Molecular screening of cat and dog ectoparasites for the presence of Bartonella spp. in Attica, Greece.

The purpose of this study was the molecular detection of Bartonella spp. in fleas and ticks parasitizing cats and dogs from 39 locations in Attica, Greece. One hundred and forty five ectoparasites (104 fleas and 41 ticks) from 92 cats and 53 dogs were investigated individually using PCRs targeting the 16S-23S ribosomal RNA intergenic spacer (ITS) and the citrate synthase (gltA) genetic loci. Bartonella spp. were detected in 14 out of 104 fleas (13.5%) and in none of the ticks examined. Consequent sequence analysis of the amplicons from the two loci identified 3 strains as Bartonella henselae, and 11 as Bartonella clarridgeiae. Οur study demonstrates the presence of B. henselae and B. clarridgeiae in Ctenocephalides felis fleas from cat and dog in Greece. We also report a novel ITS sequence for B. clarridgeiae. Considering that fleas could pose a risk for human bartonellosis from their infected hosts, further studies on the public health risk of Bartonella presence in animal ectoparasites are warranted.

Genetic Spatiotemporal Anatomy of Plasmodium vivax Malaria Episodes in Greece, 2009-2013.

An influx of immigrants is contributing to the reemergence of Plasmodium vivax malaria in Greece; 1 persistent focus of transmission is in Laconia, Pelopónnese. We genotyped archived blood samples from a substantial proportion of malaria cases recorded in Greece in 2009-2013 using 8 microsatellite markers and a PvMSP-3α gene fragment and plotted their spatiotemporal distribution. High parasite genetic diversity with low multiplicity of infection was observed. A subset of genetically identical/related parasites was restricted to 3 areas in migrants and Greek residents, with some persisting over 2 consecutive transmission periods. We identified 2 hitherto unsuspected additional foci of local transmission: Kardhítsa and Attica. Furthermore, this analysis indicates that several cases in migrants initially classified as imported malaria were actually locally acquired. This study shows the potential for P. vivax to reestablish transmission and counsels public health authorities about the need for vigilance to achieve or maintain sustainable malaria elimination.

Phlebotomine sandflies and factors associated with their abundance in the leishmaniasis endemic area of Attiki, Greece.

Leishmaniasis is a parasitic disease of animals and humans caused by several Leishmania species and transmitted by phlebotomine sandflies. The aim of the present study was to identify the species of field collected phlebotomine sandflies in the endemic area of the Attiki during 4 consecutive years, to isolate the Leishmania parasites from the infected sandflies, and identify possible factors associated with sandfly abundance in the area. A total of 542 trappings were made in 46 collection sites, in purely urban areas, periurban areas, and purely rural areas in Attiki. Out of the 3254 sandflies trapped, 1448 (44.43%) were female and 241 (16.64%) of the females were blood fed while Leishmania infantum DNA was detected in the 0.41% of them. Regarding sandfly species, the most prevalent was Phlebotomus tobbi (41.52%) followed by Sergentomyia minuta (27.44%), P. neglectus (14.83%), P. simici (11.08%), P. papatasi (3.68%), P. similis (0.89%), and P. alexandri (0.56%). Periurban areas were found to have the highest density of sandfly populations.

Occurrence of Cryptosporidium and Giardia in recycled waters used for irrigation and first description of Cryptosporidium parvum and C. muris in Greece.

Here, we present the first time findings regarding the occurrence of Cryptosporidium and Giardia in sewage waters and the first molecular characterization of Cryptosporidium species in Greece. Biological treatment plants from three regions in Greece have been investigated. The detection of Cryptosporidium oocysts was by modified Ziehl-Neelsen acid fast (MZN-AF) and by immunofluorescence microscopy (IFT) for Cryptosporidium and Giardia (oo)cysts, whereas nested PCR based on the SSU rDNA assay was used for molecular detection of Cryptosporidium followed by sequencing for the genetic characterization of the species. In total, 73 samples (37 raw sewage samples and 38 of treated water samples) were collected and analyzed. Of the 73 water samples, 4 samples were Cryptosporidium-positive by IFT and staining, 12 samples were Cryptosporidium-positive by nested PCR; 9 samples were Giardia-positive by IFT. We showed that Cryptosporidium cysts are found both in the input and the discharge of the biological treatment plants. Molecular characterization of Cryptosporidium based on the small subunit ribosomal DNA gene resulted in the determination of Cryptosporidium parvum and Cryptosporidium muris Greek isolates. This is the first report of Cryptosporidium and Giardia occurrence in wastewaters and the first molecular identification of Cryptosporidium species in Greek environments. As the treated water is used for irrigation, or it is discharged into the sea, our findings indicate that biological treatment facilities constitute a possible risk for public health because the related species are prevalent in humans; the results invite for further epidemiological investigations to evaluate the real public health risk in Greece.

Genotyping Plasmodium vivax isolates from the 2011 outbreak in Greece.

BACKGROUND: Plasmodium vivax malaria was common in Greece until the 1950s with epidemics involving thousands of cases every year. Greece was declared free of malaria by the World Health Organization in 1974. From 1974 to 2010, an average of 39 cases per year were reported, which were mainly imported. However, in 2009 and 2010 six and one autochthonous cases were reported culminating with a total of 40 autochthonous cases reported in 2011, of which 34 originated from a single region: Laconia of Southern Peloponnese. In this study the genotypic complexity of the P. vivax infections from the outbreak in Greece during 2011 is described, to elucidate the possible origin and spread of the disease. METHODS: Three polymorphic markers of P. vivax were used; Pvmsp-3α and the microsatellites m1501 and m3502 on P. vivax isolates sampled from individuals diagnosed in Greece. Thirty-nine isolates were available for this study (20 autochthonous and 19 imported), mostly from Evrotas municipality in Laconia region, in southern Greece, (n = 29), with the remaining representing sporadic cases originating from other areas of Greece. RESULTS: Genotyping the Evrotas samples revealed seven different haplotypes where the majority of the P. vivax infections expressed two particular Pvmsp-3α-m1501-m3502 haplotypes, A10-128-151 (n = 14) and A10-121-142 (n = 7). These haplotypes appeared throughout the period in autochthonous and imported cases, indicating continuous transmission. In contrast, the P. vivax autochthonous cases from other parts of Greece were largely comprised of unique haplotypes, indicating limited transmission in these other areas. CONCLUSIONS: The results indicate that several P. vivax strains were imported into various areas of Greece in 2011, thereby increasing the risk of re-introduction of malaria. In the region of Evrotas ongoing transmission occurred exemplifying that further control measures are urgently needed in this region of southern Europe. In circumstances where medical or travel history is scarce, methods of molecular epidemiology may prove highly useful for the correct classification of the cases.

Visceral leishmaniasis and COVID-19 coinfection - A case report.

As the COVID-19 pandemic spreads across the globe, it will undoubtedly cross paths with long endemic infectious diseases in different areas. Interactions between SARS-CoV2 and well-known pathogens will likely give rise to unfamiliar clinical presentations, depending on complex and as yet unknown immunological interactions. We present a case of coinfection with COVI19 and visceral leishmaniasis and discuss recent reports regarding coexistence of SARS-CoV2 and Leishmania spp. to date.

Characterization of Enterobius vermicularis in a human population, employing a molecular-based method from adhesive tape samples.

Human infection with the parasitic nematode Enterobius vermicularis occurs worldwide, particularly in children. Although its prevalence may exceed 35% in some parts of the world, molecular studies of E. vermicularis in humans are limited. The aim of the present study was to investigate the genetic variation within E. vermicularis in a human population. For this purpose, 77 adhesive tape samples taken from Greek children infested with E. vermicularis were tested. New primers were designed to amplify a segment of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of E. vermicularis from adhesive tape samples. Thirty-six amplicons were sequenced and eleven different haplotypes were identified. All sequences clustered within the type previously characterized (type B), only reported to date from captive chimpanzees. To the best of our knowledge, this is the first study of E. vermicularis genotypes from a human population.

Claw histopathology and parasitic load in natural cases of canine leishmaniosis associated with Leishmania infantum.

Histological lesions and the presence of Leishmania spp. amastigotes in claw tissues were investigated in 40 dogs with leishmaniosis, with (16/40--group A) or without (24/40--group B) generalized onychogryphosis. Following euthanasia, the entire third phalanx with intact claw was amputated, formalin fixed, decalcified in a formic acid solution, embedded in paraffin, sectioned longitudinally and stained with haematoxylin and eosin, and acid orcein-Giemsa. Nested polymerase chain reaction (PCR) was used for the detection of Leishmania amastigotes. Lichenoid mononuclear infiltration (all dogs in group A, 21 of 24 dogs in group B), basal keratinocyte vacuolation (nine of 16 dogs in group A, 15 of 24 dogs in group B) and dermoepidermal clefting (13 of 16 dogs in group A, 18 of 24 dogs in group B) were the most prominent histopathological findings. There was no difference in the frequency and severity of these lesions between the two groups. Leishmania amastigotes could not be visualized in the dermis of any of the H&E sections, but their presence was demonstrated by nested PCR in three of 16 dogs in group A and two of 24 dogs in group B. However, the frequency of positive nested PCRs was not significantly different between the two groups. In conclusion, claw histopathology in symptomatic dogs with leishmaniosis, either with or without onychogryphosis is mainly characterized by mononuclear lichenoid dermatitis with or without interface dermatitis and dermoepidermal clefting, and is not accompanied by substantial local parasitism.

Complement Receptor 1 availability on red blood cell surface modulates Plasmodium vivax invasion of human reticulocytes.

Plasmodium vivax parasites preferentially invade reticulocyte cells in a multistep process that is still poorly understood. In this study, we used ex vivo invasion assays and population genetic analyses to investigate the involvement of complement receptor 1 (CR1) in P. vivax invasion. First, we observed that P. vivax invasion of reticulocytes was consistently reduced when CR1 surface expression was reduced through enzymatic cleavage, in the presence of naturally low-CR1-expressing cells compared with high-CR1-expressing cells, and with the addition of soluble CR1, a known inhibitor of P. falciparum invasion. Immuno-precipitation experiments with P. vivax Reticulocyte Binding Proteins showed no evidence of complex formation. In addition, analysis of CR1 genetic data for worldwide human populations with different exposure to malaria parasites show significantly higher frequency of CR1 alleles associated with low receptor expression on the surface of RBCs and higher linkage disequilibrium in human populations exposed to P. vivax malaria compared with unexposed populations. These results are consistent with a positive selection of low-CR1-expressing alleles in vivax-endemic areas. Collectively, our findings demonstrate that CR1 availability on the surface of RBCs modulates P. vivax invasion. The identification of new molecular interactions is crucial to guiding the rational development of new therapeutic interventions against vivax malaria.

Detection and species identification of Old World Leishmania in clinical samples using a PCR-based method.

The aim of this study was to develop a simple, low-cost method for the detection and species differentiation of Leishmania directly from clinical samples, for routine use in a parasitology laboratory. A total of 87 samples was used, including 60 peripheral blood, seven bone marrow and 17 skin lesion material samples, derived from Greek patients with visceral or cutaneous leishmaniasis, and three reference strains. PCR was performed using primers designed to amplify the internal transcribed spacer 1 (ITS1) region of the rRNA gene. Identification of the Leishmania species studied was achieved by digestion with a single restriction endonuclease (RFLP), single-strand conformational polymorphism (SSCP) and DNA sequencing of the PCR-generated fragments. Typing identified all visceral and one cutaneous leishmaniasis strains as L. infantum, twelve of the cutaneous leishmaniasis strains as L. tropica and four as L. major. The described PCR method proved efficient for the detection of pathogenic Leishmania species in various clinical samples, most importantly in peripheral blood samples. Furthermore, PCR followed by a simple RFLP using a single restriction endonuclease was capable of identifying all Leishmania species commonly encountered in Greece.

Fatal human anaplasmosis associated with macrophage activation syndrome in Greece and the Public Health response.

Human granulocytic anaplasmosis (HGA) is a tick-borne disease caused by Anaplasma phagocytophilum that has the potential to spread in new geographical areas. The first fatal case of HGA in Greece is presented. Fever of unknown origin, renal and respiratory insufficiency and development of macrophage activation syndrome characterized the clinical presentation. Amplification and sequencing of a fragment of the groEL gene revealed the presence of A. phagocytophilum. The epidemiological and clinical features were collected during an epidemiological investigation. Public health measures were instituted by the Hellenic Centre for Disease Control and Prevention. The Public Health intervention required the collaboration of epidemiologists, veterinarians and microbiologists. Emphasis was given to communication activities and misconceptions concerning canines and their role in the disease. The emergence of human anaplasmosis in a new geographical area highlights the importance of disease awareness and of the need for continued support for tick and tick-borne disease surveillance networks.

Molecular and morphological characterization of Aedes albopictus in northwestern Greece and differentiation from Aedes cretinus and Aedes aegypti.

The presence of Aedes albopictus (Skuse) was recently confirmed for the first time in northwestern Greece. This location is within the distribution range of a morphologically similar species, Aedes cretinus Edwards, and is a potentially favorable region for the reintroduction of Aedes aegypti (L.). It was thus compelling to use methods in addition to morphology-based keys to correctly identify specimens badly damaged, rubbed, or otherwise altered in their external characteristics. It was decided to use molecular techniques as a novel and reliable method for differentiating the three Stegomyia species. The nuclear internal transcribed spacer 2 (ITS2) fragments from morphologically identified Ae. albopictus and Ae. cretinus specimens were amplified, and their sequences were compared with those in GenBank for Ae. albopictus, Ae. cretinus, and Ae. aegypti. Also, mitochondrial cytochrome oxidase I (COI) fragments were amplified for Ae. albopictus and Ae. cretinus (so far not available in GenBank) and compared with Ae. aegypti fragments. ITS2 and COI sequences generated in our study were deposited in GenBank and could be useful in future studies of mosquitoes by other research workers.

Cutaneous Disease as Sole Clinical Manifestation of Protothecosis in a Boxer Dog.

Prototheca wickerhamii is ubiquitous, saprophytic achlorophyllous algae that cause opportunistic infections in the dog and cat and disseminated disease usually in immunocompromised animals. In this report an uncommon case of canine cutaneous protothecosis is presented. A 6-year-old female boxer was brought in with skin lesions that consisted of nodules and generalized footpad hyperkeratosis, depigmentation, and erosion. Cytology and histopathology showed pyogranulomatous inflammation along with organisms containing round sporangia with spherical sporangiospores. PCR and sequencing identified the causal organism as Prototheca wickerhamii. Therapy applied in this patient with either fluconazole alone or combination of amphotericin B and itraconazole proved effective only for footpad lesions but not for skin nodules. Systemic therapy seems to be ineffective for skin nodules, at least in chronic cases of canine cutaneous protothecosis. Although canine protothecosis usually presents with the disseminated form, cutaneous disease as sole clinical manifestation of the infection may also be witnessed.

West Nile Virus Circulation in Mosquitoes in Greece (2010-2013).

Background of the Study. Following a large West Nile virus (WNV) epidemic in Northern Greece in 2010, an active mosquito surveillance system was implemented, for a 3-year period (2011, 2012, and 2013). Description of the Study Site and Methodology. Using mainly CO2 mosquito traps, mosquito collections were performed. Samples were pooled by date of collection, location, and species and examined for the presence of WNV. Results. Positive pools were detected in different areas of the country. In 2010, MIR and MLE values of 1.92 (95% CI: 0.00-4.57) and 2.30 (95% CI: 0.38-7.49) were calculated for the Serres Regional Unit in Central Macedonia Region. In 2011, the highest MIR value of 3.71(95% CI: 1.52-5.91) was recorded in the Regions of Central Greece and Thessaly. In 2012, MIR and MLE values for the whole country were 2.03 (95% CI: 1.73-2.33) and 2.15 (95% CI: 1.86-2.48), respectively, for Cx. pipiens. In 2013, in the Regional Unit of Attica, the one outbreak epicenter, MIR and MLE values for Cx. pipiens were 10.75 (95% CI: 7.52-13.99) and 15.76 (95% CI: 11.66-20.65), respectively. Significance of Results/Conclusions. The contribution of a mosquito-based surveillance system targeting WNV transmission is highlighted through the obtained data, as in most regions positive mosquito pools were detected prior to the date of symptom onset of human cases. Dissemination of the results on time to Public Health Authorities resulted in planning and application of public health interventions in local level.

Current clinical, laboratory, and treatment outcome characteristics of visceral leishmaniasis: results from a seven-year retrospective study in Greece.

OBJECTIVES: Visceral leishmaniasis (VL) is re-emerging in endemic areas. The epidemiological, clinical, laboratory, and treatment outcome characteristics in a large cohort of VL patients is described herein. METHODS: The cases of 67 VL patients (57% male, mean age 56 years) treated in two Greek hospitals over the last 7 years were identified and evaluated retrospectively. RESULTS: Forty-six percent of patients reported contact with animals. Seventeen patients (25%) were immunocompromised, and 22% were co-infected with another pathogen. Sixty-four percent of patients had fever, 57% had weakness, 37% had sweats, 21% had weight loss, and 13% had a dry cough, while 6% developed haemophagocytic syndrome. The median duration of symptoms was 28 days. Fifty-eight percent of patients had splenomegaly, 49% had hepatomegaly, and 36% had lymphadenopathy. The diagnosis was established by positive PCR in peripheral blood (73%) and/or bone marrow specimens (34%). Sixty-one patients (91%) received liposomal amphotericin (L-AMB). Six patients (10%) did not respond or relapsed but were eventually cured after a second cycle of L-AMB. During a 6-month follow-up, the overall mortality was 9%, although none of these deaths was attributed to VL. CONCLUSIONS: VL is still a common disease in endemic areas, affecting immunocompetent and immunocompromised patients. Its diagnosis is challenging, and molecular techniques are valuable and helpful tools to achieve this. Treatment with L-AMB is safe and very effective.

Genotypic assignment of infection by Dirofilaria repens.

Dirofilariasis is a parasitic disease, which if treated inappropriately due to misdiagnosis, can cause unwanted complications particularly when the infection is located in the breast. The numerous obstacles that can cause misdiagnosis of dirofilariases by standard morphological procedures prompted the development of a Dirofilaria repens-specific direct polymerase chain reaction (PCR)-based diagnostic approach using freshly infected dog blood. Reliable amplification of nematode DNA from formalin-fixed infected human specimens by this method is only possible from relatively fresh biological material, preserved in the fixative for up to 20 days. We report here our first case of dirofilariasis since the development of PCR genotyping, where the pathogen was morphologically unrecognizable and the diagnosis was based exclusively on DNA amplification. We complete our methodological contribution to the clinical laboratory diagnosis of dirofilariasis by presenting two more cases, where the primary genotypic assignment of infection by D. repens was further confirmed by conventional morphological means.

Oseltamivir-resistant influenza A(H1N1) 2009 virus in Greece during the post-pandemic 2010-2011 season.

Information on the drug susceptibility of influenza epidemic strains is important for antiviral resistance monitoring. In Greece, the 2009-2010 pandemic waves were very mild and seroprevalence rates remained low after this influenza season, resulting in exclusive detection of the pandemic strain during the 2010-2011 influenza season. In the present study during the post-pandemic 2010-2011 season, 50 consecutive influenza A(H1N1) 2009 virus-positive samples from patients hospitalised in Greek hospitals were analysed for resistance to the neuraminidase inhibitor oseltamivir. All patients were hospitalised with severe influenza complications and had previously received oseltamivir. Influenza A(H1N1) 2009 virus detection and testing for oseltamivir resistance were performed with real-time PCR amplification assays. The H275Y substitution associated with resistance to oseltamivir was identified in two immunocompetent patients who received oseltamivir treatment for 3 days and 5 days, respectively. In both cases, patients were discharged in good condition despite development of resistance to antiviral treatment.

Differences in clinical significance and morphologic features of Blastocystis sp subtype 3.

Blastocystis is a polymorphic intestinal parasite that is common in humans. A total of 51 asymptomatic and symptomatic patients positive for Blastocystis only were included in the study. Symptoms were mainly nonspecific gastrointestinal symptoms. Blastocystis isolates were xenically cultured and subtyped. Blastocystis species subtype 3 was the predominant subtype. Intrasubtype differences (vacuolar/amoeboid presence) in subtype 3 morphotypes were observed in 32 asymptomatic and symptomatic subtype 3 cases and could possibly be related to Blastocystis pathogenic potential. Diverse morphologic features (vacuolar transiting to amoeboid), probably reflecting the progression from an asymptomatic to a symptomatic state, were observed in an asymptomatic subtype 3 carrier who later had symptoms. Searching for amoeboid forms might be helpful to presumptively screen symptomatic patients with subtype 3 or to follow up an asymptomatic subtype 3 carrier in case symptoms become evident before antiprotozoal treatment was attempted. Further studies on the roles of morphologic features and variation within Blastocystis species subtypes as predictors of symptoms are encouraged.

Direct detection of Blastocystis sp. in human faecal samples and subtype assignment using single strand conformational polymorphism and sequencing.

Blastocystis is an anaerobic parasitic microorganism, which has been found in the intestinal tract of many vertebrates including humans. Recently, members of Blastocystis sp. were classified into nine subtypes, based on phylogenetic trees derived from sequence analysis of the small subunit ribosomal RNA (SSU rRNA) gene. The role of Blastocystis in human disease remains uncertain and the existence of pathogenic and non-pathogenic subtypes is under investigation. We report the development of a polymerase chain reaction (PCR)-based assay that is able to detect Blastocystis directly from human faeces. Furthermore, combined with single strand conformational polymorphism (SSCP) analysis and/or sequencing of the respective PCR product, the protocol can classify Blastocystis among the nine established subtypes. The method was applied to 45-positive and 30-negative faecal samples and proved to be highly sensitive and specific. Genotyping using SSCP analysis and sequencing revealed that subtype 3 is the most frequent in Greece, while subtypes 1, 2, 4, 6 and 7 are also present but in lower frequencies. Hopefully, the simplicity of the proposed method will contribute toward large-scale epidemiological studies for prompt clarification of the role of the parasite.