Comparative performance evaluation of four different methods for diagnosing Giardia infection in dogs and zoonotic assemblages' identification.

Giardia duodenalis (syn. G. intestinalis or G. lamblia) is a parasitic protozoan that infects the upper intestinal tract of a broad range of hosts, including humans and domestic animals. Thus, it has raised concerns about the public health risk due to companion animals. Recently, with the improvement of living standards and increasing contacts between pets and humans, the zoonotic transmission of Giardia has dramatically increased. From a genetic point of view, G. duodenalis should be viewed as a complex species that includes eight different species-specific genetic assemblages. The laboratory diagnosis is mainly based on the finding of microscopic cysts in stool samples by coprological examination. Other methods include the detection of antigens, immunoassays or PCR protocols, which allow the identification of Giardia assemblages. The study aimed to compare the performance of Direct Fluorescence Antibody test (DFA), zinc sulfate flotation technique (ZnSO4), rapid diagnostic test (RDT), end-point PCR amplification (PCR) for the detection of Giardia and to identify the concerning assemblages in a canine population from Central Italy. Direct fluorescence antibody test is the reference standard for laboratory diagnosis of Giardia in fecal samples from dogs, despite the microscopic examination after flotation remains the most useful method in many veterinary diagnostic centers. The present findings demonstrate the high performance of DFA and ZnSO4 in detecting Giardia, while RDT may be useful as alternative or complementary method to the DFA and ZnSO4. PCR performance was low, but it allowed determining Giardia B zoonotic assemblage in 25% of the PCR-positive specimens (15 out of 60), while the remaining PCR-positive isolates belonged to the dog-specific assemblage C. The 26% prevalence of G. duodenalis detected by DFA in owned dogs and the identification of potentially zoonotic assemblages underline the potential risk for public health and indicate frequent cross-species transmission of the parasite between humans and dogs.

Laboratory associated zoonotic parasitic infections: a review of main agents and biosecurity measures.

Laboratory workers are exposed to the risk of acquiring infections due to the manipulation of infectious materials. The biological hazard for researchers is seven times higher when compared with hospital and public health laboratory workers. Despite the implementation of standardized practices to control infections, multiple cases of Laboratory Associated Infections (LAIs) usually go unreported. There has been a lack of comprehensive epidemiological data regarding the situation of LAIs for parasitic zoonosis and besides, the available sources are not completely updated. Since most accounts of laboratory infections are organism-specific, this study has focused on common pathogenic/zoonotic species handled at parasitological laboratories and summarising the standard biosecurity protocols for the infectious agents. The main characteristics of Cryptosporidium spp., Entamoeba spp, Giardia duodenalis, Toxoplasma gondii, Leishmania spp., Echinococcus spp., Schistosoma spp., Toxocara canis, Ancylostoma caninum, Strongyloides stercoralis are considered in this review in order to assess the potential risk of developing occupational infections in the workplace along with stating prevention and prophylactic measures for each species. It was concluded that the LAIs from these agents can be prevented by using personal protective measures and good laboratory practices. However, further studies are necessary to better understand the environmental resistance of cysts, oocysts and eggs, with a view to select the most suitable disinfection methods. Furthermore, it is fundamental to constantly update epidemiological data of infection acquired by laboratory workers, to develop accurate risk indicators.

New cases of human dirofilariosis on the Balkan Peninsula - "Masked intruders" uncovered by a surgeon.

Dirofilaria spp. nematodes are accidental parasites of humans causing mild to serious, superficial or visceral infections. Superficial dirofilariosis is rather common in Europe and is typically manifested as subcutaneous form. Herein we report 46 new cases of human dirofilariosis (19 patients with subcutaneous, 18 patients with ocular, 4 patients wih genital, 2 patients with submucosal, 2 patients with pulmonary and 1 patient with intramuscular form of infection) that were recorded from the beginning of 2015 to May 2021 on the Balkan Peninsula with a goal to update the prevalence of this parasitosis and point out potential problems in diagnosis and treatment. Besides, given the high possibility of misinterpretation as tumor, our second aim was to encourage the inclusion of this pathogen in the differential diagnosis of subcutaneous nodules. Although quite common forms, subcutaneous and ocular dirofilariosis can be very often misdiagnosed in clinical practice due to the absence of specific clinical manifestations. Therefore, raising awareness of clinicians about this zoonosis is needed as well as closer collaboration between physicians and veterinarians.

Sarcocystis gigantea infection associated with granulomatous eosinophilic myositis in a horse.

The only Sarcocystis species currently known to inhabit the fibers of skeletal and cardiac muscles in horses are S. fayeri, S. bertrami, and S. asinus. We describe herein the invasion of myofibers in a horse by S. gigantea, a sheep-specific species with low virulence in the original host. A hunter gelding was referred to a veterinary surgeon in Newmarket (UK). The anamnestic data reported that the horse had an initial history of swelling of the right forelimb with fluid on the front of the carpus and edema spreading up the forearm. Subsequently, 2 firm lumps were found on the left pectoral muscle adjacent to the axilla of the left forelimb. Histologic examination of biopsies from the lumps revealed multifocal granulomatous eosinophilic myositis associated with intact and degenerate encysted parasites, consistent with Sarcocystis spp. Based on amplification and DNA sequencing of the 18S rRNA gene obtained from formalin-fixed, paraffin-embedded tissue blocks, S. gigantea was identified. The presence of sarcocysts in equine skeletal muscles has been considered an incidental finding, and there are only sporadic associated reports of myositis. Our finding suggests that some Sarcocystis spp. have a wider intermediate host range than believed previously, and that Sarcocystis of other species (not considered horse-associated) can invade the muscle fibers of equids, leading to myositis.

Decline in Total Serum IgE and Soluble CD30 in the Context of Soil-Transmitted Helminth Decline in Bolivia.

In the Bolivian Chaco, recent surveys documented a dramatic decrease in the prevalence of soil-transmitted helminth (STH) infections as compared with the 1980s after thirty years of preventive chemotherapy (PC). Concomitant immunological rearrangements are expected. Because nematode infections are associated with increased levels of circulating IgE and glycoprotein CD30 soluble form (sCD30), this study aims to evaluate changes in serological markers of T helper (Th)2-cells activity between 1987 (high STH prevalence) and 2013 (low STH prevalence) in rural communities in the Bolivian Chaco area. We collected 151 sera during two different surveys in 1987 (n = 65) and 2013 (n = 86) and measured the concentration of total IgE and sCD30 by immunoassays. We found a statistically significant age-independent decrease in the total IgE (P < 0.0001) and sCD30 (P < 0.0001) from 1987 to 2013. The significant decrease in serological Th2 markers (IgE and sCD30) between 1987 and 2013 is consistent with the drop in STH prevalence in this geographical area during the same period of time. Further studies might elucidate the clinical and epidemiological impact of these serological rearrangements.

Occurrence of intestinal parasites among asylum seekers in Italy: A cross-sectional study.

BACKGROUND: In recent years Europe has experienced a dramatic increase in migration flows. Nevertheless, limited data is available about the occurrence of neglected parasitic diseases among migrant population. The purpose of the present study was to evaluate the prevalence of intestinal and urinary parasites in newly arrived asylum seekers. METHODS: A total of 364 newly arrived migrants hosted at the Asylum Seekers Centre of Castelnuovo di Porto (Italy) were screened during 8 months period for intestinal and urinary parasites. Each enrolled subject was interviewed using a standardized questionnaire, with focus on socio-demographical data and risk factors of parasitic infections. RESULTS: Stool analysis showed a prevalence of intestinal parasites of 20.6%. The travel route did not affect the prevalence of intestinal parasites (p = 0.096), while a significant negative correlation was found between the length of travel and the prevalence of parasite infection (p = 0.019). No statistically significant correlation between gastrointestinal symptoms and the presence of intestinal parasites was detected. CONCLUSION: The prevalence of intestinal parasitosis reported in asylum seekers does not necessarily reflect the prevalence of the parasitosis in the motherland. An anamnestic and syndromic approach may not be sufficient to highlight the problem of intestinal parasitic infestations in a screening setting.

Cervical cancer prevention in Senegal: an International Cooperation Project Report.

BACKGROUND: Cervical cancer is the second most common women's cancer in less developed regions and it is the leading cause of cancer mortality among women in Senegal. Because of the high costs, cervical cancer prevention is very rare in developing countries and it is often based on visual inspection methods. The University of Parma (CUCI)  and the MANI association have developed a cervical cancer screening project addressed to rural Senegal communities. The aim of the project was to disseminate the cytocervical sampling technique among Senegalese nurses and midwives, as a completion to the local visual inspection method. Other objectives were to verify the prevalence of precancerous lesions and to evaluate cervical cancer knowledge and attitudes among women who participated at the screening campaign. INTERVENTIONS: A theoretical and practical training plan on cervical cancer screening was provided to the local health workers at various levels. The screening was performed through a first step visual inspection exam to 203 women, followed by a Pap smear in case of positive outcome. 20 women participated also to a structured interview. RESULTS: The Pap smear material was adequate in all the samples collected where this indicates that the objective to disseminate cervical cancer screening techniques has been achieved.  Of the 203 women involved in the screening project, 68 were tested positive at the first step and 38 of them were submitted to Pap tests. CONCLUSION: The difficulties encountered confirm how Pap tests have overall proved inordinately complex and expensive for developing countries. It seems more sensible to deal first with global health in terms of basic hygienic sanitation, including an educational component in screening programs in order to sensitize women to the risk factors of cervical cancer.

Scaling down of a deworming programme among school-age children after a thirty-year successful intervention in the Bolivian Chaco.

OBJECTIVE: Preventive chemotherapy is the WHO-recommended control method for soil-transmitted helminthiases. In the Bolivian Chaco, 6-monthly single-dose mebendazole delivery to school-age children achieved a dramatic decrease in soil-transmitted helminthiases prevalence between 1987 and 2013. Consequently, in September 2016, preventive chemotherapy delivery was interrupted in nine rural communities. In compliance with WHO recommendations, we intensified surveillance to monitor soil-transmitted helminthiases prevalence and detect potential changes that would require interventions. METHODS: We conducted two cross-sectional parasitology surveys 12 months apart (September 2016-2017) among school-age children living in the communities where preventive chemotherapy delivery had been halted. Study design, methods of sampling and sample analysis technique (direct microscopy, Kato-Katz technique) followed WHO recommendations, aiming to obtain data representative of the Bolivian Chaco ecological zone. RESULTS: We collected 426 samples in 2016 and 520 in 2017. Soil-transmitted helminthiasis prevalence was unremarkable: 0.7% (95% CI 0-1.5%) in 2016 and 0.8% (0-1.5%) in 2017. Conversely, the prevalence of tapeworms (13% in 2016, 12% in 2017) and intestinal protozoan infections (81% in 2016 and 75% in 2017) continued to be high. CONCLUSIONS: Our findings support the role of preventive chemotherapy in reducing soil-transmitted helminthiases transmission, as otherwise poor hygienic and health conditions persist in the Bolivian Chaco. A national survey, involving areas from all the ecological zones of Bolivia, is now warranted.

Molecular genotyping of Echinococcus granulosus in the North of Iraq.

Cystic echinococcosis/hydatidosis is an important cosmopolitan zoonotic disease that causes large economic losses and human suffering. The larval stages of Echinococcus granulosus are the etiological agents of cystic echinococcosis that showed different genotypes in different regions in the world. The present study was aimed at the detection of E. granulosus strains circulating in two cities from north of Iraq (Kirkuk and Sulaimania). A total of 47 specimens of hydatid cysts were collected from patients and from different domestic intermediate hosts including cattle, sheep, goat and buffalo from slaughterhouses. Molecular characterization was performed by direct sequencing of the mitochondrial DNA (mtDNA) genes coding for the cytochrome c oxidase I (cox1) and the small subunit ribosomal RNA (rrnS). The results showed a high prevalence for the sheep strain (G1), an isolated finding of the buffalo strain (G3) and the presence of seven and three different microvariants for cox1 and rrnS, respectively. This is the first contribution on molecular genotyping of E. granulosus in Iraq with the observation of genotypes other than G1.

Dirofilaria repens microfilariae from a human node fine-needle aspirate: a case report.

BACKGROUND: Human dirofilariosis is still a little known infection even in endemic areas. Dirofilariosis is zoonotic infection usually abortive in humans; instead, we report a very rare case (the 4th in the world), the first in Italy, in which at least two infective larvae became mature adults that mated and produced active microfilariae even though they did not reach peripheral blood. CASE PRESENTATION: A 30-year-old Italian woman presented with a transient oedematous swelling on the left abdominal wall with a creeping eruption followed by the occurrence of a subcutaneous nodular painless mass in the iliac region. One month later, a similar temporary swelling appeared on the contralateral inguinal region associated with intermittent joint discomfort in both knees. The patient had recently travelled abroad, therefore many possible diagnoses were to be ruled out. Routine laboratory investigations revealed eosinophilia. An ultrasound examination of the iliac swelling evidenced a well-defined cyst with a big filamentous formation in continuous movement. A fine-needle aspiration of the lesion was performed for parasitological, cytological and histological exams. The prompt microscopic examination of the aspired material showed the presence of numerous microfilariae that were initially morphologically attributed to Mansonella ozzardi. Subsequently, the revision of the Giemsa stained film and molecular analyses of the biological material, allowed to identify Dirofilaria repens as etiological agent of infection. CONCLUSIONS: We report of a case in whom microfilariae were detected in fine-needle aspirate of subcutaneous node, without evidence of microfilaraemia, and the infection failed to become fully patent. Therefore we confirm that complete development and fertilization of D. repens worms in human hosts may occur, at variance with what is commonly believed, that Dirofilaria worms cannot fully develop in humans.

Seroreactivity to Dirofilaria antigens in people from different areas of Serbia.

BACKGROUND: The Northern part of Serbia is hyperendemic-endemic for canine dirofilarioses. Considering this fact, many human dirofilarial infections could be expected, however only about 30 cases in Serbia have been described until today. Aims of this survey were to assess the people reactivity to the antigens of D. repens and D. immitis and to identify risk factors for the contact exposure. METHODS: Investigation included sera taken from 297 people (179 women and 118 men) living in different areas of Serbia (Pancevo, Novi Sad, Zajecar, Leskovac, Vranje, Nis, Pirot). Sera were analysed by means of two indirect enzyme-linked immunosorbent (ELISA) home-designed that use as antigens adult somatic/metabolic polyproteins of D. repens (DR) and D. immitis (DI), respectively. The results were elaborated using the statistical method of descriptive and quantitative analysis. RESULTS: Significant differences by area in the reactivity of human sera to dirofilarial antigens were not observed (p = 0.056). A high seroreactivity was demonstrated in people from the towns of northern Serbia (Pancevo = 27,1%; Novi Sad = 16,3%), as well as in people from Zajecar (eastern Serbia = 15,8%) and Vranje (southern Serbia = 15,1%). No differences were evidenced between people reactivity to polyproteins of the two dirofilarial species, nor differences related to the gender of examinees. Factor risks evidenced were: i) place of residence; ii) spending work time outdoors during the mosquito season; iii) spending time outdoors and nearby rivers, lakes, swamps or canals; unespectedly, iv) cat owning. CONCLUSION: The findings emerging from this investigation indicate that clinicians and public health authorities should pay greater attention to this zoonosis. Continuing education and training of physicians will greatly contribute to the knowledge of the actual impact of filarial worms on animal and public health, and allow for the planning of suitable measures to prevent the infections.

Canine Dirofilaria infections in two uninvestigated areas of Serbia: epidemiological and genetic aspects.

In 2009 canine filarial infections were investigated in two northern areas of Serbia (Pancevo and Veliko Gradiste), applying morphometry, biochemical staining, and immunological kit to detect Dirofilaria immitis antigens, and two home-made ELISAs to detect antibodies to D. repens and D. immitis somatic/metabolic polyproteins. Moreover, molecular tools were applied to analyze the phylogenetic relationships of the isolates. The microfilariae detected in 21/122 dogs (17.2%) were identified as D. repens (n=21) and D. immitis (n=2). D. immitis antigens were found in another 13 animals with occult infection. All of the 15 heartworm-positive dogs also had antibodies to this parasite, which were detected in another 13 subjects, indicating an overall D. immitis seroprevalence rate of 22.9%. Serology for D. repens revealed evidence of antibodies in 42.6% of the dogs, but was negative for 4 microfilaremic dogs. As for the two different areas, the prevalence of microfilariae and/or D. immitis antigens, mainly due to D. repens microfilaremic animals, was not significantly higher in Veliko Gradiste (33.3%) than in Pancevo (22%). However, serology showed a different epidemiological picture. Heartworm infection occurred more often in both areas, and antibodies to dirofilarial nematodes were detected in 72.9% of dogs living in Pancevo, a rate higher than in those living in Veliko Gradiste (57.1%). No risk factors for infection were found, confirming the uselessness of prophylactic drugs against D. repens, and suggesting the presence in these areas of sunrise- or sunset-biting mosquitoes as important vectors. The results indicate the need for both appropriate entomological studies and further research on the intra-species variability shown by D. repens.

Is the goat a new host for the G3 Indian buffalo strain of Echinococcus granulosus?

Four goats bred in Central Italy (province of Rieti) revealed, in the liver, metacestodes of Echinococcus granulosus. The cysts, unilocular and fertile, were examined by microscopy and molecular diagnostics. Morphological data on the rostellar hooks are in agreement with the original description of the strain found in buffaloes and are largely compatible with those reported in Europe for cattle and humans. Specific PCR followed by DNA sequencing of the mitochondrial cox1 gene revealed for all the isolates 99.5% identity to the reference strain G3 genotype and 99.3% and 99.1% to G2 and G1, respectively. Further genetic markers (nad1 and 12S rRNA) confirmed the identity of the goat isolates to the G3 strain. This genotype, here reported for the first time in goats, proved to have a wider than previously supposed host range, therefore its relevance in human hydatidosis is expected to be more often evidenced.

Investigation on the occurrence of Echinococcus multilocularis in Central Italy.

BACKGROUND: Recent studies on geographic distribution of Echinococcus multilocularis in Europe show that it has a wider range than previously thought. It is unclear, however, if the wider distribution is due to its recent spreading or to a lack of previous data from the new areas. Italy, previously considered E. multilocularis-free, is now part of these new areas: infected foxes (the main definitive host of the tapeworm) have been observed in a Northern Alpine territory. Thus, more surveys need to be done in other Italian regions in order to monitor the spreading of E. multilocularis. The aim of the present study was to look for this parasite in 283 foxes collected in an Apennine area of Central Italy by different diagnostic methods. RESULTS: The foxes were heavily parasitized by 11 helminthic genera, but none of the animals was infected by E. multilocularis neither by E. granulosus (harboured adult worms or their DNA). Low specificity was observed in commercially available ELISA kits for the detection of E. multilocularis antigens in the faeces. Molecular diagnostics were sensitive and specific for the detection and identification of tapeworm eggs in faeces, but less sensitive, although specific, to adult tapeworms in the intestinal content. CONCLUSION: Preliminarily, we can say that no E. multilocularis could be found in the study area. These data will enable us to follow temporal changes of the spatial distribution of the parasite in the study area of the Central Apennines. Due to its low specificity the ELISA kit for E. multilocularis coproantigens is not suitable for epidemiological surveys, whereas molecular diagnostics applied to faecal samples give useful results. Finally, absence of E. granulosus in foxes living in the endemic areas studied confirms the thought that this tapeworm prefers a different definitive host.

Muscular cystic hydatidosis: case report.

BACKGROUND: Hydatidosis is a zoonosis caused by Echinococcus granulosus, and ingesting eggs released through the faeces from infected dogs infects humans. The location of the hydatid cysts is mostly hepatic and/or pulmonary, whereas musculoskeletal hydatidosis is very rare. CASE PRESENTATION: We report an unusual case of primary muscular hydatidosis in proximity of the big adductor in a young Sicilian man. The patient, 34 years old, was admitted to the Department of Infectious and Tropical Diseases for ultrasonographic detection, with successive confirmation by magnetic resonance imaging, of an ovular mass (13 x 8 cm) in the big adductor of the left thigh, cyst-like, and containing several small cystic formations. Serological tests for hydatidosis gave negative results. A second drawing of blood was done 10 days after the first one and showed an increase in the antibody titer for hydatidosis. The patient was submitted to surgical excision of the lesion with perioperatory prophylaxis with albendazole. The histopathological examination of the bioptic material was not diriment in the diagnosis, therefore further tests were performed: additional serological tests for hydatidosis for the evaluation of IgE and IgG serotype (Western Blot and REAST), and molecular analysis of the excised material. These more specific serological tests gave positive results for hydatidosis, and the sequencing of the polymerase chain reaction products from the cyst evidenced E. granulosus DNA, genotype G1. Any post-surgery complications was observed during 6 following months. CONCLUSION: Cystic hydatidosis should always be considered in the differential diagnosis of any cystic mass, regardless of its location, also in epidemiological contests less suggestive of the disease. The diagnosis should be achieved by taking into consideration the clinical aspects, the epidemiology of the disease, the imaging and immunological tests but, as demonstrated in this case, without neglecting the numerous possibilities offered by new serological devices and modern day molecular biology techniques.