Microfilaremic Dirofilaria repens Infection in Patient from Serbia.

We report a case of Dirofilaria repens infection causing microfilaremia in a patient from Serbia. Serum samples tested positive for D. repens IgG by ELISA. Our findings and those of others suggest the parasite's progressive adaptation to humans. Clinicians should be aware that microfilaremia can develop during Dirofilaria spp. infections.

Usual or unusual presentations of Dirofilaria repens in two sibling dogs: a case report.

This study describes two different manifestations of Dirofilaria repens infection in sibling dogs with microfilaremia. Dog 1, asymptomatic, harbored a gravid female of D. repens on the parietal layer of tunica vaginalis of one testicle and showed a marked circulating eosinophilia (3.3·103/µL). Both testicles were normal in shape and size without any gross lesions. Dog 2 had a pyotraumatic dermatitis. The cases were confirmed by PCR and sequencing. The sequences obtained showed 100% identity with those of D. repens isolated from human scrotum in Croatia. The treatment with moxidectin 2.5% and imidacloprid 10%/kg was effective in eliminating microfilariae after just one application, as demonstrated by negative modified Knott's tests and PCR analyses of blood samples. This status was maintained during the post-treatment observation period. The classical localization of D. repens in dogs is in subcutaneous tissues, within nodules or free; however, it can also occur with some frequency in testicles, as described in humans. The infection can be associated with circulating eosinophilia or pyotraumatic dermatitis, as reported in this study. Thus, in endemic areas, it is advisable to carefully inspect the removed testicles at neutering since parasite localization can take place without any macroscopic changes. Moreover, in the case of circulating eosinophilia or pyotraumatic dermatitis, investigations should include modified Knott's test and PCR to ensure that D. repens is not the cause of these alterations. Rapid and sensitive tests for the early detection of infected animals would help to prevent or limit the spread of this zoonosis.

Molecular characterization of Blastocystis subtypes in HIV-positive patients and evaluation of risk factors for colonization.

BACKGROUND: Blastocystis is one of the most common intestinal protozoa in human faecal samples with uncertain impact on public health. Studies on the prevalence of Blastocystis in HIV-positive patients are limited and dated. METHODS: A cross-sectional study was carried out involving 156 HIV-positive patients to evaluate the prevalence of Blastocystis-subtypes by molecular amplification and sequencing the small subunit rRNA gene (SSU rDNA), to identify the risk factors for its transmission, to examine the relationship between the presence of the protist and gastrointestinal disorders. Furthermore, the evaluation of the faecal calprotectin by immunoassay from a sample of subjects was performed to evaluate the gut inflammation in Blastocystis-carriers. RESULTS: Blastocystis-subtypes ST1, ST2, ST3, ST4 were identified in 39 HIV-positive patients (25%). No correlation was found between the presence of the protist and virological or epidemiological risk factors. Blastocystis was more frequently detected in homosexual subjects (p = 0.037) infected by other enteric protozoa (p = 0.0001) and with flatulence (p = 0.024). No significant differences in calprotectin level was found between Blastocystis-carriers and free ones. CONCLUSIONS: Blastocystis is quite common in HIV-positive patients on ART showing in examined patients 25% prevalence. Homosexual behaviour may represent a risk factor for its transmission, while CD4 count and viremia didn't correlate with the presence of the protist. The pathogenetic role of Blastocystis remains unclear and no gut inflammation status was detected in Blastocystis-carriers. The only symptom associated with Blastocystis was the flatulence, evidencing a link between the presence of the protist and the composition and stability of gut microbiota.

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.

Seroepidemiological trend of strongyloidiasis in the Bolivian Chaco (1987-2013) in the absence of disease-specific control measures.

OBJECTIVES: The prevalence of Strongyloides stercoralis infections is grossly underestimated because infections go mostly undetected, although they can persist for a lifetime due to the auto-infective cycle. In the Bolivian Chaco, the prevalence of soil-transmitted nematodes dropped dramatically in the past 25 years, but the mebendazole used for preventive chemotherapy has no effect on S. stercoralis. Meanwhile, the prevalence of intestinal protozoan infections remains unchanged. We compared S. stercoralis seroprevalence in rural communities of the Bolivian Chaco from 1987 to 2013. METHODS: Sera collected during two previous serosurveys, conducted in the Chaco region in 1987 and 2013, were tested for S. stercoralis using a commercial assay (Bordier-ELISA, Bordier Affinity Products, Switzerland). RESULTS: Overall, 355 sera were analysed, 122 from the 1987 survey and 233 from the 2013 survey. Seropositivity for S. stercoralis was significantly more prevalent in 1987 (19/122, 16% in 1987 vs. 15/233, 6% in 2013, P = 0.006), accounted for by a drop from 17% to 3% in people under 26 years of age. Multivariate analysis showed a significant association between seropositivity for S. stercoralis and age in the 2013 population (OR 1.03 for each one-year increase, 95%CI 1.00-1.05, P = 0.04), but none in 1987. CONCLUSIONS: The significant reduction in S. stercoralis seroprevalence in Bolivian Chaco cannot be explained by preventive chemotherapy or improved social-sanitary conditions. As the drop is seen in younger generations, it is consistent with little transmission occurring. However, the risk of transmission still exists, as prevalence is persistently high in older individuals, who present a potential reservoir due to the lifelong nature of S. stercoralis infections.

An appraisal of canine and human cases reveals an endemic status of dirofilariosis in parts of Serbia.

Dirofilaria immitis and D. repens are mosquito-borne nematodes that primarily infect canids, and can also cause mild to serious superficial or visceral infection in humans. In the present survey, peripheral blood from 150 asymptomatic dogs from Serbia were examined using the modified Knott's technique. Dirofilaria immitis, identified based on morphological and morphometric characteristics, was prevalent in dogs not receiving preventative treatment (in 44% and 60% of pound and pet dogs, respectively). These results, together with findings of autochthonous cases of subcutaneous D. repens infection in human patients from Southeastern Serbia emphasize the need for further investigations of this veterinary and public health problem.

Comparison of PCR assays targeting the multi-copy targets B1 gene and 529 bp repetitive element for detection of Toxoplasma gondii in swine muscle.

The comparison of the sensitivities of two molecular assays designed to target the multi-copy sequences of the Toxoplasma gondii genomic B1 region and 529 bp-RE respectively, in detecting T. gondii in swine muscle was assessed. Diaphragm pillars were obtained from 498 slaughtered pigs managed in intensive farms in Central Italy. Genomic DNA was extracted from the tissues and T. gondii-B1 and 529 bp-RE sequences were amplified by specific PCR protocols. Toxoplasma gondii DNA was detected in 165 samples (33.13%). There was a good correlation (κ = 0.77) between the results obtained targeting the two different genetic markers, however the 529 bp RE-PCR assay overall detected a significantly higher (P < 0.05) number of T. gondii-positive samples (150 samples) than the B1-PCR protocol (134). Our results show that: i) standardized B1 and 529 bp-RE PCRs applied to muscle tissues can detect a high rate of T. gondii-infection; ii) a multi-target PCR approach is recommended for the accurate diagnosis of infection in swine and can also be used in food testing.

Detection of Toxoplasma gondii in faeces of privately owned cats using two PCR assays targeting the B1 gene and the 529-bp repetitive element.

Toxoplasma gondii infection is a worldwide parasitic zoonosis with a high-health risk for humans. The key epidemiological role played by felids is related to oocyst shedding. The present study compared two amplification protocols for the molecular diagnosis of Toxoplasma infection in owned cats. A total of 78 owned cats referred to an Italian university-teaching hospital and exposed to various T. gondii-associated risk factors were sampled for blood and faeces. Faecal specimens were processed by flotation and tested using 2 copro-PCRs targeting the widely used B1 gene and the 529-bp repetitive element (RE). The sera were tested by the indirect immunofluorescent antibody test (IFAT) for the detection of immunoglobulins against T. gondii. Sixteen faeces (20.52%) tested positive for T. gondii DNA; 12 samples were positive only at B1-PCR, two at 529-bp RE-PCR and two at both genetic targets (overall agreement = 82.11%). The amplicons obtained were sequenced, and the Basic Local Alignment Search Tool analysis showed a high homology with the T. gondii strains available in reference databases. Two stool samples were microscopically positive for T. gondii-like oocysts and also tested positive by both B1 and 529-bp RE-PCRs. Thirty-three (42.3%) sera tested positive for antibodies; of which, seven were found to have T. gondii DNA-positive results using the B1 genetic target (overall agreement = 57.77%). The amplification sets targeting B1 and 529-bp RE showed substantially different yields. Further research is needed to better understand the significance and the sensitivities of using these multi-copy-targeted molecular methods from cat faeces before being used for routine diagnosis.

Seroprevalence and Risk Factors for Toxocara canis Infection in Serbia During 2015.

We report data on the Toxocara seroprevalence evidenced in 2015 from samples of 40 children and 298 adults of the population living in different areas of Serbia, and on possible association of certain variables with infection. Detection of specific antibodies was performed using an enzyme-linked immunosorbent assay; all ambiguous results and part of the positive and negative sera were further analyzed by confirmatory Western blot test. An overall 23.5% seroprevalence was noticed, which was confirmed in 13.0% of the examined population with no significant difference regarding the age (children = 10.0%; adults = 13.4%) or by country area (East = 18.2%; North = 15.5%, Southeastern = 9.5%; p = 0.005). In contrast, the group of adult women proved more reactive than men (p = 0.001), and subjects both who spend spare time in square/parks (p = 0.041) and with positive onychophagy (p = 0.001) habit turned out more exposed to the infection. Possible reasons of these differences were analyzed, and the medical, veterinary, and economic impact of this soil-transmitted zoonosis were discussed.

Human Babesiosis, Bolivia, 2013.

To investigate human babesiosis in the Bolivian Chaco, in 2013 we tested blood samples from 271 healthy persons living in 2 rural communities in this region. Microscopy and PCR indicated that 3.3% of persons were positive for Babesia microti parasites (US lineage); seroprevalence was 45.7%. Appropriate screening should mitigate the risk for transfusion-associated babesiosis.

Malaria in children of Tshimbulu (Western Kasai, Democratic Republic of the Congo): epidemiological data and accuracy of diagnostic assays applied in a limited resource setting.

BACKGROUND: The literature data on malaria in Western Kasai, DRC, are limited and inadequate. A recent molecular survey there has detected Plasmodium ovale and Plasmodium malariae as mixed infections with Plasmodium falciparum. In Tshimbulu, Western Kasai, during a humanitarian initiative designed to provide children with free preventive screening and to reduce the local high malaria death rate, accurate species identification was performed, in order to collect unambiguous epidemiological data and to evaluate the reliability of locally applied diagnostics. METHODS: Finger pricks provided fresh blood for microscopic analysis (MA), for rapid diagnostic test (RDT) and for molecular diagnostics (MD). MA and RDT were first performed by the local team and then a re-interpretation of the results (on the same slides and on RDT's taken pictures) was conducted in Italy, where MD were performed. RESULTS: The analysis was conducted on 306 children; RDT found 80.9 % as P. falciparum-positive (37.4 % as two-band positive, P. falciparum single infection). MA identified a further four children as positive to P. falciparum and six co-infections with P. ovale. The second RDT evaluation confirmed a similar infection rate (78.2 %) but interpreted as two-band positive a significantly higher share of tests (56.8 %). MA confirmed 80.0 % of the children as malaria positive and, in addition to P. falciparum, identified P. malariae (13.8 %), P. vivax (3.4 %) and P. ovale (2.4 %), and detected Babesia microti in 19 smears. MD confirmed all of the species found (Babesia microti included), classified as mono-infection with P. falciparum a rate of spots comparable to MA revision, and identified all P. ovale as Plasmodium ovale wallikeri. The RDT used locally proved 93.1 % sensitive and 92.1 % specific for P. falciparum. CONCLUSIONS: The malaria prevalence among the children and the presence of four Plasmodium species, highlighted in this study, identified a sanitary issue which proved to be more alarming than expected, as it was worsened by the unpredictable presence of P. vivax and Babesia microti (never before reported in DRC). Each diagnostic tool showed its point of weakness. Therefore, the most correct approach is by the combined use of different, locally available, diagnostic tools.

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.

Molecular detection of Rickettsia, Borrelia, and Babesia species in Ixodes ricinus sampled in northeastern, central, and insular areas of Italy.

The aim of the present study was to provide insight into the diversity of tick-borne pathogens circulating in Italy, carried/transmitted by Ixodes ricinus, one of the most abundant tick species in the country. A total of 447 specimens sampled in five areas of northeastern, central and insular Italy were analysed by polymerase chain reaction and sequencing for the presence of rickettsiae, borreliae and babesiae. Several rickettsial species of the spotted fever group of zoonotic concern and other zoonotic pathogens were found, such as Borrelia burgdorferi s.s., Borrelia afzelii, Borrelia garinii, and Babesia venatorum. These findings confirm a wide distribution of tick-borne bacterial and protozoan species in Italy, and highlight the sanitary importance of I. ricinus, often recorded as feeding on 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.

Human oestriasis acquired in Florence and review on human myiasis in Italy.

Myiases-causing flies are widely observed in tropical countries, whereas in Italy, a temperate country, their epidemiology and clinical presentation are poorly known. We report three cases of human conjunctival myiasis recently observed at our hospital, and the results of a review of the literature on human myiasis in Italy. In August 2012, a case of Oestrus ovis conjunctival myiasis acquired in the city centre of Florence, Italy was diagnosed at our hospital. In the early fall of 2013, two additional cases, acquired in the neighbouring areas, occurred. The review of literature showed that, up to the middle of 1990s, myiasis in Italy was mainly an occupational disease of shepherds, caused by O. ovis. Recently, cases of travel acquired furuncular myiasis emerged, together with "opportunistic" autochthonous cases of wound myiasis in patients with underlying health conditions. Considering the causative agents of human of myiasis in Italy, among the 703 autochthonous cases reported, 98.1% were caused by O. ovis, while among the 42 imported cases described, 59.5% were due to Cordylobia spp. and 40.5% to Dermatobia hominis. Our findings suggest that O. ovis conjunctival myiasis may still be observed in urban setting in Italy. Health care providers should know and implement the basic rules of entomoprophylaxis for myiasis in the facilities where they are working and use these indications to educate patients and care givers in both pretravel care and geriatric outpatient settings.

Parasitological and molecular observations on a little family outbreak of human fasciolosis diagnosed in Italy.

In the year 2010, three children who were born in a Romanian cattle farmer family went to Italy to join their mother. One of them was admitted to an Italian pediatric hospital for severe anemia that, when she was in her country, had been treated with blood transfusion. Blood tests and an abdominal ultrasound study triggered the suspicion of biliary parasitosis. The child underwent a cholangiopancreatography that caused the release of parasitic material microscopically identified as Fasciola hepatica. All children and their mother were submitted to coproparasitological analyses, which identified F. hepatica eggs only in the patient and in her twin sister. Parasitic materials recovered and flatworm specimens by us ad hoc obtained from Italian and Romanian cattle were genetically (ITS and COI genes) analyzed, and their sequences were compared with those deposited in GenBank. Specimens from children clustered with the Romanian strain examined and showed remarkable genetic differences with flatworm specimens from Italy. Anamnesis, parasite biology, and genetic data strongly suggest that twin sisters became infected in Romania; however, human fasciolosis is an emerging sanitary problem, favored by climate changes and global drivers; therefore, it deserves more attention on behalf of physicians working in both developing and developed countries.

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.

Parasitic Effects on the Congenital Transmission of Trypanosoma cruzi in Mother-Newborn Pairs.

Maternal parasitemia and placental parasite load were examined in mother-newborn pairs to determine their effect on the congenital transmission of Trypanosoma cruzi. Parasitemia was qualitatively assessed in mothers and newborns by the microhematocrit test; parasite load was determined in the placental tissues of transmitting and non-transmitting mothers by the detection of T. cruzi DNA and by histology. Compared to transmitter mothers, the frequency and prevalence of parasitemia were found to be increased in non-transmitter mothers; however, the frequency and prevalence of parasite load were higher among the transmitter mothers than among their non-transmitter counterparts. Additionally, serum levels of interferon (IFN)-γ were measured by an enzyme-linked immunosorbent assay (ELISA) in peripheral, placental, and cord blood samples. Median values of IFN-γ were significantly increased in the cord blood of uninfected newborns. The median IFN-γ values of transmitter and non-transmitter mothers were not significantly different; however, non-transmitter mothers had the highest total IFN-γ production among the group of mothers. Collectively, the results of this study suggest that the anti-T. cruzi immune response occurring in the placenta and cord is under the influence of the cytokines from the mother's blood and results in the control of parasitemia in uninfected newborns.

Genetic variability of Dirofilaria repens isolates from humans and dogs in Italy.

Dirofilaria repens is a paradigmatic example of an emerging vector-borne pathogen (VBP) in both human and veterinary fields. The spatial expansion and the increasing zoonotic impact of this VBP can be related to several drivers including the genetic structure of parasite populations. Italy is one of the European countries traditionally endemic with the highest incidence of canine and human cases of subcutaneous dirofilariosis. The present study aimed to assess the genetic identity and variability of D. repens isolates of human and canine origin from areas of Central Italy, compared with those isolated from different areas of Europe by sequence analysis of mtDNA genes (i.e., 12 S rDNA and cox1). A total of twenty isolates of D. repens were obtained from biopsies of subcutaneous and ocular cases of dirofilariosis occurring in 10 dogs and 10 humans. The sequence analysis of 12 S rDNA showed that all the sequences obtained clustered as a monophyletic group with a strong nodal support, indicating that all sequence types represented D. repens. The cox1 and the 12 S sequence analysis did not show host-related polymorphisms between human and dog-derived specimens. The sequence analysis of cox1 was performed including 8 additional sequences previously obtained from human and canine isolates in the same areas. Out of the 28 sequences analyzed, 20 were grouped in a haplogroup comprising 15 haplotypes (i.e., DR1, DR2, DR4, DR5, DR7, DR8, DR10-DR18), 2 sequences matched to DR9, reported for the first time in Italy, and 6 showed peculiar polymorphisms that were not previously described. The results obtained have implications for a better understanding of the epidemiology and phylogeography of this emerging vector-borne zoonotic parasite.