Inherited metabolic disorders in Cyprus.

Selective screening for inherited metabolic disorders (IMD) began in Cyprus in 1990. Over the last thirty-three years 7388 patients were investigated for IMD and 200 diagnoses were made (diagnostic yield 2.7%). The existence of a single laboratory of Biochemical Genetics for the whole island facilitated the creation of a national registry for IMD. The minimal prevalence of IMD in Cyprus is 53.3 cases per 100,000 live births. The most common group are disorders of amino acid metabolism (41.0%), followed by disorders of carbohydrate metabolism (16.5%), disorders of complex molecule degradation (16.5%), mitochondrial disorders (10.5%) and disorders of vitamin and co-factor metabolism (5.5%). Hyperphenylalaninaemia is the most common IMD (14.0%) followed by galactosaemia (7.0%), glutaric aciduria type I (5.5%) and MSUD (4.0%). Some disorders were found to have a relatively high incidence in specific communities, for example Sandhoff disease among the Cypriot Maronites and GM1 gangliosidosis in one particular area of the island. Other disorders were found to have a relatively higher overall incidence, compared to other Caucasian populations, for example galactosaemia, glutaric aciduria type I and MSUD, while fatty acid oxidation defects, Gaucher disease and classic PKU were found to have a relatively lower incidence. Molecular characterization of selected disorders revealed many novel genetic variants, specific to the Cypriot population.

Thoracoscopic vs open repair of congenital diaphragmatic hernia after extracorporeal membrane oxygenation: a comparison of intra-operative data.

PURPOSE: ECMO is an escalation treatment for hypoxic respiratory failure in patients with CDH. Open repair has been advocated after ECMO indicating that physiological changes associated to thoracoscopic repair were not well tolerated. METHODS: We have performed a retrospective review of all patients who underwent ECMO prior CDH repair over a 7 year period (2015-2021). Outcome measures were intra-operative Ph, PCO2, PO2 and FiO2 at 30 min, 1 h 30 min, and 2 h 30 min of surgery, operative time and recurrence rate. Data are shown in median (range). RESULTS: Eleven patients required ECMO prior CDH repair. Six of eleven (55%) were done thoracoscopically (Group A) and five of eleven (45%) via laparotomy (Group B). Two of six (33%) patients (Group A) were converted to a laparotomy, one of six (16%) patient developed a recurrence, and there was no recurrence in Group B. Two of five (40%) patients died within the first 60 days of life, whilst there was no death in Group A. Intra-operative values are shown below. CONCLUSION: Whilst this is a preliminary report of a limited number of patients, there is no obvious difference of intra-operative blood gas parameters during surgical repair in patients after ECMO. Thoracoscopic CDH repair may be considered in patients after ECMO.

Spontaneous assembly of redox-active iron-sulfur clusters at low concentrations of cysteine.

Iron-sulfur (FeS) proteins are ancient and fundamental to life, being involved in electron transfer and CO2 fixation. FeS clusters have structures similar to the unit-cell of FeS minerals such as greigite, found in hydrothermal systems linked with the origin of life. However, the prebiotic pathway from mineral surfaces to biological clusters is unknown. Here we show that FeS clusters form spontaneously through interactions of inorganic Fe2+/Fe3+ and S2- with micromolar concentrations of the amino acid cysteine in water at alkaline pH. Bicarbonate ions stabilize the clusters and even promote cluster formation alone at concentrations >10 mM, probably through salting-out effects. We demonstrate robust, concentration-dependent formation of [4Fe4S], [2Fe2S] and mononuclear iron clusters using UV-Vis spectroscopy, 57Fe-Mössbauer spectroscopy and 1H-NMR. Cyclic voltammetry shows that the clusters are redox-active. Our findings reveal that the structures responsible for biological electron transfer and CO2 reduction could have formed spontaneously from monomers at the origin of life.

Spatial genetic structure and Ovis haplogroup as a tool for an adaptive conservation management of the endangered Cyprus mouflon.

The mouflon (Ovis gmelini ophion) is the largest wild terrestrial mammal of Cyprus. Regarded as the local flagship species, its population range is limited to the mountainous Paphos Forest and adjacent areas including part of Troodos National Forest Park (> 700 Km2). This species, protected by both national and international legislation, is classified as Endangered by the IUCN, with livestock intrusion, poaching and habitat loss as the main threatening factors. We determined the spatial genetic structure by investigating sexes separately and shed further light on the Cyprus mouflon placement among Ovis haplogroups (HGs) to enforce its protection within an adaptive conservation framework. We genotyped 108 samples collected across the entire species' range at both14 loci of the microsatellite DNA and the mitochondrial DNA Control Region (CR). Microsatellite genotyping indicated that the wild population retained low genetic variability, which, however, was not associated with a level of inbreeding raising particular concern (FIS = 0.12). An overall weak spatial genetic structure was disclosed, consistently with the limited extension of the mouflon range, the lack of significant physical barriers to dispersal and the intense gene flow mostly occurring along a northeast-southwest axis across the forest. Nevertheless, evidences of structure were found for females in compliance with their philopatric behaviour. The disclosure of unique features compared to Mediterranean and Near East conspecifics, such as the occurrence of a six-fold 76 bp-long repeated motif in the Cyprus mouflon CR, along with the outcome of a phylogenetic reconstruction (based on a far higher number of Ovis GenBank records and Cyprus haplotypes than in previous studies) inspired the proposal of a new haplogroup (HGC2) which included the Anatolian mouflon (O. g. anatolica, former HGX) as sister taxon. While both high habitat heterogeneity and low human disturbance were the main drivers in determining the overall spatial structure, future conservation efforts to preserve this valuable genetic resource should focus on avoiding possible introgressive hybridisation with co-grazing livestock to the edge of its range.

Collaborative study on the effect of grinding on the detection of bones from processed animal proteins in feed by light microscopy.

Bone fragments are essential structures for the detection of processed animal proteins (PAPs) in feed by light microscopy for official controls according to Annex VI of European Union Regulation EC/152/2009. The preparation of samples submitted for analysis requires a grinding step to make them suitable for microscopic slide preparation and observation. However, there are no technical guidelines set down for this step despite the fact that it can lead to an increase in bone numbers due to fragmentation. This was demonstrated by an in-house study carried out by the Irish National Reference Laboratory (NRL) for animal protein detection. The present collaborative study investigated the possible effects of three different grinding conditions on the final result for a feed adulterated with 0.05 and 0.01% (w/w) of PAP. The microscopic analysis either combined or not with an Alizarin Red staining was carried out by 10 different laboratories. The results demonstrated that although a large variation in the numbers of bone fragments was noted, five of the six different grinding/staining combinations applied at two levels of PAP adulteration did not significantly (at p = 0.05) differ from one another. The only exception occurred when grinding the feed containing 0.05% of PAP with a rotor mill equipped with a 0.5-mm sieve and combined with a staining which resulted in a greater number of bone fragments by forced fragmentation. Overall, the impact of the grinding/staining combinations on the final results was shown to be negligible when considering the regulatory limit of detection (LOD) requirement for the method and the current rules of implementation of the light microscopic method. From a total of 180 analyses carried out on the feed matrix containing 0.05% of PAP no false-negative result was observed, and at a level of 0.01% PAP only 10 false-negative results occurred.

Species composition, distribution, ecological preference and host association of ticks in Cyprus.

Tick population and species depend on the effect of biotic and abiotic factors, especially vegetation, climate and host density; Cyprus, due to the mild climate, favors the appearance and spread of tick-borne infections. Our objective was to identify the tick species present in the island, to investigate their geographical distribution and their epidemiological implications. During a three-year study (2004-2006) we collected ticks from domestic and wild animals over the island of Cyprus. Data on temperature, humidity, altitude and vegetation, were also recorded. Each tick was identified by species using existing taxonomic keys. The results were mapped on a county level. During the current study 3057 ticks belonging to 11 tick species and four genera were collected from 441 (24.6%) infested animals. Rhipicephalus sanguineus was the predominant species (38.5%), followed by R. turanicus (21.3%) and R. bursa (17.8%). Most infestations occurred in May (24.0%), followed by March (13.6%) and June (12.2%). Rhipicephalus sanguineus had a positive correlation with humidity and temperature, R. bursa and Ixodes gibbosus had a positive correlation with altitude and a negative correlation with temperature. Contrary, Hyalomma excavatum had a negative correlation with altitude. Climate and the availability of hosts are among the major factors influencing ticks.

Genetic, histochemical and biochemical studies on goat TSE cases from Cyprus.

Scrapie and bovine spongiform encephalopathy (BSE) are transmissible spongiform encephalopathies (TSE's) affecting sheep and goats. Susceptibility of goats to scrapie is influenced by polymorphisms of the prion protein gene (PRNP) of the host. Five polymorphisms are associated with reduced susceptibility to TSE's. In the study presented here caprine samples from a scrapie eradication program on Cyprus were genotyped and further characterized using BioRad TeSeE rapid test, histological, immunohistochemical and biochemical methods. In total 42 goats from 20 flocks were necropsied from which 25 goats showed a positive result in the rapid test, a spongiform encephalopathy and an accumulation of pathological prion protein (PrPSc) in the obex. PrPSc deposits were demonstrated in the placenta, peripheral nervous and lymphoreticular system. Two animals showed PrPSc-accumulations in peripheral tissues only. By discriminatory immunoblots a scrapie infection could be confirmed for all cases. Nevertheless, slight deviations in the glycosylation pattern might indicate the presence of different scrapie strains. Furthermore scrapie samples from goats in the current study demonstrated less long term resistance to proteinase K than ovine or caprine BSE control samples. Reduced scrapie susceptibility according to the PRNP genotype was demonstrated (Fishers Exact test, p < 0.05) for the goats with at least one polymorphism (p = 0.023) at the six codons examined and in particular for those with polymorphisms at codon 146 (p = 0.016). This work characterizes scrapie in goats having implications for breeding and surveillance strategies.

Presence of Coxiella burnetii in fleas in Cyprus.

Over 40 tick species are naturally infected by Coxiella burnetii. However, little is known about the presence of C. burnetii in other ectoparasites such as fleas. During a 6-year (2000-2006) study, 1147 fleas were collected from 652 animals (252 rats, 118 foxes, and 282 hares) captured from different areas of Cyprus. Three flea species-Xenopsylla cheopis, Ctenocephalides felis, and C. canis-were identified. Fleas were pooled (153 pools) and tested by PCR for the presence of C. burnetii. The pathogen was identified in 25 (16.3%) pools. None of the fleas parasitizing hares was positive for C. burnetii, as opposed to fleas collected from rats (12% pool positivity) and foxes (47.6% pool positivity). The highest prevalence of positive pools was recorded in C. canis (38%) compared to C. felis (16.6%) and X. cheopis (10.8%). All pools of C. canis positive for C. burnetii were removed from foxes (44.4%), whereas all positive X. cheopis (10.8%) were removed from rats. The role of fleas in the maintenance and transmission of C. burnetii among wild vertebrates remains to be determined.

Coxiella burnetii in wildlife and ticks in an endemic area.

BACKGROUND: Ticks are considered to be a natural reservoir of Coxiella burnetii and are responsible for the spread of infection in wild animals and for the transmission to domestic animals. More than 40 tick species are naturally infected with C. burnetii. In Cyprus, few studies have been carried out on the distribution and incidence of C. burnetii infection in wildlife and the threat that infected wild animals pose to humans and domestic animals remains uncertain. METHODS: During 3 studies in Cyprus, lasting 7 years (2000-2006), ticks were collected from rats (98 Rattus norvegicus and 38 R. rattus), 74 mouflons (Ovis orientalis ophion), 32 foxes (Vulpes vulpes indutus), 247 hares (Lepus europaeus), 557 birds (endemic and migratory) and 10 different tick species. All samples were tested for the presence of Coxiella burnetii using molecular assays. RESULTS: In total, 31% (23/74) of mouflons, 28% (9/32) of foxes, 48% (15/31) of hares and 31% (41/131) of birds were positive for C. burnetii. We tested 1315 ticks (195 pools) and C. burnetii was detected in 28.9% (56/195) of them. Forty percent (24/60) of ticks collected from hares and 25.2% (27/107) of tick pools collected from mouflons were positive for C. burnetii. However, C. burnetii was detected in only one tick from foxes and one from birds. Positive samples were prevalent all over the island and did not show a specific geographic distribution pattern. CONCLUSIONS: Several animal and tick species collected from wildlife are potential sources of C. burnetii in Cyprus. These species are abundant in the area and may represent a risk for domestic livestock that share grazing environments.

Isolation and analysis of tetracycline-resistant Mycoplasma agalactiae strains from an infected goat herd in Cyprus - short communication.

A major concern with the use of tetracycline against mycoplasmas is the development of resistance. Infections in small ruminants due to tetracyclineresistant Mycoplasma agalactiae strains are becoming a frequent problem worldwide. In the present paper the detection and analysis of three tetracycline-resistant M. agalactiae strains, isolated from infected goats in Cyprus, are reported. The three field isolates were identified as M. agalactiae by polymerase chain reaction (PCR) showing 98% identity to the M. agalactiae PG2 reference strain. Furthermore, they were found sensitive to tylosin, enrofloxacin, spiramycin and lincomycin. In contrast, they were resistant to tetracycline. None of the putative genes [tet(M), tet(O) and tet(S)] that commonly contribute to high-level resistance to tetracycline could be amplified from their genome. Contrarily, the field isolates were found to carry ISMag1, an insertion sequence related to the IS30 family of mobile elements. Although ISMag1 is widely believed to induce high-frequency chromosomal rearrangements resulting in phenotypic changes of microorganisms, its potential role in tetracycline resistance of mycoplasmas requires further studies.

Identification of a novel uncultured Rickettsia species strain (Rickettsia species strain Tselenti) in Cyprus.

In a previous study conducted in Cyprus, various spotted fever group Rickettsia species were detected and identified in ticks by molecular analysis. Among them, a partially characterized Rickettsia species was detected in Hyalomma anatolicum excavatum and Rhipicephalus turanicus ticks. We report characterization of this rickettsial strain by using polymerase chain reaction sequencing analysis of partial citrate synthase A, outer membrane protein A, outer membrane protein B, and 17-kD protein genes. We propose a provisional name Rickettsia sp. strain Tselenti for this strain until it is isolated and further characterized.

Murine typhus in Cyprus: a 9-year survey.

Epidemiological and clinical data of 193 human cases of murine typhus in Cyprus were recorded and analysed during a 9-year period (2000-2008). The incidence rate was estimated at 24.5 cases/100,000 population/year. The incidence rate varied considerably between rural, urban and semi-urban areas, with residents in rural areas accounting for 79.3% of the total cases. Most (72.5%) of the cases occurred in late summer (July and August) and early autumn (September to October) with a peak in September. Well-established persistent endemic foci with clusters of cases were identified and characterised as 'high risk' areas. Presence of or contact with rats and fleas, presence of domestic/peridomestic animals and residence in rural areas, especially locations near the 'green line' (a narrow zone patrolled by UN forces that separates the northern and southern parts of the island), increased the possibility of murine typhus infection. The results of the current study enhance the belief that murine typhus is a serious public health problem in Cyprus.

Spotted fever group Rickettsiae in ticks in Cyprus.

In two surveys conducted from March 1999 to March 2001 and from January 2004 to December 2006, a total of 3,950 ticks (belonging to ten different species) were collected from seven domestic and wild animals (goat, sheep, cattle, dog, fox, hare, and mouflon) from different localities throughout Cyprus. In order to establish their infection rate with Spotted Fever Rickettsiae (SFG), ticks were pooled and tested by polymerase chain reaction targeting gltA and ompA genes, followed by sequencing analysis. When tick pools tested positive, individual ticks were then tested one by one, and of the 3,950 ticks screened, rickettsial DNA was identified in 315 ticks (infection rate, 8%). Five SFG Rickettsiae were identified: Rickettsia aeschlimannii in Hyalomma marginatum marginatum, Rickettsia massiliae in Rhipicephalus turanicus and Rhipicephalus sanguineus, Rickettsia sibirica mongolotimonae in Hyalomma anatolicum excavatum, and a Rickettsia endosymbiont of Haemaphysalis sulcata (later described as Rickettsia hoogstraalii) in Haemaphysalis punctata. Two additional genes, 17 kDa and ompB, were targeted to characterize a new genotype of "Candidatus Rickettsia barbariae" genotype in R. turanicus, designated here as "Candidatus Rickettsia barbariae" Cretocypriensis. These results confirm the presence of a spectrum of SFG Rickettsiae on the island. Further studies are necessary to gain better knowledge on the epidemiology of SFG Rickettsiae in Cyprus.

Neural network approach to retrieve the inherent optical properties of the ocean from observations of MODIS.

Retrieving the inherent optical properties of water from remote sensing multispectral reflectance measurements is difficult due to both the complex nature of the forward modeling and the inherent nonlinearity of the inverse problem. In such cases, neural network (NN) techniques have a long history in inverting complex nonlinear systems. The process we adopt utilizes two NNs in parallel. The first NN is used to relate the remote sensing reflectance at available MODIS-visible wavelengths (except the 678 nm fluorescence channel) to the absorption and backscatter coefficients at 442 nm (peak of chlorophyll absorption). The second NN separates algal and nonalgal absorption components, outputting the ratio of algal-to-nonalgal absorption. The resulting synthetically trained algorithm is tested using both the NASA Bio-Optical Marine Algorithm Data Set (NOMAD), as well as our own field datasets from the Chesapeake Bay and Long Island Sound, New York. Very good agreement is obtained, with R² values of 93.75%, 90.67%, and 86.43% for the total, algal, and nonalgal absorption, respectively, for the NOMAD. For our field data, which cover absorbing waters up to about 6 m⁻¹, R² is 91.87% for the total measured absorption.

Tick-borne bacteria in mouflons and their ectoparasites in Cyprus.

The Cypriot mouflon (Ovis orientalis ophion), a once almost extirpated species of wild sheep, is under strict surveillance because it can be threatened by likely transmission of pathogenic bacteria, such as Anaplasma spp., Rickettsia spp., and Coxiella burnetii, primarily from domestic ungulates. We collected 77 blood samples from Cypriot mouflons and 663 of their ectoparasites (Rhipicephalus turanicus, Rhipicephalus sanguineus, Rhipicephalus bursa, Hyalomma anatolicum excavatum, Hyalomma marginatum, Haemaphysalis punctata, Haemaphysalis sulcata, and Ixodes gibossus) and tested them by polymerase chain reaction and sequencing. Twenty-three mouflon blood samples (30%) were positive for C. burnetii, 23 (30%) for Rickettsia spp., and 8 (10%) for Anaplasma ovis. Of 109 pools of ectoparasites, 32.1% were positive for C. burnetii, 28.4% for Rickettsia spp., and 10.9% for A. ovis; 11.9% were positive for both C. burnetii and Rickettsia spp., 6.4% for both Rickettsia spp. and A. ovis, and 2.8% for all three pathogens. This is the first survey that records the presence of tick-borne pathogens, both in the Cypriot mouflon and in ticks parasitizing it.

The effect of locally delivered doxycycline in the treatment of chronic periodontitis. A clinical and microbiological cohort study.

OBJECTIVES: To evaluate the efficacy of locally delivered doxycycline as an adjunct to non-surgical treatment with the use of an ultrasonic device compared to scaling and root planing using hand instruments, by means of clinical and microbiological criteria. MATERIAL AND METHODS: Thirty three patients with chronic periodontitis participated in this cohort study and were divided into two groups. Patients in control group received scaling and root planing using hand instruments, whereas patients in control group received ultrasonic debridement and 8.8% doxycycline gel was applied after initial therapy and at 3 months at preselected sites. Clinical recordings concerning probing pocket depth, clinical attachment level, plaque index and gingival bleeding index were performed at baseline, 3 and 6 months after baseline. Subgingival samples were analysed using the "checkerboard" DNA-DNA hybridisation technique for Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola. RESULTS: Both treatments resulted in significant improvement in all clinical recordings. Six months after the treatment a statistically significant decrease was observed for Porphyromonas gingivalis in both of groups and Treponema denticola in the control group (P < 0.05). No inter-group differences were observed (P < 0.05). CONCLUSIONS: Both treatment modalities provided comparable clinical and microbiological results in the treatment of chronic periodontitis.

Algorithms for remote estimation of chlorophyll-a in coastal and inland waters using red and near infrared bands.

Remote sensing algorithms that use red and NIR bands for the estimation of chlorophyll-a concentration [Chl] can be more effective in inland and coastal waters than algorithms that use blue and green bands. We tested such two-band and three-band red-NIR algorithms using comprehensive synthetic data sets of reflectance spectra and inherent optical properties related to various water parameters and a very consistent in situ data set from several lakes in Nebraska, USA. The two-band algorithms tested with MERIS bands were Rrs(708)/Rrs(665) and Rrs(753)/Rrs(665). The three-band algorithm with MERIS bands was in the form R3=[Rrs(-1)(665)-Rrs(-1)(708)]×Rrs(753). It is shown that the relationships of both Rrs(708)/Rrs(665) and R3 with [Chl] do not depend much on the absorption by CDOM and non-algal particles, or the backscattering properties of water constituents, and can be defined in terms of water absorption coefficients at the respective bands as well as the phytoplankton specific absorption coefficient at 665 nm. The relationship of the latter with [Chl] was established for [Chl]>1 mg/m3 and then further used to develop algorithms which showed a very good match with field data and should not require regional tuning.

Rickettsia typhi and Rickettsia felis in Xenopsylla cheopis and Leptopsylla segnis parasitizing rats in Cyprus.

Fleas collected from rats during a three-year period (2000-2003) in 51 areas of all provinces of Cyprus were tested by molecular analysis to characterize the prevalence and identity of fleaborne rickettsiae. Rickettsia typhi, the causative agent of murine typhus, was detected in Xenopsylla cheopis (4%) and in Leptopsylla segnis (6.6%). Rickettsia felis was detected in X. cheopis (1%). This is the first report of R. typhi in X. cheopis and L. segnis from rats, in Cyprus, and the first report of R. felis in X. cheopis in Europe. The role of fleas (mainly X. cheopis) was confirmed in the epidemiologic cycle of murine typhus in Cyprus by interrelation of current results with those of previous studies. The geographic distribution of fleas coincided with the geographic distribution of the pathogen they can harbor, which emphasizes the potential risk of flea-transmitted infections in Cyprus.

Rats as indicators of the presence and dispersal of six zoonotic microbial agents in Cyprus, an island ecosystem: a seroepidemiological study.

A total of 622 rats (402 Rattus norvegicus and 220 R. rattus frugivorus) were collected in 51 different areas in Cyprus during 2000-2003 and used as indicators of the presence and dispersal of six zoonotic microbial agents. IgG antibodies against Rickettsia typhi (241/496, 48.6%), R. conorii (209/500, 41.8%), Toxoplasma sp. (138/494, 27.9%), Coxiella burnetti (63/494, 12.8%), Bartonella henselae (52/494, 10.5%) and Leishmania infantum (36/494, 7.3%) were detected by indirect immunofluorescence test. There was variation in the association between the seropositivity of the six microbial agents and other factors. Rat species affected R. typhi and R. conorii seropositivity, the prefecture where the rats were caught affected R. typhi, C. burnetii, B. henselae, T. gondii and L. infantum, the sampling season impacted on R. typhi, R. conorii, T. gondii and L. infantum, and the flea species affected R. typhi, R. conorii and B. henselae. These results were analysed using geographical information system (GIS) technology and the seropositivity in rats against the pathogens tested appeared to follow the occurrence of these pathogens in humans. This suggests that rats could be used as disease sentinels and, together with GIS technology, they could be a useful tool for the identification of endemic foci and high-risk areas for each pathogen.