Malaria in Greece, 1975 to 2010.

Malaria, which was endemic in Greece in the past, was officially eliminated in 1974. Since that time and up to 2010, a number of imported cases (ranging from 19 to 76) have been annually reported. The total number of reported laboratory-confirmed cases between 1975 and 2010 was 1,419. Plasmodium falciparum was identified in 628 (44%) of these cases, while P. vivax was found in 524 (37%). Of the total cases, 1,123 (79%) were male (ratio males vs. females: 3.78). Age was only available for 490 cases, of which 352 (72%) belonged to the 18-40 year-age group. Of the 382 malaria cases reported from 1999 to 2010 for which the region/country of acquisition was known, 210 (55%) were from Africa and 142 (37%) from Asia. The massive introduction of economic migrants, in the period from 1990 to 1991 and from 2006 onwards, mainly from countries where malaria is endemic, resulted in the appearance of introduced sporadic cases. In Peloponnese, Central and East Macedonia, Thrace and East Attica, mosquitoes of the genus Anopheles (e.g. Anopheles sacharovi, A. superpictus and A. maculipenis) that can act as plasmodia vectors are abundant and during the summer of 2011, 27 P. vivax cases were reported in Greek citizens residing in the agricultural area of Evrotas in Lakonia and without travel history. As further P. vivax malaria cases occurred in the Lakonia and East Attica areas in 2012, it is becoming urgent to strengthen surveillance and perform integrated mosquito control that will help eliminate the potential risk of malaria reintroduction and reestablishment.

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.

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.

Expression of mRNA for the LH and FSH receptors in mouse oocytes and preimplantation embryos.

The gonadotrophins LH and FSH are known to regulate gonadal growth, and differentiation, endocrine function and gametogenesis. The LH receptor is expressed in ovarian theca, granulosa and luteal cells, and in testicular Leydig cells. The FSH receptor is expressed only in ovarian granulosa cells and in testicular Sertoli cells. The expression of the FSH and LH receptors was analysed by RT-PCR to study the role of these receptors in early mouse development. After reverse transcription, strategically designed nested primers were used for amplification from cDNA. Transcripts for the receptors were present in mouse oocytes and preimplantation embryos. The presence of mRNA for FSH and LH receptors in oocytes, zygotes and preimplantation embryos indicates a potential role for the gonadotrophins in the modulation of meiotic resumption and completion of oocyte maturation, as well as a beneficial effect on early embryonic development in mice.

Birth of two infants who were seronegative for human immunodeficiency virus type 1 (HIV-1) after intracytoplasmic injection of sperm from HIV-1-seropositive men.

OBJECTIVE: To report two cases of live births after intracytoplasmic sperm injection (ICSI) in two women who were seronegative for human immunodeficiency virus type 1 (HIV-1) after the use of processed semen from their seropositive husbands. DESIGN: Case reports. SETTING: University hospital IVF center. PATIENT(S): Two HIV-1 seropositive men and their HIV-1 seronegative female partners; all gave their informed consent in writing before undergoing the ICSI procedures. INTERVENTION(S): The men provided semen samples that were processed with the use of Percoll and swim-up techniques. Ovarian stimulation in the women was performed with the long protocol using GnRH analogs and recombinant FSH. ICSI was performed. MAIN OUTCOME MEASURE(S): Oocytes were fertilized by ICSI, and the resulting embryos were transferred to the patients. The mothers and babies were tested for HIV-1 antibodies. RESULT(S): In the first case, seven mature oocytes were collected and fertilized with ICSI, and three embryos were transferred; the woman became pregnant and gave birth to a healthy boy. Six months after the birth, testing for HIV-1 antibodies in the woman and the baby gave negative results. In the second case, 10 mature oocytes were collected and fertilized with ICSI, and four embryos were transferred; the second woman became pregnant and also gave birth to a healthy boy. Testing for HIV-1 antibodies at the baby's delivery also gave negative results. CONCLUSION(S): In women who are infertile because of fallopian tube obstruction or in men who have poor quality semen for artificial insemination, ICSI can be performed using processed semen. This method, which involves the use of only one spermatozoon per oocyte, provides HIV-1 seropositive men with the opportunity to have children with a minimal risk-if any-of infecting their female partners.

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.

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.