hospitals in this region were reviewed for CE human cases during the period of 2006–2021. Out of 251 (0.00071%) CE patients identified during the considered period, 142 (56.6%) were females, and 109 (43.4%) were males. The highest number of CE cases was recorded in the 21–30 (27.9%) age group, followed by 31–40 (23.1%) and 41–50 (16.3%). Most of the CE patients in KPK province were members of the Afghani ethnic group (17.1%); secondarily, they were Pakistani (6.4%), while for 76.5% ethnicity data were not available. The liver (41%) and the lungs (4.8%) were the most infected organs identified among CE patients in KPK province. The present study identified CE as a significant public health problem in KPK province, and the current findings demonstrated a constant endemicity of CE during the last 15 years. Further filed studies on the active search of CE carriers by means of ultrasound population-based surveys are needed to fill knowledge gaps on clinical and molecular epidemiology of human CE in Pakistan.

Recent reports have demonstrated that the new commercially available immobilized-type chiral stationary phases (CSPs) containing amylose tris(3-chloro-5-methylphenylcarbamate) (ACMPC) as a selector exhibit not only an exceptionally high enantioselectivity in high-performance liquid chromatography (HPLC) but they are also applicable to a wide range of chiral analytes. Herein, we report the results obtained in the HPLC analysis of omeprazole and its impurities B and E on the ACMPC-based Chiralpak IG-3 CSP (CSP) under polar organic conditions. A systematic evaluation of the retention characteristics of the selected benzimidazole chiral probes was carried out by changing the composition of the mobile phase and the column temperature. It is worth emphasizing that the high affinity of both enantiomers of all analytes recorded in pure methanol mode dramatically decreased incorporating small volumes of either basic or acid additives in the mobile phase. Unspecified sites of the IG-3 CSP presumably involved in strong and non-stereoselective H-bonding contacts with chiral analytes are assumed responsible for the unproductive retention process. Keywords: Chiralpak® IG-3, Retention, Enantioselective HPLC, Omeprazole, Column temperature, Basic/acid additives

Among echinococcoses diseases of human interest, two have a global public health impact: cystic and alveolar echinococcosis caused by Echinococcus granulosus sensu lato and Echinococcus multilocularis, respectively. Cystic and alveolar echinococcosis are neglected infectious diseases epidemiologically and are clinically vastly different with distinct microscopic features. Because of the rareness of these zoonotic diseases, pathologists have limited diagnostic experience in the analysis of the lesions caused by Echinococcus tapeworms. Here, we describe the main microscopic features to be considered to characterize these lesions: laminated layer, central necrosis, growth pattern, and delineation from adjacent tissue. Moreover, immunohistology using monoclonal antibodies is of great diagnostic help in reaching a definitive diagnosis by identifying the laminated body and small particles of E. multilocularis (spems) and small particles of E. granulosus (spegs).

Human alveolar echinococcosis (AE), caused by the tapeworm Echinococcus multilocularis, is one of the most dangerous zoonoses in the Northern hemisphere. In Europe, the parasite's life cycle is sylvatic, involving small rodents as intermediate hosts and red foxes as the major definitive hosts. Given the severity of this disease in humans and the high levels of environmental contamination with E. multilocularis in endemic areas, it seems crucial to implement control measures in order to prevent human AE. This systematic review identifies previous anthelmintic control programs targeting E. multilocularis in wild and domestic carnivores and evaluates the effectiveness of the different strategies implemented. A search through six databases identified 302 scientific papers for the period 1950–2015, of which only 17 were retained according to the inclusion criteria set. These 17 papers focused on control of E. multilocularis by baiting foxes in highly endemic areas of Europe or Japan, with the exception of one study focused on dogs in Alaska. The papers highlighted differences in baiting types, baiting frequency, choice of control areas and length of treatment period. Overall, these studies resulted in a sharp and statistically significant decrease in parasite prevalence, confirmed by the absence of overlap between confidence intervals for the pooled risk differences of control and treated areas. A monthly baiting frequency was proven to be highly effective at decreasing E. multilocularis prevalence in foxes, even in highly endemic areas and in a short period of time. Nevertheless, when foxes were not fully dewormed, the parasite showed a strong capacity to rapidly recover its initial prevalence.The fox baiting approach appears to be the most useful method for controlling the sylvatic life cycle of E. multilocularis, but it require a cost/benefit analysis before it is likely to be accepted by stakeholders. Keywords: Echinococcus multilocularis, systematic review, control programs, baiting

Repurposing of approved non-antitumor drugs represents a promising and affordable strategy that may help to increase the repertoire of effective anticancer drugs. Benzimidazole-based anthelmintics are antiparasitic drugs commonly employed both in human and veterinary medicine. Benzimidazole compounds are being considered for drug repurposing due to antitumor activities displayed by some members of the family. In this study, we explored the effects of a large series of benzimidazole-based anthelmintics (and some enantiomerically pure forms of those containing a stereogenic center) on the viability of different tumor cell lines derived from paraganglioma, pancreatic and colorectal cancer. Flubendazole, parbendazole, oxibendazole, mebendazole, albendazole and fenbendazole showed the most consistent antiproliferative effects, displaying IC50 values in the low micromolar range, or even in the nanomolar range. In silico evaluation of their physicochemical, pharmacokinetics and medicinal chemistry properties also provided useful information related to the chemical structures and potential of these compounds. Furthermore, in view of the potential repurposing of these drugs in cancer therapy and considering that pharmaceutically active compounds may have different mechanisms of action, we performed an in silico target prediction to assess the polypharmacology of these benzimidazoles, which highlighted previously unknown cancer-relevant molecular targets.

Cystic echinococcosis (CE) or hydatid cyst (HC) caused by the larval stage of the tapeworm Echinococcus granulosus continues to be a substantial cause of morbidity and mortality in many parts of the world. Since Kurdistan region is considered as endemic area, an epidemiological study was conducted to investigate the occurrence of CE isolated from sheep, cattle and goats from July 2013 to June 2014. The total rate of infection was 11.17(457/4092), with the highest rate (9.07%) in sheep and the lowest (0.54%) in goats. Regarding the sex of the infected animals, female showed slightly a higher rate than males (5.99% versus 5.18%) which statistically non-significant (P>0.05). Among the three intermediate host sheep showed the highest rate of infection (14.51%) regarding the sex females of sheep and cattle showed higher rate of infection versus males (27.36 and 20.35 versus 9.37 and 3.86). Furthermore, the highest incidence of CE in sheep, cattle and goats were found among the age group over three years (18.75, 5.41 and 1.69%, respectively), while the lowest rate was noted in the age groups less than one year (3.96 and 0.25%, o.33, respectively). On the other hand, sheep showed high rate of infection throughout the year with peaks during May to July which were 19.44%, 17.59 and 18.14%,respectively. This was followed by cattle and goats (11.67% and 5.26%) in April and December, respectively. Regarding the size, the higher numbers of small sized cysts were found in the liver (98/175), while the highest numbers of large sized cysts were found in both liver and lung which were 69/165. The liver and lungs appeared to be the site of predilection, since highest rate of infection was reported in these organs. Regarding the fertility of CE, the highest was in sheep (87.6%), followed by goats (77.27%) and then the cattle (40.63%).

Infections with eggs of Echinococcus granulosus sensu lato (s.l.) can cause cystic echinococcosis in intermediate host animals and humans. Upon ingestion of viable eggs, oncospheres hatch from the eggs and subsequently develop into fluid-filled larval cysts, most frequently in the liver or the lungs. The slowly growing cysts progressively interfere with organ function. The risk of infection is determined by the host range of the parasite, its pathogenicity and other epidemiologically relevant parameters, which differ significantly among the five species within the E. granulosus s.l. complex. It is therefore essential to diagnose the correct species within E. granulosus s.l. to help understand specific disease epidemiology and to facilitate effective implementation of control measures. For this purpose, simple, fast and cost-effective typing techniques are needed. We developed quantitative real-time polymerase chain reactions (qPCRs) to target polymorphic regions in the mitochondrial genome of E. granulosus s.l. In a single-step typing approach, we distinguished E. granulosus s.l. members in four epidemiologically relevant subgroups. These were E. granulosus sensu stricto, E. equinus, E. ortleppi and the E. canadensis cluster. The technique also allowed identification and differentiation of these species from other Echinococcus or Taenia taxa for samples isolated from cysts or faeces.

Cystic echinococcosis (CE) is one of the most widespread helminthic zoonoses and is caused by the tapeworm Echinococcus granulosus complex. CE diagnosis and monitoring primarily rely on imaging techniques, complemented by serology. This is usually approached by the detection of IgG antibodies against hydatid fluid (HF), but the use of this heterogeneous antigenic mixture results in a variable percentage of false positive and negative results, and has shown to be useless for follow-up due to the long persistence of anti-HF antibodies in cured patients. To improve test performances and standardization, a number of recombinant antigens mainly derived from HF have been described, among them the B2t and 2B2t antigens. The performance of these antigens in the diagnosis and follow up of patients with CE has been so far evaluated on a limited number of samples. Here, we evaluated the performances of tests based on B2t and 2B2t recombinant antigens compared to HF in IgG-ELISA and immunochromatography (IC) for the diagnosis and follow-up of patients with CE in a retrospective cohort study. A total of 721 serum samples were collected: 587 from 253 patients with CE diagnosed by ultrasonography (US), 42 from patients with alveolar echinococcosis and 92 from healthy donors from Salamanca (Spain). The highest overall sensitivity was obtained with HF in ELISA (85.5%), followed by IC containing HF and 2B2t-HF (83.0% and 78.2%, respectively). The lowest sensitivity was obtained with B2t and 2B2t in ELISA (51.8%). The highest specificity was obtained with IC containing 2B2t-HF (100%), and the lowest with HF-ELISA (78.0%). The lowest cross-reactivity with sera from patients with alveolar echinococcosis was detected with the recombinant antigens in ELISA (9.5% - 16.7%) and the highest with the HF-IC (64.3%). The results of B2t and 2B2t-ELISA were influenced by cyst stage, as classified by US according to the WHO-Informal Working Group on Echinococcosis (WHO-IWGE), with low sensitivity for inactive (CE4 and CE5) cysts, and by the drug treatment, with higher sensitivity in patients after drug treatment compared with patients not subjected to drug treatment. The two recombinant antigens in ELISA provided promising results for monitoring patients in follow-up, although their use is limited to patients with positive serology against them at the beginning of the follow-up. Potential biological reasons behind the low sensitivity of the recombinant antigens and possible strategies to enhance the performance of CE serology are discussed.

Human cystic and alveolar echinococcosis are helmintic zoonotic diseases caused by infections with the larval stages of the cestode parasites Echinococcus granulosus and E. multilocularis, respectively. Both diseases are progressive and chronic, and often fatal if left unattended for E. multilocularis. As a treatment approach, chemotherapy against these orphan and neglected diseases has been available for more than 40 years. However, drug options were limited to the benzimidazoles albendazole and mebendazole, the only chemical compounds currently licensed for treatment in humans. To compensate this therapeutic shortfall, new treatment alternatives are urgently needed, including the identification, development, and assessment of novel compound classes and drug targets. Here is presented a thorough overview of the range of compounds that have been tested against E. granulosus and E. multilocularis in recent years, including in vitro and in vivo data on their mode of action, dosage, administration regimen, therapeutic outcomes, and associated clinical symptoms. Drugs covered included albendazole, mebendazole, and other members of the benzimidazole family and their derivatives, including improved formulations and combined therapies with other biocidal agents. Chemically synthetized molecules previously known to be effective against other infectious and non-infectious conditions such as anti-virals, antibiotics, anti-parasites, anti-mycotics, and anti-neoplastics are addressed. In view of their increasing relevance, natural occurring compounds derived from plant and fungal extracts are also discussed. Special attention has been paid to the recent application of genomic science on drug discovery and clinical medicine, particularly through the identification of small inhibitor molecules tackling key metabolic enzymes or signalling pathways.

Abstract Background Following the first finding of Echinococcus multilocularis in Sweden in 2011, 2985 red foxes (Vulpes vulpes) were analysed by the segmental sedimentation and counting technique. This is a labour intensive method and requires handling of the whole carcass of the fox, resulting in a costly analysis. In an effort to reduce the cost of labour and sample handling, an alternative method has been developed. The method is sensitive and partially automated for detection of E. multilocularis in faecal samples. The method has been used in the Swedish E. multilocularis monitoring program for 2012–2013 on more than 2000 faecal samples. Methods We describe a new semi-automated magnetic capture probe DNA extraction method and real time hydrolysis probe polymerase chain reaction assay (MC-PCR) for the detection of E. multilocularis DNA in faecal samples from red fox. The diagnostic sensitivity was determined by validating the new method against the sedimentation and counting technique in fox samples collected in Switzerland where E. multilocularis is highly endemic. Results Of 177 foxes analysed by the sedimentation and counting technique, E. multilocularis was detected in 93 animals. Eighty-two (88%, 95% C.I 79.8-93.9) of these were positive in the MC-PCR. In foxes with more than 100 worms, the MC-PCR was positive in 44 out of 46 (95.7%) cases. The two MC-PCR negative samples originated from foxes with only immature E. multilocularis worms. In foxes with 100 worms or less, (n = 47), 38 (80.9%) were positive in the MC-PCR. The diagnostic specificity of the MC-PCR was evaluated using fox scats collected within the Swedish screening. Of 2158 samples analysed, two were positive. This implies that the specificity is at least 99.9% (C.I. = 99.7 -100). Conclusions The MC-PCR proved to have a high sensitivity and a very high specificity. The test is partially automated but also possible to perform manually if desired. The test is well suited for nationwide E. multilocularis surveillance programs where sampling of fox scats is done to reduce the costs for sampling and where a test with a high sensitivity and a very high specificity is needed.

Human alveolar echinococcosis (AE) is a severe zoonotic disease caused by the metacestode stage of Echinococcus multilocularis. AE is commonly associated with a long incubation period that may last for more than ten years. The objective of this systematic literature review was to identify and summarize the current knowledge on statistically relevant potential risk factors (PRFs) associated with AE in humans.Six bibliographic databases were searched, generating a total of 1,009 publications. Following the removal of duplicate records and the exclusion of papers that failed to meet the criteria of a previously agreed a priori protocol, 23 publications were retained; however, 6 of these did not contain data in a format that allowed their inclusion in the meta-analysis. The remaining 17 publications (6 case-control and 11 cross-sectional studies) were meta-analysed to investigate associations between AE and PRFs. Pooled odds ratios (OR) were used as a measure of effect and separately analysed for case-control and cross-sectional studies. In the case-control studies, the following PRFs for human AE showed higher odds of outcome: "dog ownership", "cat ownership", "have a kitchen garden", "occupation: farmer", "haymaking in meadows not adjacent to water", "went to forests for vocational reasons", "chewed grass" and "hunting / handling foxes". In the cross-sectional studies, the following PRFs showed higher odds of outcome: "dog ownership", "play with dogs", "gender: female", "age over 20 years", "ethnic group: Tibetan", "low income", "source of drinking water other than well or tap", "occupation: herding" and "low education". Our meta-analysis confirmed that the chance of AE transmission through ingestion of food and water contaminated with E. multilocularis eggs exists, but showed also that food- and water-borne PRFs do not significantly increase the risk of infection.This systematic review analysed international peer-reviewed articles that have over the years contributed to our current understanding of the epidemiology of human AE. The identification of potential risk factors may help researchers and decision makers improve surveillance and/or preventive measures that aim at decreasing human infection with E. multilocularis. More primary studies are needed to confirm potential risk factors and their role in the epidemiology of human AE.

Cystic echinococcosis (CE) is classified by the WHO as a neglected disease inflicting economic losses on the health systems of many countries worldwide. The aim of this case-series study was to investigate the burden of human CE in Palestine during the period between 2010 and 2015.Records of surgically confirmed CE patients from 13 public and private hospitals in the West Bank and Gaza Strip were reviewed. Patients' cysts were collected from surgical wards and formalin-fixed paraffin-embedded (FFPE) blocks were collected from histopathology departments. Molecular identification of CE species /genotypes was conducted by targeting a repeat DNA sequence (EgG1 Hae III) within Echinococcus nuclear genome and a fragment within the mitochondrial cytochrome c oxidase subunit 1, (CO1). Confirmation of CE species/genotypes was carried out using sequencing followed by BLAST analysis and the construction of maximum likelihood consensus dendrogram. CE cases were map-spotted and statistically significant foci identified by spatial analysis. A total of 353 CE patients were identified in 108 localities from the West Bank and Gaza Strip. The average surgical incidence in the West Bank was 2.1 per 100,000. Spot-mapping and purely spatial analysis showed 13 out of 16 Palestinian districts had cases of CE, of which 9 were in the West Bank and 4 in Gaza Strip. Al-Khalil and Bethlehem were statistically significant foci of CE in Palestine with a six-year average incidence of 4.2 and 3.7 per 100,000, respectively.To the best of our knowledge, this is the first confirmation of human CE causative agent in Palestine. This study revealed that E. granulosus sensu stricto (s.s.) was the predominating species responsible for CE in humans with 11 samples identified as G1 genotype and 2 as G3 genotype. This study emphasizes the need for a stringent surveillance system and risk assessment studies in the rural areas of high incidence as a prerequisite for control measures.

Evolution and dispersion history on Earth of organisms can best be studied through biological markers in molecular epidemiological studies. The biological diversity of the cestode Echinococcus multilocularis was investigated in different cladistic approaches. First the morphological aspects were explored in connection with its ecology. More recently, molecular aspects were investigated to better understand the nature of the variations observed among isolates. The study of the tandemly repeated multilocus microsatellite EmsB allowed us to attain a high genetic diversity level where other classic markers have failed. Since 2006, EmsB data have been collected on specimens from various endemic foci of the parasite in Europe (in historic and newly endemic areas), Asia (China, Japan and Kyrgyzstan), and North America (Canada and Alaska). Biological data on the isolates and metadata were also recorded (e.g. host, geographical location, EmsB analysis, citation in the literature). In order to make available the data set of 1,166 isolates from classic and aberrant domestic and wild animal hosts (larval lesions and adult worms) and from human origin, an open web access interface, developed in PHP, and connected to a PostgreSQL database, was developed in the EmsB Website for the Echinococcus Typing (EWET) project. It allows researchers to access data collection, perform genetic analyses online (e.g. defining the genetic distance between their own samples and the samples in the database), consult distribution maps of EmsB profiles, and record and share their new EmsB genotyping data. In order to standardize the EmsB analyses performed in the different laboratories throughout the world, a calibrator was developed. The final aim of this project was to gather and arrange available data to permit to better understand the dispersion and transmission patterns of the parasite among definitive and intermediate hosts, in order to organize control strategies on the ground.

To characterize the circulating E. granulosus genotype in Duhok province, a total of 17 human (14 female and 3 male) hydatid cysts (10 from lung and 7 from liver) were subjected to molecular analysis using mitochondrial cox1 gene sequencing. Total DNA was extracted using Qiagene DNeasy blood and tissue mini kit then it was electrophoresed on 1.5% Agarose gel in TBE 0.5% buffer for 20 minutes and sequenced. Variable sequences of 418bp were obtained for 15 out of 17 samples. The sequences were aligned with old sequences of G1, G2, G3 genotypes and the new Turkish and Iranian genotypes. As a result, the Duhok isolates were more identical to the newer Turkish and Iranian G1 common sheep genotype sequences than the old ones due to the occurrence of different multiple nucleotide substitutions among nearly half of Duhok isolates. In conclusion, the results of this study showed the presence of the G1-G3 cluster sensu stricto cluster genotype in Duhok province, therefore preventive measures should be arranged accordingly.

BACKGROUND:Scientific literature on cystic echinococcosis (CE) reporting data on risk factors is limited and to the best of our knowledge, no global evaluation of human CE risk factors has to date been performed. This systematic review (SR) summarizes available data on statistically relevant potential risk factors (PRFs) associated with human CE. METHODOLOGY/PRINCIPAL FINDINGS:Database searches identified 1,367 papers, of which thirty-seven were eligible for inclusion. Of these, eight and twenty-nine were case-control and cross-sectional studies, respectively. Among the eligible papers, twenty-one were included in the meta-analyses. Pooled odds ratio (OR) were used as a measure of effect and separately analysed for the two study designs. PRFs derived from case-control studies that were significantly associated with higher odds of outcome were "dog free to roam" (OR 5.23; 95% CI 2.45-11.14), "feeding dogs with viscera" (OR 4.69; 95% CI 3.02-7.29), "slaughter at home" (OR 4.67; 95% CI 2.02-10.78) or at "slaughterhouses" (OR 2.7; 95% CI 1.15-6.3), "dog ownership" (OR 3.54; 95% CI 1.27-9.85), "living in rural areas" (OR 1.83; 95% CI 1.16-2.9) and "low income" (OR 1.68; 95% CI 1.02-2.76). Statistically significant PRFs from cross-sectional studies with higher odds of outcome were "age >16 years" (OR 6.08; 95% CI 4.05-9.13), "living in rural areas" (OR 2.26; 95% CI 1.41-3.61), "being female" (OR 1.38; 95% CI 1.06-1.8) and "dog ownership" (OR 1.37; 95% CI 1.01-1.86). CONCLUSIONS/SIGNIFICANCE:Living in endemic rural areas, in which free roaming dogs have access to offal and being a dog-owner, seem to be among the most significant PRFs for acquiring this parasitic infection. Results of data analysed here may contribute to our understanding of the PRFs for CE and may potentially be useful in planning community interventions aimed at controlling CE in endemic areas.

The diagnosis of cystic echinococcosis (CE) is based primarily on imaging, in particular with ultrasound for abdominal CE, complemented by serology when imaging results are unclear. In rural endemic areas, where expertise in ultrasound may be scant and conventional serology techniques are unavailable due to lack of laboratory equipment, Rapid Diagnostic Tests (RDTs) are appealing.We evaluated the diagnostic accuracy of 3 commercial RDTs for the diagnosis of hepatic CE. Sera from 59 patients with single hepatic CE cysts in well-defined ultrasound stages (gold standard) and 25 patients with non-parasitic cysts were analyzed by RDTs VIRapid HYDATIDOSIS (Vircell, Spain), Echinococcus DIGFA (Unibiotest, China), ADAMU-CE (ICST, Japan), and by RIDASCREEN Echinococcus IgG ELISA (R-Biopharm, Germany). Sensitivity, specificity and ROC curves were compared with McNemar and t-test. For VIRapid and DIGFA, correlation between semiquantitative results and ELISA OD values were evaluated by Spearman's coefficient. Reproducibility was assessed on 16 randomly selected sera with Cohen's Kappa coefficient. Sensitivity and Specificity of VIRapid (74%, 96%) and ADAMU-CE (57%, 100%) did not differ from ELISA (69%, 96%) while DIGFA (72%, 72%) did (p = 0.045). ADAMU-CE was significantly less sensitive in the diagnosis of active cysts (p = 0.019) while DIGFA was significantly less specific (p = 0.014) compared to ELISA. All tests were poorly sensitive in diagnosing inactive cysts (33.3% ELISA and ADAMU-CE, 42.8% DIGFA, 47.6% VIRapid). The reproducibility of all RDTs was good-very good. Band intensity of VIRapid and DIGFA correlated with ELISA OD values (r = 0.76 and r = 0.79 respectively, p

The island of Cyprus was a historical endemic area for cystic echinococcosis (CE) in the Mediterranean. During the last decades, Cyprus has been an open-air laboratory and a model for testing and implementing control measures aiming to eliminate CE as a public health problem. Despite control and surveillance measures implemented during last 50 years, molecular characterization of Echinococcus granulosus sensu lato specimens has been never provided. In February 2023, the carcass of a stray dog collected in the Nicosia district was examined by the Veterinary Services and found infected with Echinococcus spp. worms. The worms were sent to the European Union Reference Laboratory (EURLP) for species/genotype identification. The sequences analyses of nad2 and nad5 genes allowed us to identify the tapeworms as Echinococcus canadensis, genotype G7b. In November 2023, a parasitic liver cyst was observed during the post-mortem examination of a mouflon from the same area of the dog's finding. The cyst sample was also referred to EURLP for identification and comparison with tapeworms previously collected from the dog. The sequences analysis of cox1 gene allowed to identify the cyst as E. granulosus sensu stricto, genotype G1. The finding of 2 different species of E. granulosus s.l. in a limited area raises epidemiological questions on the origin of the samples: whether distinct transmission cycles are present or a recent introduction event have occurred. From a public health perspective, it will be essential to conduct further molecular epidemiology studies to clarify the recent transmission dynamics of Echinococcus species in Cyprus.