Towards a Semi-Automatic Early Warning System for Vector-Borne Diseases.

The emergence and spread of vector-borne diseases (VBDs) is a function of biotic, abiotic and socio-economic drivers of disease while their economic and societal burden depends upon a number of time-varying factors. This work is concerned with the development of an early warning system that can act as a predictive tool for public health preparedness and response. We employ a host-vector model that combines entomological (mosquito data), social (immigration rate, demographic data), environmental (temperature) and geographical data (risk areas). The output consists of appropriate maps depicting suitable risk measures such as the basic reproduction number, R0, and the probability of getting infected by the disease. These tools consist of the backbone of a semi-automatic early warning system tool which can potentially aid the monitoring and control of VBDs in different settings. In addition, it can be used for optimizing the cost-effectiveness of distinct control measures and the integration of open geospatial and climatological data. The R code used to generate the risk indicators and the corresponding spatial maps along with the data is made available.

A spatial predictive model for malaria resurgence in central Greece integrating entomological, environmental and social data.

Malaria constitutes an important cause of human mortality. After 2009 Greece experienced a resurgence of malaria. Here, we develop a model-based framework that integrates entomological, geographical, social and environmental evidence in order to guide the mosquito control efforts and apply this framework to data from an entomological survey study conducted in Central Greece. Our results indicate that malaria transmission risk in Greece is potentially substantial. In addition, specific districts such as seaside, lakeside and rice field regions appear to represent potential malaria hotspots in Central Greece. We found that appropriate maps depicting the basic reproduction number, R0, are useful tools for informing policy makers on the risk of malaria resurgence and can serve as a guide to inform recommendations regarding control measures.

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.

Attitudes and beliefs on the establishment of a national food safety authority in Cyprus. A population-based survey.

Cyprus does not have a National Food Safety Authority (NFSA), but a multi-level, fragmented system with responsibilities divided among different ministries and governmental agencies, frequently impeding efforts to effectively manage food risks by duplication and overlapping of responsibilities. A population-based survey was carried out to determine the beliefs and attitudes of interested parties concerning the establishment of a NFSA in Cyprus. Information was collected using a random stratified sampling design and a structured questionnaire. A total of 868 questionnaires were collected (704 from regular consumers, 154 from food businesses' representatives, and 10 from public services' directors or acting head officers). About 11% of food businesses' representatives and 45% of consumers reported that they did not know which public authorities are responsible for food control. Moreover, 2 out of 10 (17%) of responders from public agencies, 70% from food businesses and 91% from consumers, although not aware of ongoing efforts to establish a food safety authority in Cyprus (currently under consideration), were supportive of the idea [8 out of 10 (83%) of responders from public services, 93% from food businesses, and 89% of consumers]. Finally, 7 out of 10 (67%) from the public agencies and 84% of representatives from food businesses agreed with the separation of risk assessment from risk management activities. Public opinion in Cyprus as well as public agencies and food businesses' representatives support the establishment of a single independent national food safety authority in Cyprus based on the European paradigm including the division of risk activities.

Study of the whole cell lysate of two Coxiella burnetii strains using N-terminomics.

The etiological agent of Q fever is Coxiella burnetii , an obligate intracellular Gram-negative bacterium and the only bacterium known to date that survives and replicates within a vacuole of phagolysosomal characteristics. In humans, Q fever is characterized by a wide spectrum of clinical manifestations. Of note is that genetic diversity among C. burnetii strains has been reported. To further investigate C. burnetii's diversity, but now at the proteome level, we compared the proteomes of whole cell lysates from two reference strains, Nine Mile and Q212. Proteomes were isolated from each strain and subjected MS-driven combined fractional diagonal chromatography (COFRADIC), a peptide-centered proteomics technique, with a total of 322 proteins that were unambiguously identified. On the basis of their identified neo-N-terminal peptides that are highly likely generated upon in vivo processing by proteases, the most proteolytical sensitive proteins in these strains were identified, and a consensus cleavage pattern was obtained. Further, with the use of differential proteomics based on the here-identified N-terminal peptides, 44 proteins were found to be differentially expressed between the two C. burnetii strains, representing 13.6% of the here-identified C. burnetii proteome. Among these proteins, 10 proteins were found uniquely expressed in the NM strain including proteins with unknown functions as well as housekeeping enzymes, suggesting that strain-related proteins might be present among such uncharacterized proteins.

Investigation of rifampicin resistance mechanisms in Brucella abortus using MS-driven comparative proteomics.

Mutations in the rpoB gene have already been shown to contribute to rifampicin resistance in many bacterial strains including Brucella species. Resistance against this antibiotic easily occurs and resistant strains have already been detected in human samples. We here present the first research project that combines proteomic, genomic, and microbiological analysis to investigate rifampicin resistance in an in vitro developed rifampicin resistant strain of Brucella abortus 2308. In silico analysis of the rpoB gene was performed and several antibiotics used in the therapy of Brucellosis were used for cross resistance testing. The proteomic profiles were examined and compared using MS-driven comparative proteomics. The resistant strain contained an already described mutation in the rpoB gene, V154F. A correlation between rifampicin resistance and reduced susceptibility on trimethoprim/sulfamethoxazole was detected by E-test and supported by the proteomics results. Using 12 836 MS/MS spectra we identified 6753 peptides corresponding to 456 proteins. The resistant strain presented 39 differentially regulated proteins most of which are involved in various metabolic pathways. Results from our research suggest that rifampicin resistance in Brucella mostly involves mutations in the rpoB gene, excitation of several metabolic processes, and perhaps the use of the already existing secretion mechanisms at a more efficient level.

Re-emergence of visceral and cutaneous leishmaniasis in the Greek Island of Crete.

Leishmaniases are vector-borne diseases transmitted by phlebotomine sand flies. Three species of Leishmania are found in the Mediterranean basin: Leishmania infantum, the most common species responsible for both visceral (VL) and cutaneous leishmaniasis (CL); Leishmania major, found in North Africa and Middle East causing CL; Leishmania tropica with a limited presence in Europe, causing CL. During the last 25 years, Crete has become an endemic zone for L. infantum with a high number of infected dogs and an increasing number of human cases every year; in the last 4 years, the incidence has reached an average of seven VL patients per year in a population of 600,000. At the same time, CL has re-emerged in Crete due to L. tropica, with an average of three CL cases per year in the last 4 years. Isolates were typed as L. infantum MON-1 and MON-98 and L. tropica MON-300, a zymodeme not reported before. Both VL and CL have spread to the whole of the island during the last 25 years, primarily in semi-urban and urban areas with altitudes of 0-50 m. The prevailing Phlebotomus species were Phlebotomus neglectus (proven vector of L. infantum) and Phlebotomus similis (suspected vector of L. tropica).

A proteomic approach to investigate the differential antigenic profile of two Coxiella burnetii strains.

Q fever is a widespread zoonosis caused by Coxiella burnetii, an obligate intracellular Gram-negative bacterium. Current diagnostics of Q fever is based on serological testing of patient serum. Biological distinction among C. burnetii strains has been referred at the genetic level as well as in virulence in animal models of Q fever. Disclosure of strain specific antigens might show insight into the biology and pathogenesis of this query pathogen, as well as it can provide the literature with potential serodiagnostic markers. In the present study, we sought to obtain an outer membrane enriched fraction of two C. burnetii reference strains, which originate from different sources, in order to investigate the way in which their antigenic profile is differentiated against a patient serum. We systematically analyzed the sarcosyl-insoluble fraction, enriched in outer membrane proteins, of the two C. burnetii strains using doubled SDS-PAGE combined with MS/MS analysis. In total, twenty-two outer membrane proteins were identified, representing 26% of the overall 86 identified proteins. The sarcosyl-insoluble fraction was then separated on 2DE IEF/SDS-PAGE and probed with serum from an infected patient. Different immuno-reactive proteins between the two C. burnetii strains were identified and included 2 outer membrane proteins, a hypothetical protein (CBU_0937) with unknown function and OmpH (CBU_0612), a previously identified marker for Q fever endocarditis. This approach can be used to reveal strain-specific proteins involved in pathogenesis and new serodiagnostic markers.

Protective effect of exclusive breastfeeding against infections during infancy: a prospective study.

OBJECTIVE: To prospectively investigate the effects of breastfeeding on the frequency and severity of infections in a well-defined infant population with adequate vaccination coverage and healthcare standards. STUDY DESIGN: In a representative sample of 926 infants, successfully followed up for 12 months, feeding mode and all infectious episodes, including acute otitis media (AOM), acute respiratory infection (ARI), gastroenteritis, urinary tract infection, conjunctivitis and thrush, were recorded at 1, 3, 6, 9 and 12 months of life. RESULTS: Infants exclusively breastfed for 6 months, as per WHO recommendations, presented with fewer infectious episodes than their partially breastfed or non-breastfed peers and this protective effect persisted after adjustment for potential confounders for ARI (OR 0.58, 95% CI 0.36 to 0.92), AOM (OR 0.37, 95% CI 0.13 to 1.05) and thrush (OR 0.14, 95% CI 0.02 to 1.02). Prolonged exclusive breastfeeding was associated with fewer infectious episodes (r(s)=-0.07, p=0.019) and fewer admissions to hospital for infection (r(s)=-0.06, p=0.037) in the first year of life. Partial breastfeeding was not related to protective effect. Several confounding factors, including parental age and education, ethnicity, presence of other siblings, environmental tobacco smoke exposure and season of birth were demonstrated to have an effect on frequency of infections during infancy. CONCLUSIONS: Findings from this large-scale prospective study in a well-defined infant population with adequate healthcare standards suggest that exclusive breastfeeding contributes to protection against common infections during infancy regarding and lessens the frequency and severity of infectious episodes. Partial breastfeeding did not seem to provide this protective effect.

Predisposing factors for acute otitis media in infancy.

SUMMARY BACKGROUND: Factors predisposing to acute otitis media (AOM) are not thoroughly understood. OBJECTIVES: In this study we prospectively investigated incidence rates and risk factors of AOM in a systematic sample of infants in a well-defined population. DESIGN AND SUBJECTS: Following interview soon after delivery in a representative sample of 1049 mother-infant pairs, information was collected 1, 3, 6, 9 and 12 months after birth. Episodes of AOM were recorded as by parental reports of diagnosis made by a physician. AOM frequency was further analyzed against 21 social, maternal, paternal, prenatal, perinatal and infantile factors. RESULTS: Successful 12-month follow-up was achieved for 926/1049 infants (88.3%), of whom 265 (28.6%) were reported with episodes of AOM; 153 with a single, 55 with two, and 57 with three to six episodes (16.5%, 5.94%, and 6.16% respectively). Factors founded by multivariate analysis to predispose to AOM included presence of siblings (P<0.001), out-of-home daycare (P<0.001), ill health in pregnancy (P<0.01), and suboptimal breastfeeding (P<0.01). Multiple (3 or more) episodes were related to presence of siblings (P<0.0001), out-of-home daycare (P<0.001), ill health in pregnancy (P<0.001), and low parental education (P<0.001). 28 infants were hospitalized (10.6% of infants with AOM, 3.02% of the total cohort). Hospitalization was related to young maternal age (P<0.05). CONCLUSIONS: Our findings confirm the high morbidity of AOM during infancy and point to several infant- and family-related predisposing factors, some of which are liable to intervention.

Proteomic screening for possible effector molecules secreted by the obligate intracellular pathogen Coxiella burnetii.

Coxiella burnetii is a Gram-negative, gamma-proteobacteria with nearly worldwide distribution, and it is the pathogenic agent of Q-fever in man. It is an obligate intracellular parasite that is highly adapted to reside within the eukaryotic phagolysosome. In fact, it is the only known intracellular bacterium that manages to survive and replicate within a fully formed, acidic phagolysosome. C. burnetti possesses a functional Type 4 Secretion System (T4SS), similar to the Dot/Icm system of Legionella pneumophila. Up to date there have been no reports for the effector molecules secreted by Coxiella's T4SS. These are speculated to have quite different roles than the effectors of other intracellular pathogens, since there is no need for phagosomal arrest or escape in the case of Coxiella. In this study, we have investigated the cytoplasm of Vero cells infected with C. burnetti strain Nine Mile Phase II. We have identified by mass spectrometry (ESI-MS/MS) several C. burnetti proteins that bear typical characteristics of effector molecules. Most of the identified proteins were also very alkaline, something which is supportive for a protective strategy that has evolved in this bizarre pathogen against acidic environments.

A rare case of leishmaniasis recidiva cutis evolving for 31 years caused by Leishmania tropica.

A 64-year-old woman presented with erythematous, infiltrative plaques with a central atrophic area on both zygomatic regions. Several yellow-reddish papules were seen at the periphery of the plaques and showed an "apple-jelly" color on diascopy (Fig. 1). No visceral involvement was detected. The past medical history revealed that, at 3 years of age, she had developed an "Oriental sore" on both cheeks that healed with permanent scars. Thirty years later, she noticed an erythematous patch around the scars. She reported a hospital admission 22 years earlier for cutaneous leishmaniasis (CL); this was treated with pentavalent antimonial therapy for 10 days with partial improvement, when she refused further treatment. The lesions worsened in the summer and gradually became disfiguring, which prompted her to seek medical consultation. Laboratory findings were normal. Leishmania antibody titers were negative. Tissue samples were obtained by biopsy from the border of the lesion for culture, polymerase chain reaction (PCR), and histopathologic examination. Histology revealed a dermal infiltrate with tuberculoid granulomas surrounded by lymphocytes, histiocytes, and some plasma cells, but no caseation necrosis. A few Leishmania organisms were found on careful searching (Fig. 2). Leishmania tropica was identified by culture and PCR. A diagnosis of leishmaniasis recidiva cutis (LRC) was made on the basis of the anamnestic data together with the clinical, histopathologic, biologic, and molecular findings. Complete regression was achieved with meglumine antimoniate (Glucantime) given intramuscularly (15 mg Sb(V)/kg/day for 15 days) and cryosurgery with liquid nitrogen. No recurrence was noted during a 12-month follow-up period.

Outcome of toxoplasmosis acquired during pregnancy following treatment in both pregnancy and early infancy.

OBJECTIVES: Congenital toxoplasmosis is associated with clinical dilemmas as untreated infants may have a guarded prognosis and as treatment may induce severe side effects. The aim of this study was to investigate the outcome of infants born to mothers with toxoplasmosis acquired during pregnancy, following administration of appropriate regimens both during pregnancy and early infancy. STUDY DESIGN: All 35 infants, born to mothers with toxoplasmosis acquired during pregnancy, and referred to the major Neonatal Department in Crete, Greece, during the 7-year period 1997-2003 were included. All neonates were evaluated soon after birth and on a regular follow-up. RESULTS: Almost all mothers received spiramycin from diagnosis through labor and 2 received pyrimethamine and sulfadiazine. At birth, infants had IgG antibody titers ranging from 1/1,350 to 1/109,350. All infants initially received pyrimethamine, sulfadiazine and folinic acid but in only 4 cases treatment was continued beyond the second month of life. Transient neutropenia was commonly observed. A follow-up period of 1.2-8.2 years did not reveal any remarkable sequelae. CONCLUSIONS: Our findings suggest that effective treatment both during pregnancy and early infancy is safe and may contribute to a good outcome of infants born to mothers with toxoplasmosis acquired during pregnancy.

Invasive non-typhoidal salmonellosis in immunocompetent infants and children.

OBJECTIVE: To investigate the extraintestinal manifestations of non-typhoidal Salmonellae (NTS) infection in immunocompetent infants and children. METHOD: The study took place at the University General Hospital at Heraklion, Crete. Over a 10-year period from 1993-2002 we studied 1087 patients, of whom 443 were children less than 14 years old, with a culture-proven diagnosis of NTS infection. Stool and blood cultures were routinely obtained in patients presenting with fever and diarrhea. The cases of invasive infection in otherwise well children, including bacteremia and/or extraintestinal focal infections were further analyzed. RESULTS: Invasive cases were less common in children than adults (4.06% vs. 8.7%; relative risk 0.467; 95% confidence intervals (CI) 0.279-0.784; p=0.0033). Furthermore, invasive cases were much less common in the otherwise well than in immunocompromised children (3.5% vs. 21.4%; relative risk 0.163; 95% CI 0.053-0.500; p=0.0008). The 15 otherwise well children with invasive NTS infection were aged from 3 weeks to 7.5 years, and nine were aged less than 12 months. Among them, 11 presented with bacteremia, and four with focal extraintestinal infections (rectal abscess, deep neck abscess, urinary tract infection, elbow arthritis). Salmonella enterica subsp. enterica serovars Enteritidis and Virchow were the most common invasive serotypes. All invasive strains were susceptible to beta-lactams including ampicillin, and to cotrimoxazole. All patients made a complete recovery with intravenous antibiotics and did not present with relapses or major infections during long-term follow-up. CONCLUSION: Invasive non-typhoidal salmonellosis in immunocompetent children is less frequent than in both immunocompromised children and in adulthood. However, invasive cases may well occur in otherwise healthy children, especially during infancy. In these patients, prompt appropriate treatment leads to favorable outcomes.

In vitro susceptibilities of Brucella melitensis isolates to eleven antibiotics.

BACKGROUND: Brucellosis is an endemic disease present in many countries worldwide, but it is rare in Europe and North America. Nevertheless brucella is included in the bacteria potentially used for bioterrorism. The aim of this study was the investigation of the antibiotic susceptibility profile of brucella isolates from areas of the eastern Mediterranean where it has been endemic. METHODS: The susceptibilities of 74 Brucella melitensis isolates derived from clinical samples (57) and animal products (17) were tested in vitro. The strains originate from Crete (59), Cyprus (10), and Syria (5). MICs of tetracycline, rifampicin, streptomycin, gentamicin, norfloxacin, ciprofloxacin, levofloxacin, trimethoprim/sulfamethoxazole, ampicillin, amoxicillin/clavulanic acid, and erythromycin were detected by E-test method. The NCCLS criteria for slow growing bacteria were considered to interpret the results. RESULTS: All the isolates were susceptible to tetracycline, streptomycin, gentamicin, ciprofloxacin, norfloxacin, and levofloxacin. Two isolates presented reduced susceptibility to rifampicin (MIC value: 1.5 mg/l) and eight to SXT (MIC values: 0.75-1.5 mg/l). Erythromycin had the highest (4 mg/l) MIC90value and both norfloxacin and erythromycin the highest (1.5 mg/l) MIC50 value. CONCLUSION: Brucella isolates remain susceptible in vitro to most antibiotics used for treatment of brucellosis. The establishment of a standardized antibiotic susceptibility method for Brucella spp would be useful for resistance determination in these bacteria and possible evaluation of bioterorism risks.

Nocardia cyriacigeorgica pleural empyema in an immunocompromised patient.

A case of fatal Nocardia cyriacigeorgica pleural empyema in a patient with chronic obstructive pulmonary disease and long-term corticosteroid therapy is described. The organism was isolated from the pleural fluid in pure culture and was identified by conventional and molecular methods. Despite the early administration of the appropriate antibiotic treatment, the patient died 4 days after hospital admission.

Use of the GenoType Mycobacterium CM and AS assays to analyze 76 nontuberculous mycobacterial isolates from Greece.

Seventy-six nontuberculous mycobacterial isolates obtained from patients living in Greece were analyzed with the GenoType Mycobacterium CM (for common mycobacteria) and AS (for additional species) assays. GenoType correctly identified all but one of the mycobacterial species. For this species, additional probes should be designed and added to the strip.

Yersinia enterocolitica infection mimicking surgical conditions.

Yersinia enteritis may present with alarming gastrointestinal manifestations. The aim of this study was to review the cases of children admitted to a general hospital with a preliminary diagnosis of surgical nature and subsequently proven to be infected by Yersinia enterocolitica. All cases of children aged less than 14 years with stool cultures positive for Y. enterocolitica during the 12-year period January 1993 through December 2004 were analyzed. Y. enterocolitica was isolated from the stools of 71 children with gastrointestinal manifestations; 27 children were treated as outpatients and 44 were hospitalized. Six were admitted to the Pediatric Surgery Department (13.6% of the total hospitalizations and 8.4% of all Y. enterocolitica cases). Four of the Pediatric Surgery patients presented with abdominal pain and right lower quadrant tenderness. The preliminary diagnosis of appendicitis was excluded during hospitalization and none of them underwent appendectomy. The other two children were admitted for vomiting initially attributed to a preceding head injury and for diarrhea and a perianal abscess. Two children were given antibiotics and all had an excellent outcome. Y. enterocolitica enteritis manifestations can infrequently mimic appendicitis or other surgical conditions but should remain in the differential diagnosis of children presenting with an acute abdomen.

Qualitative versus quantitative blood cultures in the diagnosis of catheter-related bloodstream infections in children with malignancy.

BACKGROUND: Paired quantitative and qualitative blood cultures have been introduced for the diagnosis of catheter-related bloodstream infections (CRBI) with the catheter in situ. The aim of the study was to compare the diagnostic performance and the prognostic value of the two methods in the evaluation of febrile episodes without an apparent source in children with cancer. PROCEDURE: During a 4-year period, in every febrile episode without an apparent focus, blood was drawn simultaneously from the catheter lumen and a peripheral vein in order to perform paired quantitative (Isolator) as well as qualitative (BacT/Alert) blood cultures. The diagnosis of a CRBI was defined as either a case of greater (at least 10 fold) or earlier (differential time to positivity >2 h) bacterial growth from the catheter compared to the peripheral blood sample, respectively. RESULTS: Nineteen febrile episodes manifested in 16 children (total period of observation 11,150 catheter-days) were evaluated with both methods. A concordant diagnosis of CRBI was stated with both methods in six episodes; one episode was diagnosed as CRBI only with qualitative culture criteria. Treatment failure resulted in catheter removal in five out of the seven episodes defined as CRBI with either method. Episodes where a CRBI was ruled out with both methods had a favorable outcome. CONCLUSIONS: In this study the two methods showed comparable results in the diagnosis of CRBI and both were of prognostic significance, regarding the outcome of the treatment. However, large scale studies are required in order to evaluate the clinical relevance and the cost effectiveness of performing routinely paired blood cultures with either method.

Incidence of toxoplasmosis in 5532 pregnant women in Crete, Greece: management of 185 cases at risk.

OBJECTIVES: To study the incidence of toxoplasmosis in pregnant women in Crete and to test a designed protocol for handling those at risk of delivering congenitally infected infants. STUDY DESIGN: Pregnant women were screened serologically over a period of 5 years. Cases with suspected acute toxoplasmosis were treated, peripheral blood (PB), and amniotic fluid (AF) tested by polymerase chain reaction (PCR) and culture, and fetuses monitored by ultrasonography. The absence of congenital infection in infants was confirmed by serology and clinical evaluation. RESULTS: Of the 5532 pregnant women followed, 70.57% remained seronegative, 29.45% were seropositive, and there was direct evidence of seroconversion in six cases. Acute toxoplasmosis was suspected in 185 cases, maternal parasitemia was detected in five cases and positive amniotic fluid in one case. Congenital infection was excluded in all infants followed, based on the absence of ultrasound findings in utero, lack of clinical symptoms at birth, negative Western blotting (WB) at birth and 3 months later, and descending serology for a year. CONCLUSION: Overall, 29.45% of the pregnant women followed were seropositive, 3.3% with suspected acute toxoplasmosis, and in 0.02% cases there was evidence of maternofetal transmission. The protocol tested allowed differentiation between acute and latent toxoplasmosis, safe management of the cases at risk and assisted in avoidance of unwarranted pregnancy terminations.