Towards harmonisation of entomological surveillance in the Mediterranean area.

BACKGROUND: The Mediterranean Basin is historically a hotspot for trade, transport, and migration. As a result, countries surrounding the Mediterranean Sea share common public health threats. Among them are vector-borne diseases, and in particular, mosquito-borne viral diseases are prime candidates as (re)emerging diseases and are likely to spread across the area. Improving preparedness and response capacities to these threats at the regional level is therefore a major issue. The implementation of entomological surveillance is, in particular, of utmost importance. Guidance in designing entomological surveillance systems is critical, and these systems may pursue different specific objectives depending on the disease. The purpose of the proposed review is to draw up guidelines for designing effective and sustainable entomological surveillance systems in order to improve preparedness and response. However, we make it clear that there is no universal surveillance system, so the thinking behind harmonisation is to define evidence-based standards in order to promote best practises, identify the most appropriate surveillance activities, and optimise the use of resources. Such guidance is aimed at policymakers and diverse stakeholders and is intended to be used as a framework for the implementation of entomological surveillance programmes. It will also be useful to collaborate and share information with health professionals involved in other areas of disease surveillance. Medical entomologists and vector control professionals will be able to refer to this report to advocate for tailored entomological surveillance strategies. The main threats targeted in this review are the vectors of dengue virus, chikungunya virus, Zika virus, West Nile virus, and Rift Valley fever virus. The vectors of all these arboviruses are mosquitoes. METHODS: Current knowledge on vector surveillance in the Mediterranean area is reviewed. The analysis was carried out by a collaboration of the medical entomology experts in the region, all of whom belong to the MediLabSecure network, which is currently funded by the European Union and represents an international effort encompassing 19 countries in the Mediterranean and Black Sea region. FINDINGS: Robust surveillance systems are required to address the globalisation of emerging arboviruses. The prevention and management of mosquito-borne viral diseases must be addressed in the prism of a One Health strategy that includes entomological surveillance as an integral part of the policy. Entomological surveillance systems should be designed according to the entomological and epidemiological context and must have well-defined objectives in order to effect a tailored and graduated response. We therefore rely on different scenarios according to different entomological and epidemiological contexts and set out detailed objectives of surveillance. The development of multidisciplinary networks involving both academics and public authorities will provide resources to address these health challenges by promoting good practises in surveillance (identification of surveillance aims, design of surveillance systems, data collection, dissemination of surveillance results, evaluation of surveillance activities) and through the sharing of effective knowledge and information. These networks will also contribute to capacity building and stronger collaborations between sectors at both the local and regional levels. Finally, concrete guidance is offered on the vector of the main arbovirus based on the current situation in the area.

Facing of Family Doctor with Hantavirus Infection.

BACKGROUND: Hantavirus infection is manifested as an urgent, severe and life-threatening disease caused by Hantavirus. The virus affects human endothelial cells. The natural reservoir of the Hantaviruses is chronically infected rodents. Human infection is accidental. Occurs by intake of contaminated food or inhalation of contaminated secretion from infected rodents' excretions have an increased risk of contamination. The most affected persons are people who work in nature. The virus causes haemorrhages, fever and acute renal failure. The disease appears more frequently in endemic regions with the lethality of 6-15%. The disease can surprise doctors with severity, urgency and undefined clinical picture. Fast clinical evaluation, proper and urgent diagnosis and treatment can improve the safe life of these patients. CASE REPORT: We report a case of 45 -year-old male patient worked as a shepherd on mountain Babuna near the city of Veles in the Republic of Macedonia at the end of the summer in the year 2017, presented with prolonged hemorrhagic fever with renal syndrome. The clinical presentation and lab findings support the diagnosis of Hantavirus infection with acute renal failure. CONCLUSION: It is necessary to raise the awareness of the family doctors for the hantavirus disease, especially in countries with sporadic cases, as in our country. It needs for prompt and timely diagnosis, timely hospitalisation and initiation of therapy.

Identification of mosquitoes (Diptera: Culicidae): an external quality assessment of medical entomology laboratories in the MediLabSecure Network.

BACKGROUND: Identification of vectors is of prime importance in the field of medical entomology for both operational and research purposes. An external quality assessment of mosquito identification capacities was carried out within the MediLabSecure Network, which is composed of laboratories located in 19 countries close to the European Union around the Mediterranean and Black seas. METHODS: A set of blind samples consisting of 7 or 8 adult mosquitoes and 4 larvae was given to each participant laboratory. In all, 138 adult mosquitoes and 76 larvae of different species were distributed for genus and species identification. RESULTS: All identifications were exclusively morphology based. Overall, 81% of identifications were correct at the genus level, 64% at the species level. The results were highly varied among the 19 participating laboratories. The levels of correct identifications were: 100% (three laboratories), 90-95% (four laboratories), 50-75% (six laboratories) and < 50% (six laboratories). CONCLUSIONS: This evaluation showed the need to maintain efforts in capacity building and quality control in the field of medical entomology and, more specifically, in the morphological identification of the Culicidae.

Hyperfibrinogenemia in Peripheral Arterial Disease: Coexistent and Independent Risk Factor (A Report of Two Cases and Review of Literature).

These case reports aim to show that hyperfibrinogenemia is a risk factor for the progression and prognosis of peripheral arterial disease (PAD), in patients with and without diabetes mellitus type 2. We present a patient with PAD who has type 2 diabetes mellitus, who has previously been repeatedly treated for lower limb ischemia with multiple vascular surgeries performed. A few weeks before admission the patient developed critical lower limb ischemia, which was treated with an iliaco-popliteal and femorofemoral bypass. The patient had elevated serum fibrinogen values. In the current admission, renewed left limb ischemia was diagnosed, and surgically evaluated with a recommendation for amputation of the left limb as a surgical recommendation. Our second patient had a stable intermittent claudication, dyslipidemia and hyperfibrinogenemia. He was successfully treated for those risk factors. Regular monitoring of the patient showed improved claudication distance and quality of life Our case reports, supported by a literature review, demonstrate that hyperfibrinogenemia is a possible risk factor for progression and the prognosis of PAD.

National Laboratory Surveillance of Influenza in Macedonia during Four Seasons.

OBJECTIVE: The aim of the study is to comprehend results of the influenza lab surveillance system in the Republic of Macedonia after the 2009 pandemic and to determine the main characteristics of four consecutive epidemic seasons (from 2010/2011 until 2013/2014). METHODS: As part of the universal surveillance system, nasal and throat specimens were collected from patients. After extraction of RNA, the CDC real-time RT-PCR assays for the detection of influenza types and subtypes were performed. RESULTS: Out of 920 tested samples, 406 (44.1%) laboratory confirmed cases of influenza were found. Influenza activity begins as early as December and continues until the end of April with peaks in January or February with predominant influenza A and A/H1N1pdm. Influenza A viruses start their activity at week 49 to 52 and subside at week 17. Usually two peaks appear, the first one between week 2 and 4 and the second one between week 6 and 9. Subtype A/H1N1pdm was dominant among influenza A types in the 2010/2011 and 2012/2013 seasons. A/H3N2 was the only circulating influenza virus in the 2011/2012 season. Influenza B season is shorter and has only one peak, between weeks 2-5. Usually the influenza B viruses emerge in later stages than influenza A viruses, except for the first post-pandemic season. CONCLUSION: Results revealed that post-pandemic influenza seasons in Macedonia were rather different. Although the influenza season pattern is similar to patterns in some countries of the WHO European region, some unique characteristics were observed.

Comparison of Directigen Flu A+B with Real Time PCR in the Diagnosis of Influenza.

UNLABELLED: Early diagnosis and treatment of patients with influenza is the reason why physicians need rapid high-sensitivity influenza diagnostic tests that require no complex lab equipment and can be performed and interpreted within 15 min. The Aim of this study was to compare the rapid Directigen Flu A+B test with real time PCR for detection of influenza viruses in the Republic of Macedonia. MATERIALS AND METHODS: One-hundred-eight respiratory samples (combined nose and throat swabs) were routinely collected for detection of influenza virus during influenza seasons. Forty-one patients were pediatric cases and 59 were adult. Their mean age was 23 years. The patients were allocated into 6 age groups: 0-4 yrs, 5-9 yrs, 10-14 yrs, 15-19 yrs, 20-64 yrs and > 65 yrs. Each sample was tested with Directigen Flu A+B and CDC real time PCR kit for detection and typisation/subtypisation of influenza according to the lab diagnostic protocol. RESULTS: Directigen Flu A+B identified influenza A virus in 20 (18.5%) samples and influenza B virus in two 2 (1.9%) samples. The high specificity (100%) and PPV of Directigen Flu A+B we found in our study shows that the positive results do not need to be confirmed. The overall sensitivity of Directigen Flu A+B is 35.1% for influenza A virus and 33.0% for influenza B virus. The sensitivity for influenza A is higher among children hospitalized (45.0%) and outpatients (40.0%) versus adults. CONCLUSION: Directigen Flu A+B has relatively low sensitivity for detection of influenza viruses in combined nose and throat swabs. Negative results must be confirmed.

Methods for molecular surveillance of influenza used in Macedonia.

THE AIM: To present and compare different Nucleic Acid Testing assays used for laboratory diagnosis of influenza virus infection in our country. MATERIALS AND METHODS: Respiratory samples used were nose and throat swabs. The RNA extraction was performed with a QIAamp viral RNA kit. During the season 2009­2010 the first 25 samples were tested with: conventional gel-based RT-PCR and CDC rtRT-PCR using published specific matrix and HA gene primers and probes for influenza virus typing and subtyping. RESULTS: Of 25 samples tested with conventional RT-PCR 7(28%) were positive for influenza A, but negative for A/H1seasonal and A/H3. Retested with rtRT-PCR 9(36%) were positive for influenza A, 8(32%) were positive for А/H1pdm and 1(4%) was А/H3. Two samples positive with rtRT-PCR for influenza A were negative with RT-PCR. The sensitivity of the RT-PCR in comparison with rtRT-PCR is 100% and the specificity is 88.89%. Positive predictive value for RT-PCR is 77.78%, and negative predictive value is 100%. RT-PCR is a four-step and rtRT-PCR a one-step procedure. The turn-around time of RT-PCR is 6 hours and for rtRT-PCR it is 2 hours. DISCUSSION AND CONCLUSION: For surveillance purposes nose and throat swabs are the more easy and practical to collect. It was proved that RT-PCR is too laborious, multi-step and time-consuming. The sensitivity of both assays is equal. The specificity of rtRT-PCR is higher. NAT assays for detection of influenza viruses have become an integral component of the surveillance programme in our country. They provide a fast, accurate and sensitive detection of influenza.

Antibiotics for upper respiratory infections: public knowledge, beliefs and self-medication in the Republic of Macedonia.

Self-medication as part of the irrational use of antibiotics contributes to the spread of antimicrobial resistance. The aim of this community-based survey in Macedonia was to determine public knowledge, beliefs and self-medication with antibiotics for upper respiratory infections. A cross-sectional study was conducted in three administrative regions in Macedonia in April 2012. 402 eligible participants answered an anonymous questionnaire. The analysis of answers involved descriptive quantitative statistics (frequencies and percentages). We also tested for significant associations between demographic characteristics and non-prescription use of antibiotics. Our respondents demonstrated a relatively low level of public knowledge about antibiotics and upper respiratory infection treatments in comparison to the EU countries. The study found that 71.4% of participants stored antibiotics at home, and 43.3% purchased antibiotics over-the-counter in the last year, despite national regulation that restricts antibiotics as prescription-only medicines. Actual self-medication with antibiotics for a recent upper respiratory infection episode was reported in 17.8% of adults and 1.8% of children aged 0-4 years. We did not find any significant association between participants demography and non-prescription use of antibiotics. Our results put in the group of eastern and southern EU countries with the highest rates for non-prescription use of antibiotics in Europe. Multifaceted interventions are needed to prevent self-medication with antibiotics, including: enforcement of regulations that restrict over-the-counter sales of antibiotics, monitoring of antibiotic use and antimicrobial resistance rates and combined public education strategies.

Laboratory surveillance during pandemic influenza season 2009/2010 in Republic of Macedonia.

OBJECTIVE: To provide virological and epidemiological information on patients laboratory-tested for influenza A/ H1N1pdm during the pandemic season April 2009/May 2010. MATERIALS AND METHODS: Demographic and other data were obtained from the request form arriving with the samples of patients whose symptoms met the clinical definition of influenza A infection. The RNA was tested for the presence of influenza virus using the CDC real-time RT-PCR assay. A total of 3010 suspect patients (pts) were tested from week 18 2009 to week 20 2010. RESULTS: 1632 pts (54.2%) were oositive for influenza. From them 1556 samples were confirmed as H1N1pdm. There was a domination of H1N1pdm positivity among young persons in age groups 5-17 (34.4%) and 18-49 (31.4%) years. The pandemic influenza was presented in two waves. The first wave started on 20 June with the first positive case and peaked early in August (week 32). The second wave started from week 44. The majority of positive cases were between week 45 and week 52. 37.7% of the positive pts were hospitalized--66.7% of pts at age 65+ and 63.3% of children in the age group 0-4 years. The highest percentage of patients with underlying medical conditions were in the age group 50-64 (49.35%) years and 65+ (88.23%) years. 1.15% of the positive pts for H1N1pdm gave data for vaccination with seasonal influenza. CONCLUSIONS: Data obtained from laboratory and epidemiological surveillance of pandemic influenza will serve public health to a full understanding of the pandemic 2009/2010 influenza in R. Macedonia and dealing with future challenges.

Measles outbreak in Macedonia: epidemiological, clinical and laboratory findings and identification of susceptible cohorts.

OBJECTIVES: Despite a 92-99% national vaccination coverage since 2000, the former Yugoslav Republic of Macedonia experienced a large measles outbreak between 2010 and 2011. Here we investigate the characteristics of patients hospitalized during this outbreak at the Clinic of Infectious Diseases in Skopje. METHODS: Epidemiological, clinical and laboratory data of 284 measles patients, including 251 from Skopje (43.80% of the 573 reported cases) and 33 from elsewhere in Macedonia were collected. RESULTS: The most affected age groups were children up to 4 years of age and adolescents/adults of 15 years and older. Most patients were unvaccinated (n=263, 92.61%) and many had non-Macedonian nationalities (n=156, 54.93%) or belonged to the Roma ethnicity (n=73, 25.70%). Bronchopneumonia and diarrhea were the most common complications. Eighty-two out of 86 tested patients (95.35%) had measles-specific IgM antibodies. The outbreak was caused by the measles variant D4-Hamburg. CONCLUSIONS: The epidemic identified pockets of susceptibles in Skopje and indicated that additional vaccination opportunities in particular for people with non-Macedonian nationality and traveler communities are warranted to ensure efficient measles control in Macedonia. The high attack rate among children of less than 1 year suggests that vaccination before 12 months of age should be considered in high risk settings.

Influence of fibrinogen and C-RP on progression of peripheral arterial disease in type 2 diabetes: a preliminary report.

BACKGROUND: Limited studies have suggested that inflammatory biomarkers play a role in the initiation and progression of atherosclerosis in diabetic patients. This study assesses the effect of inflammatory biomarkers: fibrinogen and C-reactive protein (C-RP) on the progression of peripheral arterial disease (PAD) in type 2 diabetic (T2D) patients. METHODS: Sixty two patients with T2D and PAD (mean age 60.28 ± 27 years and diabetes duration of 8.58 ± 6.17 years) were enrolled in a cohort prospective study of 36 months. Ankle-brachial index (ABI) was measured in all patients at baseline and after 36 months. Multiple linear regression analysis was used to determine the predictivity of variables for fibrinogen, C-RP, plasma lipid fractions, fasting plasma glucose, Body Mass Index (BMI), duration of diabetes status and the age on changes in ABI value. RESULTS: Linear regression analysis defined F as a predictor for endpoint value of ABI (ß = 0.469, p = 0.007). Value of C-RP determinates change of minimal value of ABI (ß = 0.449, p = 0.037) and change of mean ABI per year (ß = 0.442, p = 0.025). CONCLUSION: Our data indicate that plasma determination of fibrinogen and C-RP might have a clinical implication in defining the process of progression of PAD in T2D population.

The first survey about the antibiotic usage in the Republic of Macedonia.

Development of antibiotic resistance represents a major global and Macedonian public health problem. To assess the opinion and knowledge of the citizens of Macedonia about the usage of antibiotics, voluntary and anonymous survey was realized. A total of 239 persons (age 10-67 years) were interviewed. Following information was obtained: 73.64% get antibiotics with a medical prescription; and 87.03% receiving the antibiotic on time, dosage and prescribed duration. When asked about knowledge about antibiotics, 38% of the interviewed persons gave the right answer, 43.1% of respondents made false statements that antibiotics are effective against viral infections and 25.52% did not express any opinion.

Spread of measles virus D4-Hamburg, Europe, 2008-2011.

A new strain of measles virus, D4-Hamburg, was imported from London to Hamburg in December 2008 and subsequently spread to Bulgaria, where an outbreak of >24,300 cases was observed. We analyzed spread of the virus to demonstrate the importance of addressing hard-to-reach communities within the World Health Organization European Region regarding access to medical care and vaccination campaigns. The D4-Hamburg strain appeared during 2009-2011 in Poland, Ireland, Northern Ireland, Austria, Greece, Romania, Turkey, Macedonia, Serbia, Switzerland, and Belgium and was repeatedly reimported to Germany. The strain was present in Europe for >27 months and led to >25,000 cases in 12 countries. Spread of the virus was prevalently but not exclusively associated with travel by persons in the Roma ethnic group; because this travel extends beyond the borders of any European country, measures to prevent the spread of measles should be implemented by the region as a whole.

Clinical role of estimation metabolic syndrome's components in type 2 diabetic population with symptomatic coronary artery disease--a comparison of two criteria.

Objective of our study was to estimate the metabolic syndrome (MSy)' components in type 2 diabetic subjects (T2D) with symptomatic coronary artery disease (CAD): arterial hypertension, waist circumference, triglyceridemia and plasma level of HDL, in order to define their clinical role in angina severity status. Second objective was to compare Adult Treatment Panel (ATP III) and International Diabetes Federation (IDF) criteria in testing of association MSy with current coronary status of population. Three hundred and twenty seven pts with T2D and manifested CAD were randomly included in a survey. Angina severity was assessed with the Canadian Cardiovascular Society (CCS). Data relieved MSyand its components, defined by ATP III, with predictive role for advanced angina stages. Presence of MSy is predictive for CCS3 angina severity (OR 9,93, 95% CI 0,78-126,28). Increased waist is a predictor for CCS3 (OR 1,22, 95% CI 0,27-5,46) and CCS4 symptoms onset (OR 1,55, 95% CI 0,36-6,73). CCS4 severity symptom is independently associated with arterial hypertension (OR 3,72, 95% CI 1,03-13,40) and hypertriglyceridemia (OR 1,72, 95% CI 0,42- 7,00). MSy components: increased waist, arterial hypertension and hypertriglyceridemia have been found predictive for angina CCS4 stage in T2D subjects, when IDF criteria were used. These findings raise the question of importance of metabolic syndrome and its individual constellations in T2D subjects with CAD. Results indicate that both criteria ATP III and IDF are reliable to define MSy in predictive model for coronary clinical status in T2D population.

RT-PCR detection of HIV in Republic of Macedonia.

The aim of the study was to detect HIV RNA in seropositive patients using RT-PCR method and thus, to establish PCR methodology in the routine laboratory works. The total of 33 examined persons were divided in two groups: 1) 13 persons seropositive for HIV; and 2) 20 healthy persons - randomly selected blood donors that made the case control group. The subjects age was between 25 and 52 years (average 38,5). ELFA test for combined detection of HIV p24 antigen and anti HIV-1+2 IgG and ELISA test for detection of antibodies against HIV-1 and HIV-2, were performed for each examined person. RNA from the whole blood was extracted using a commercial kit based on salt precipitation. Detection of HIV RNA was performed using RT-PCR kit. Following nested PCR, the product was separated by electrophoresis in 1,5 % agarose gel. The result was scored positive if the band of 210bp was visible regardless of intensity. Measures of precaution were taken during all the steps of the work and HIV infected materials were disposed of accordingly. In the group of blood donors ELFA, ELISA and RT-PCR were negative. Assuming that prevalence of HIV infection is zero, the clinical specificity of RT-PCR is 100 %. The analytical specificity of RT-PCR method was tested against Hepatitis C and B, Human Papiloma Virus, Cytomegalovirus, Herpes Simplex Virus, Rubella Virus, Mycobacterium tuberculosis, Chlamydia trachomatis. None of these templates yielded amplicon. In the group of 13 seropositive persons, 33 samples were analyzed. HIV RNA was detected in 15 samples. ELISA and ELFA test were positive in all samples. Different aliquots of the samples were tested independently and showed the same results. After different periods of storing the RNA samples at -70 masculineC, RT-PCR reaction was identical to the one performed initially. The obtained amplicons were maintained frozen at -20 masculineC for a week and the subsequently performed electrophoresis was identical to the previous one. The reaction is fast, simple for manipulation; with low detection level of 60 IU/ml. RT-PCR needs a small amount of RNA, as well as a small volume of sample. HIV RNA was detected in different periods of time with different clinical presentations in patients, with or without antiretroviral therapy. RT-PCR method gives the opportunity for reliable determination of HIV-1 RNA with border of detection of 60 IU/ml. The test is reproducible and has high analytical and clinical specificity.