Intravitreal combination of dexamethasone sodium phosphate and bevacizumab in the treatment of exudative AMD.

The purpose of this study is to investigate the efficacy and safety of intravitreal dexamethasone sodium phosphate (DSP) combined with bevacizumab for the treatment of neovascular age-related macular degeneration (AMD). In this non comparative case study, 30 eyes of 27 patients with CNV due to AMD received intravitreal DSP (0.2 mg) and bevacizumab (1.25 mg) during a 6-month PRN (pro re nata) dosing regimen. Visual acuity, macular thickness and intraocular pressure (IOP) were monitored and recorded. After 6 months, mean retinal thickness decreased from 423.5 ± 75.3 to 228.2 ± 34.5 and mean visual acuity improved from 0.9 ± 0.39 logMAR to 0.53 ± 0.34 (p = 0.001) logMAR. During the trial period, 81 intravitreal injections were performed in 30 eyes, thus the mean number of injections per eye was 2.7 ± 1.1. 86.7% of the eyes required 3 or less injections while only 13.3% needed 4 or more injections. None of the patients, phakic or pseudophakic, manifested an elevation of IOP during the treatment, ranging between 12 and 22 mmHg. Combined DSP and bevacizumab offers encouraging results in the challenge of AMD treatment, providing immediate response of macular edema, reduced number of intravitreal injections and stabilization or improvement of visual acuity.

Autochthonous Plasmodium vivax malaria in Greece, 2011.

Between May and September 2011, twenty cases of Plasmodium vivax infection were reported in Greek citizens without reported travel history. The vast majority of those cases were confined to a delimited agricultural area of Evrotas, Lakonia. Conditions favouring locally acquired transmission of malaria, including the presence of competent vectors and migrants from endemic countries exist in Greece, underscoring the need for the development of an integrated preparedness and response plan for malaria prevention.

Ongoing outbreak of West Nile virus infection in humans, Greece, July to August 2011.

Between 16 July and 21 August 2011, 31 cases of West Nile neuroinvasive disease were reported from four regions in Greece. Of these, 17 occurred in districts that had not been affected in 2010. The reoccurrence of human cases in two consecutive years (following the large 2010 outbreak) and the spread of the virus in new areas suggest that West Nile virus is established in Greece, and its transmission may continue to occur in the future.

Molecular detection of Leishmania infantum in wild rodents (Rattus norvegicus) in Greece.

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.

Molecular characterization of the Anopheles maculipennis complex during surveillance for the 2004 Olympic Games in Athens.

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.

Genotyping of pathogenic Acanthamoebae isolated from clinical samples in Greece--report of a clinical isolate presenting T5 genotype.

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.

Documentation of Legionella pneumophila and Mycobacterium tuberculosis co-existence in a patient with acute respiratory distress syndrome.

BACKGROUND: Bacterial lung infections are common causes of ARDS and, despite intensive research for decades, the mortality rate remains very high. Only two reports suggest the co-existence of Legionnaires' disease and pulmonary tuberculosis based mainly on clinical presentation and serologic results for Legionella and positive cultures for Mycobacterium tuberculosis (M. tuberculosis). MATERIALS AND METHODS: A variety of specimens from a 61-year-old man was used for detection of Legionella pneumophila (L. pneumophila) and M. tuberculosis by PCR. Further identification of the pathogens was carried out by sequence analysis. RESULTS: L. pneumophila region mip was detected in bronchial washings, bronchoalveolar lavage and urine specimens of the patient. M. tuberculosis regions IS6110 and mtp40 were detected in endo-bronchial secretions and bronchoalveolar lavage. CONCLUSION: By using polymerase chain reaction (PCR) and DNA sequencing we documented L. pneumophila and M. tuberculosis co-existence, in multiple specimens of a patient presenting with acute respiratory distress syndrome (ARDS). Furthermore, the efficacy of the specific antibiotic treatment, based on the PCR results, suggest the co-existence of these two pathogens.

A single-step, PCR-based method for the detection and differentiation of Plasmodium vivax and P. falciparum.

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/microl blood.

Evolution of antibiotic resistance of non-typhoidal salmonellae in Greece during 1990-97.

Susceptibility to 15 antibiotics was determined in 1548 non-typhoidal salmonella strains isolated in Greece from l990 to l997. The overall prevalence of resistance of both Salmonella enterica serotype Enteritidis and Salmonella enterica serotype Typhimurium increased during the first years of the study. A decrease was observed from 1996, especially for S. Enteritidis, which showed the highest overall antibiotic resistance. S. Typhimurium was the serotype with the highest multiresistance to antibiotics. The rest of the serotypes had very low resistance prevalence compared with both S. Enteritidis and Typhimurium serotypes.

Microbiological quality of sewage sludge in Greece disposed for agricultural use.

For the disposal of sewage, sludge landfill is currently the practice adopted in Greece. Thirteen wastewater treatment plants located throughout Greece (islands excluded) were investigated over a period of 1 year in order to determine sludge microbiological quality. The time of exposure of the samples to environmental conditions and stress varied considerably, from a few hours to several months. The mean values for total coliforms were 4 to > 2400/100 ml and for faecal coliforms < 3 to > 2400/100. Salmonellae were isolated from all plants with the exception of two. Parasites were isolated from five treatment plants. There was no difference in the bacterial indicator counts of faecal pollution or the presence of salmonellae and parasites according to the time of disposal of the sludge to the fields. The samples taken from the soil, or from flowers and vegetables which grow in the area of the sludge disposal were mostly negative for faecal pollution bacteria indicators. Salmonellae and parasites were not recovered from any samples. Five of the 16 serotypes reported from the National Salmonella Reference Center were present in the sludge samples.

Microscopy, PCR and ELISA applied to the epidemiology of amoebiasis in Greece.

Detection and differentiation between Entamoeba histolytica and E. dispar are of great importance both for diagnosis and epidemiological studies. The present study was aimed to estimate the prevalence of these species in Greece and to assess the performance of microscopy, ELISA and PCR in the field. Our results indicate that the prevalence of these parasites is very low in Greece and that PCR and ELISA are by far better than microscopy. When it comes to the choice between ELISA and PCR, it seems that in real situations the advantages of PCR are outweighed by those of ELISA.

Evolution of antibiotic resistance of non-typhoidal salmonellae in Greece during 1990-97.

Susceptibility to 15 antibiotics was determined in 1548 non-typhoidal salmonella strains isolated in Greece from l990 to l997. The overall prevalence of resistance of both Salmonella enterica serotype Enteritidis and Salmonella enterica serotype Typhimurium increased during the first years of the study. A decrease was observed from 1996, especially for S. Enteritidis, which showed the highest overall antibiotic resistance. S. Typhimurium was the serotype with the highest multiresistance to antibiotics. The rest of the serotypes had very low resistance prevalence compared with both S. Enteritidis and Typhimurium serotypes.

Subconjunctival zoonotic Onchocerca in an Albanian man.

A case of subconjunctival infection with a zoonotic species of Onchocerca is described, in a 16-year-old Albanian man who had immigrated to Greece. This is the first report of human infection with Onchocerca in this tissue location and only the eighth report of zoonotic Onchocerca in man.

High sequence divergence in the 5' non-coding region of reference Coxsackie B and ECHO viral strains and clinical isolates revealed by restriction fragment length polymorphism analysis.

We report the restriction fragment length polymorphism (RFLP) patterns of a 440-bp-long 5' non-coding region (5' NCR) amplification target of all 34 reference Coxsackie B and ECHO (enteric cytopathic human orphan) enterovirus strains and a total of 42 serotypically pre-assigned clinical isolates, in order to afford meaningful comparisons among these patterns and those of polioviruses. The RFLP patterns of reference Coxsackie B strains differed from one another and from those of polio and ECHO reference enteroviruses except from Coxsackie B1 and B2, which, although they differed from one another, had identical RFLP patterns with ECHO 17 and 13, respectively. The 28 ECHO reference strains formed a more variable viral group including strains with RFLP patterns distinct from one another and from those of polio and Coxsackie B enteroviruses, and others with RFLP pattern identities common to other ECHO viruses and Coxsackie B1 and B2 but not polioviruses. The RFLP patterns of the clinical isolates and their corresponding serotypically assigned reference Coxsackie B and ECHO strains presented the most notable variations. The observed differences between serotype and genotype-dependent assignments within the 440-bp long 5' NCR target sequence of Coxsackie B and ECHO enteroviruses were in sharp contrast to the analogous situation with polioviruses. These findings support the specificity of the described method for clinical diagnostic genotyping of polioviruses and demonstrate that the 440-bp-long target sequence follows a different evolutionary process in polio and non-polio enteroviruses that is particularly prominent between reference non-polio strains and their serotypically assigned clinical isolates.

A nested, multiplex, PCR assay for the simultaneous detection and differentiation of Entamoeba histolytica and Entamoeba dispar in faeces.

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.

[Human pulmonary dirofilariasis: the first case observed in Greece]. / Dirofilariose pulmonaire humaine: un premier cas en Grèce.

We report a case of human pulmonary dirofilariasis in a 31-year-old man residing in the Aigion (Pelopennesus) Greece. The patient was hospitalized for fever of unknown cause. The chest x-ray showed a coin lesion in the left lung. Lung cancer was suspected and lobectomy was performed. Pathology reported presence of a nematode in an infarcted nodule. Its morphology enabled the diagnosis of an immature female Dirofaria repens. Twenty-two cases of human subcutaneous or subconjunctival human dirofilariasis have been reported. The present case is the first located in the lung.

Improved detection of Dirofilaria repens DNA by direct polymerase chain reaction.

Diagnosis of human infection by Dirofilaria repens, depends mainly on microscopic evaluation of tissue cross-sections and the macroscopic characteristics of the worm. Tissue degeneration and/or poor specimen preparation practices however, often render many cases of subcutaneous dirofilariasis elusive to such morphological diagnostic approaches. The early PCR protocols, developed to satisfy these complex diagnostic needs, failed to amplify dirofilariae DNA from formalin preserved material. To overcome these difficulties, we developed an improved PCR protocol using a set of primers designed to amplify a rather stable, highly repetitive D. repens-specific genomic DNA target. We report the performance of this protocol with a large variety of dirofilariae infected DNA specimens, including those extracted from formalin preserved biological material for up to 20 days. Our findings support its potential application to routine clinical diagnosis.

Ophthalmic dirofilariasis.

PURPOSE: To report a rare case of ophthalmic dirofilariasis in a 68-year-old man with sudden, intense pain and itching in the left eye. The nematode was located in the eye and recognized as Dirofilaria repens. METHODS: Slit-lamp examination disclosed a moving nematelminth under the bulbar conjunctiva of the left eye. The nematode was surgically removed. RESULTS: The nematelminth surgically removed from the subconjunctival space was identified as D repens on the basis of transverse sections and its macroscopic characteristics. CONCLUSION: It is important to identify the nematode as D repens, based on both its microscopic and macroscopic characteristics, to avoid treatment with anthelminthic agents. The surgical removal of the worm is the sole recommended treatment.

Human and canine dirofilariasis in Greece.

Human dirofilariasis is a disease which is considered to be rare in Greece, less than 20 cases having been reported prior to 1990. The clinical manifestations were usually subcutaneous nodules, and in only two cases there was ocular dirofilariasis. Eight new cases were recognized in humans during the last six years in Athens, two of which with ophthalmic involvement. Twelve additional cases have been detected but not published in Thessaloniki. In all human cases, the worms were identified as being Dirofilaria repens (D. conjunctivae). The identification was based on the morphological observations of the whole nematodes and on the histological sections of the subcutaneous nodules in the samples. Four different filarial nematodes (D. immitis, D. repens, Dipetalonema reconditum and D. grassii) were recovered from dogs. Infection of dogs with all types of filariae ranges from 12% to 37%. The high incidence of dirofilariasis in dogs is of great interest compared to the low incidence in humans. The occurrence of common host animals and insect vectors makes dirofilariasis a potential danger for public health.

Cases of Neisseria meningitidis in Greece.

In Greece, Neisseria meningitidis appears as sporadic cases with some seasonal local outbreaks. The notification of meningococcal disease to the local health authorities and the Ministry of Health is mandatory. In 1994, the National Reference Centre for M