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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. © 2017 The Authors

Dientamoeba fragilis is now considered a potentially emerging gastrointestinal pathogen in both developing and developed countries. We first report an autochthonous case of D. fragilis infection in Greece. A 49-year-old female with acute non-specific abdominal pain required emergency surgical admission for active observation and repeated assessment. To the best of our knowledge, this is the first reported case of acute unexplained abdominal pain finally attributed to D. fragilis infection using microscopic and molecular methods. © 2016 Indian Journal of Medical Microbiology Published by Wolters Kluwer - Medknow.

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. © 2015 The Authors.

Leishmaniosis due to Leishmania infantum (Syn: L. chagasi) is one of the most common diseases of dogs in Mediterranean countries and also has zoonotic potential. The aim of this study was to evaluate the efficacy of an optimized dosage regimen of aminosidine for the treatment of canine leishmaniosis (CanL) in terms of clinical remission, restoration of clinicopathological abnormalities, evolution of antibody titer, lymph node and bone marrow parasitic density and of PCR-based parasitological cure. Twelve non-uremic dogs without proteinuria, presenting clinical signs of CanL were included in the study. The diagnosis was confirmed by serology, microscopy and PCR of lymph node and bone marrow samples. Aminosidine was administered subcutaneously at the dose of 15 mg/kg body weight, once daily, for 21 consecutive days. A partial remission of the clinical signs, amelioration of clinicopathological abnormalities such as anemia, lymphopenia, hyperproteinemia, hyperglobulinemia, and reduced albumin/globulin ratio and reduced lymph node and bone marrow parasitic density were witnessed, although parasitological cure was not achieved. Since data are not supportive enough for the use of aminosidine as an alternative treatment, a large-scale controlled clinical trial using this optimized dosage regimen of aminosidine is warranted to compare efficacy against currently used drugs. (c) 2012 Elsevier B.V. All rights reserved.

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. © 2012 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

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. © 2011 Elsevier Ltd.

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. Am J Clin Pathol

Leishmania infantum and Leishmania tropica are the species responsible for visceral leishmaniasis and cutaneous leishmaniasis respectively. In Greece, both diseases are endemic. The dog is considered the main reservoir of L. infantum, whereas the role of other animals for both L. infantun and L. tropica is unknown. Spleens from wild Rattus norvegicus, live trapped in Greece, were examined for the presence of Leishmania parasites by PCR. Out of 16 samples examined, only one was found positive for L. infantum with scant amount of parasitic DNA present. This is the first documented case of detection of L. infantum in R. norvegicus in Greece. The results of this preliminary study indicate that R. norvegicus is unlikely to be a reservoir for Leishmania parasites in Greece.

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. Mol Cell Probes

Specimens belonging to the Anopheles maculipennis complex were collected as larvae or resting adults from May 2003 to November 2004 in the area of the Athens 2004 Olympic Rowing Centre in Schinias, Attiki, Greece, and identified by morphological and molecular analyses. Of the 201 specimens collected, 199 were found to be Anopheles sacharovi Favre and two were An. maculipennis Meigen s.s. on the basis of similarity to published sequence data for the rDNA internal transcribed spacer (ITS2) region and the mitochondrial cytochrome c oxidase I gene (COI). Sequence data from a number of specimens were obtained for both genes and compared with corresponding GenBank data derived from diverse geographical areas. A high degree of homology in ITS2 sequences was found in both species, ranging from 99.5% to 100% in An. sacharovi and 99.4% to 100% in An. Maculipennis, with no intraspecific variation in either of the two species in our study. The degree of homology in the COI sequences was 94.8-99.8% in An. sacharovi and 95.0-99.8% in An. maculipennis. The 522-bp fragment produced a rather high degree of intrapopulation polymorphism for An. sacharovi, generating nine different haplotypes, five of which were singletons. Intraspecific variation for these sequences ranged from 0.2% to 1.4%, but was much lower (0.77%) for the two An. maculipennis sequences. These findings represent the first characterization of the An. maculipennis complex in the area of Schinias.

Amoebae belonging to the genus Acanthamoeba are potentially pathogenic to humans, causing mainly amoebic keratitis. Pathogenic ability of the 15 known Acanthamoeba genotypes is under investigation. We report that four out of five cases of amoebic keratitis studied in Greece, present T4 sequence type, while the remaining one presents T5 sequence type (Acanthamoeba lenticulata), which is the second most frequent genotype found among environmental samples. Thus, it is confirmed, for the first time to our knowledge, that A. lenticulata can cause keratitis. However the reason that it is under represented in clinical samples compared to environmental ones is unknown. (c) 2006 Elsevier Ireland Ltd. All rights reserved.

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. albopictug, Ae. cretinus, and Ae. aegypti. Also, mitochondrial cytochrome oxidase 1 (001) 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.

The ability to detect and differentiate between Plasmodium falciparum and P. vivax is of great importance for the routine laboratory diagnosis of malaria, donor-blood screening and epidemiological studies. Most PCR-based methods for the discrimination of these two species require nested protocols or an additional hybridization reaction, leading to high labour costs and long turn-around times. A simple, time-effective and yet sensitive and specific technique, based on a multiplex PCR, has now been developed for the simultaneous detection and differentiation of P. falciparum and P. vivax in blood samples. Compared with the ‘gold standard’ of microscopy, this method had a sensitivity and specificity of 100%, with a detection limit of just one P. falciparum or three P. vivax parasites/mul blood.

Visceral Leishmaniasis (VL) cases reported in Mediterranean countries and in Northern Europe are becoming increasingly frequent. The past few years several studies have shown Polymerase Chain Reaction to be more effective than the classical methods for the diagnosis of VIL in clinical samples. The purpose of this study was the development of a simple, specific and sensitive PCR-based assay for the detection of Leishmania in blood samples. A specific pair of oligonucleotides was designed using conserved sequences of the ssu-rRNA Leishmania infantum gene. Of the 53 blood samples of patients suspected for leishmaniasis that were processed with the newly designed oligonucleotides, 13 were successfully diagnosed positive. The results were confirmed with sequencing and restriction fragment length polymorphism. The lower detection limit of the reported assay was 10 parasites per ml in all seeded samples tested and considered highly satisfactory for diagnosis of Leishmaniasis in blood samples. (C) 2002 Elsevier Science Ltd. All rights reserved.

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. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

A number of immunological parameters were studied in 82 DSM-III-R diagnosed schizophrenic patients (53 first drug-naive and 29 medicated chronic patients) as well as 67 healthy blood donors. The serum levels of interleukin-2 (IL-2), interleukin-1 beta (IL-1 beta) and tumour necrosis factor-alpha (TNF-alpha) were measured and correlated with cellular immunity, as assessed by the autologous mixed lymphocyte reaction (AMLR). T lymphocyte subsets were also examined. The above immune parameters were reassessed in a subgroup of ii first-episode, drug-naive patients 1 month after neuroleptic medication. IL-2 serum levels were significantly lower, and IL-1 beta and TNF-alpha were significantly higher in schizophrenic patients compared with healthy donors (P

The detection of and differentiation between Entamoeba histolytica and Entamoeba dispar are of great importance, both for diagnosis and for epidemiological studies. Most PCR-based methods for the discrimination of these two species employ complex procedures for DNA extraction and require different protocols for E. histolytica and E. dispar, leading to relatively high expenditure, labour costs and turnaround times. A simple, rapid, cost-effective and yet sensitive and specific multiplex PCR technique has now been developed for the simultaneous detection and differentiation of E. histolytica and E. dispar in faecal samples. The detection limit is 200 trophozoites of E. dispar or 1000 trophozoites of E. histolytica/g stool sample. The sensitivity of the assay remains practically unchanged, even in the presence of 20 000 trophozoites of the other species/g stool sample. Thus, this technique may also easily reveal mixed infections, without the danger of misdiagnosis caused by one strain displacing the other in culture.

We have recently reported that administration of Pro Tα to DBA/2 mice before the inoculation of syngeneic L1210 leukemic cells prolonged the survival of these animals by (a) inducing tumoricidal peritoneal macrophages, (b) enhancing natural killer (NK) and inducing lymphokine-activated killer (LAK) activities in splenocytes and (c) inducing the production of interleukin-2 and tumor necrosis factor α [Papanastasiou et al. (1992) Cancer Immunol Immunother 35:145; Baxevanis et al. (1994) Cancer Immunol Immunother 38:281]. In this report we demonstrate that Pro T α, when administered simultaneously with L1210 tumor cells, is capable of generating in DBA/2 animals tumorspecific CD8+ cytotoxic T lymphocytes (CTL). The Pro T α-induced CD8+ CTL lysed their syngeneic L1210 targets in a major histocompatibility complex (MHC)-restricted fashion since monoclonal antibodies (mAb) against the H-2Kd allelic product could inhibit the cytotoxic response. Mice receiving only Pro T α developed non-MHC-restricted cytotoxic activity (NK, and LAK activities) whereas those receiving Pro T α and L1210 tumor cells developed both MHC-restricted (CTL) and non-MHC-restricted cytotoxic activities and survived longer. The Pro T α-induced CD8+ CTL activity was regulated by Pro T α-induced L1210-specific syngeneic CD4+ cells. This was shown in two different ways: first, CD8+-cell-mediated cytotoxic responses against L1210 targets were associated with L1210-specific and MHC-restricted proliferative responses of syngeneic CD4+ cells and, second, CD4+ cells from mice that had received both Pro T α and L1210 tumor cells could enhance in vitro the otherwise weak, MHC-restricted and L1210-specific cytotoxicity of syngeneic CD8+ cells from mice that had received only L1210 cells. Our data suggest that Pro T α is capable of inducing nonspecific, as well as tumor-specific CTL responses in vivo. This is of importance since Pro T α may prove to be useful in clinical protocols aimed at cancer immunotherapy. © 1995 Springer-Verlag.