Alemka Markotić

Exaggerated platelet reactivity to physiological agonists in war veterans with posttraumatic stress disorder

An association between traumatic stress and cardiovascular disease (CVD) is supported by various epidemiological studies. Platelet activation and binding of activated platelets to leukocytes contributes to the pathophysiology of CVD. Evidence of hyperactive sympathetic nervous system, altered expression of platelet α(2)-adrenoreceptors (α(2)AR), and altered platelet adenylate cyclase activity in patients with posttraumatic stress disorder (PTSD) suggest that platelet reactivity in PTSD may be altered as well. We tested whether platelet reactivity to increasing doses of adenosine-diphosphate (ADP), epinephrine (EPI), or their combination differs between war veterans with PTSD (n=15) and healthy controls (n=12). For this purpose, citrated whole blood was incubated with increasing concentrations of ADP (0.1, 1, 10 μM), EPI alone (10 nM, 100 nM, 1000 nM), or EPI (10 nM, 100 nM, 1000 nM) in combination with 0.1 μM ADP. A subset of samples was also incubated with 10 μM yohimbine (YOH), α(2)AR antagonist, to distinguish receptor-specific effects. Platelet CD62P expression and formation of platelet-leukocyte aggregates (PLA) [platelet-monocyte (P-Mo), -lymphocyte (P-Ly), and -neutrophil (P-Ne) aggregates] were measured using three-color flow cytometry. Platelet reactivity was higher in war veterans with PTSD when compared to controls, as determined by greater CD62P expression and formation of PLA in response to ADP alone or in combination with EPI. Platelet reactivity also correlated with the severity of PTSD symptoms. Preliminary experiments with YOH indicate that stress-associated EPI elevations may contribute to platelet activation through a α(2)AR-dependent mechanism. The enhanced platelet reactivity observed in our study may be the underlying mechanism contributing to the development of CVD in PTSD patients.

Validation of high throughput sequencing and microbial forensics applications

High throughput sequencing (HTS) generates large amounts of high quality sequence data for microbial genomics. The value of HTS for microbial forensics is the speed at which evidence can be collected and the power to characterize microbial-related evidence to solve biocrimes and bioterrorist events. As HTS technologies continue to improve, they provide increasingly powerful sets of tools to support the entire field of microbial forensics. Accurate, credible results allow analysis and interpretation, significantly influencing the course and/or focus of an investigation, and can impact the response of the government to an attack having individual, political, economic or military consequences. Interpretation of the results of microbial forensic analyses relies on understanding the performance and limitations of HTS methods, including analytical processes, assays and data interpretation. The utility of HTS must be defined carefully within established operating conditions and tolerances. Validation is essential in the development and implementation of microbial forensics methods used for formulating investigative leads attribution. HTS strategies vary, requiring guiding principles for HTS system validation. Three initial aspects of HTS, irrespective of chemistry, instrumentation or software are: 1) sample preparation, 2) sequencing, and 3) data analysis. Criteria that should be considered for HTS validation for microbial forensics are presented here. Validation should be defined in terms of specific application and the criteria described here comprise a foundation for investigators to establish, validate and implement HTS as a tool in microbial forensics, enhancing public safety and national security.

Double Trouble: Hemorrhagic Fever with Renal Syndrome and Leptospirosis

The clinical picture of hemorrhagic fever with renal syndrome can closely mimic that of unicteric leptospirosis and vice versa. This is the first description of dual infection with Dobrava virus and leptospira and alteration of immune parameters in a Croatian soldier.

Multiple Co-infections of Rodents with Hantaviruses, Leptospira, and Babesia in Croatia

Hantaviruses, Leptospira spp., and Babesia spp. are rodent-borne pathogens present worldwide. We studied multiple co-infections of small rodents in Croatia with all three pathogens. Twenty-eight Apodemus flavicollis and 16 Myodes glareolus were tested for the presence of hantavirus RNA by real-time RT-PCR, Leptospira strains by renoculture method and Babesia DNA by PCR. Anti-hantavirus antibodies and anti-Leptospira antibodies were detected by serological methods. Very high infection rates with each pathogen were found in A. flavicollis: 20 of 28 rodents (71%) were infected with Dobrava virus, 13 rodents (46%) were infected with Leptospira, and 5 rodents (18%) were infected with Babesia. Multiple co-infections with all three pathogens were found in 3 of 28 (11%) A. flavicollis animals, suggesting that the same rodent host can be infected with several pathogens at the same time. Dual infections with both hantaviruses and Leptospira were found in 7 of 44 rodents (16%), with hantaviruses and Babesia in 2 rodents (5%), and double infection with both Leptospira and Babesia were found in 1 rodent (2%). Since hantaviruses, Leptospira, and Babesia have similar geographical distributions, it is to be expected that in other parts of the world multiple co-infections, representing a serious threat to public health, can be found.

Genetic evidence for the presence of two distinct hantaviruses associated with Apodemus mice in Croatia and analysis of local strains

In Europe, Dobrava‐Belgrade (DOBV), Saaremaa (SAAV), and Puumala (PUUV) viruses are known to cause hemorrhagic fever with renal syndrome (HFRS). All three hantaviruses are now found in Croatia. Lung tissue samples of 315 Apodemus mice trapped in 2003–2004 were screened for the presence of hantaviral N‐Ag and 20 mice (6.3%) were found either strongly positive or weak/suspected‐positive. Partial sequences of hantavirus M and S segments were recovered by RT‐PCR from six mice and subjected to (phylo)genetic analysis that revealed the presence of four novel strains of DOBV and one of SAAV. Curiously, one of the newly described DOBV strains was found in Apodemus agrarius mouse, that is, not in the traditional host, A. flavicollis mice, suggesting a spillover event. S segment sequences recovered previously from HFRS cases [Markotić et al., 2002 ] were confirmed as DOBV sequences; one of which appeared particularly close to the prototype Slovenian DOBV isolate. Taken together with earlier data on PUUV in Croatia, these results show a co‐circulation of three European hantavirus pathogens in this country. So far, not a single SAAV sequence has been recovered from HFRS patients either in Croatia or neighboring Slovenia and Hungary nor in Slovakia suggesting a somewhat lower fequency of acute SAAV infection in humans in this part of Europe than for example in the Baltics. J. Med. Virol. 83:108–114, 2011.

Electrocardiographic changes in patients with haemorrhagic fever with renal syndrome

The purpose of the study was to assess the incidence, type and dynamics of electrocardiography (ECG) alterations in patients with haemorrhagic fever with renal syndrome (HFRS) according to different stages of the disease. 79 patients hospitalized at the University Hospital for Infectious Diseases in Zagreb during the large HFRS outbreak in Croatia in 2002 were retrospectively analysed. HFRS diagnosis was confirmed by enzyme-linked immunosorbent assay. A 12-lead resting ECG was obtained. 30 (38%) patients had abnormal ECG findings, most frequently in the oliguric stage. Increased levels of urea and creatinine were observed in all patients with abnormal ECG, along with abnormal chest X-ray in nearly 50% of cases. Sinus tachycardia was the most frequent ECG disorder in the febrile stage, and bradycardia in the oliguric stage. During the course of disease, some other ECG disorders were recorded: bundle branch conduction defects, non-specific ventricular repolarization disturbances, supraventricular and ventricular extrasystoles, prolonged QT interval, low voltage of the QRS complexes in standard limb leads, atrioventricular block first-degree, and atrial fibrillation. Myocarditis was present in 3 patients. In conclusion, abnormal ECG was found in more than one-third of HFRS patients with the most common findings during the oliguric stage. All ECG changes were transient.

HFRS and hantaviruses in the Balkans/South-East Europe.

Hemorrhagic fever with renal syndrome is endemic in the Balkans with epidemic outbreaks and sporadic cases that have been recorded yearly since the disease was first recognized. The incidence of Balkan HFRS is modest, with approximately one hundred cases reported in most years. Seroepidemiological investigations conducted in several Balkan countries revealed an overall seroprevalence of 6% in Bosnia and Herzegovina, 1.6% in Croatia, 4% in Greece and 1.7% in Slovenia, respectively. The complex ecology of the Balkan Peninsula supports the existence of diverse rodent and insectivore species which harbor several pathogenic and non-pathogenic hantaviruses. Among them only Dobrava (DOBV) and Puumala (PUUV) viruses are associated with disease in humans. Comprehensive clinical studies compared clinical signs and symptoms between patients infected with either virus. A spectrum of clinical picture of the disease ranges from mild illness typical of PUUV infections to a severe form with fulminant hemorrhagic fever and an overall mortality rate of 9.8% among DOBV infected patients. While severe DOBV cases are recognized from Slovenia in the North to Greece in the South, PUUV infections are more frequent in northern part of the area. Balkans represent an area with a potential need for hantavirus vaccines, but due to co-existence of DOBV and PUUV causing HFRS in the same region, a universal vaccine is required.

Molecular characterization of dengue virus 1 from autochthonous dengue fever cases in Croatia

In the summer of 2010, two autochthonous dengue fever cases were detected in Croatia. Here we report the retrospective detection of an additional case of dengue fever, representing the first sustained autochthonous transmission in Europe since 1928. In addition, we present the phylogenetic analyses based on two sequences from the Pelješac peninsula, southern Croatia. The sequences were identified as dengue virus genotype 1 and recovered from two out of the three Pelješac patients in whom infection occurred.

Baseline level of platelet-leukocyte aggregates, platelet CD63 expression, and soluble P-selectin concentration in patients with posttraumatic stress disorder: A pilot study

Platelets may have an important role in the development of cardiovascular disease (CVD) as a result of chronic stress. We conducted a pilot study to evaluate the effect of posttraumatic stress disorder (PTSD) on baseline platelet activation. Platelet-leukocyte aggregates (PLA) and CD63 expression were measured by flow cytometry, and soluble (s)P-selectin concentration was determined in sera of 20 Croatian male combat veterans with PTSD and 20 healthy civilians. Groups were matched in sex, age, body mass index (BMI) and traditional CVD risk factors. Our data showed no differences in measured parameters. Other platelet activation markers should be determined and a larger sample size used in future studies.

Enumeration of Haemagglutinin‐specific CD8+ T Cells after Influenza Vaccination Using MHC Class I Peptide Tetramers

With emergence of MHC class I tetramers loaded with CD8+ T‐cell viral epitopes, it is possible to study virus‐specific CD8 cells in humans during infection and after vaccination. MHC class I tetramers was used to detect the frequency of haemagglutinin (HA)‐specific T cells in 26 healthy influenza‐vaccinated humans. Peripheral blood was collected before, and 7, 14 and 28 days after vaccination. Four‐colour flow cytometry was used for monitoring of vaccine induced T‐cell response. In 15 donors, two‐ to fivefold increase in frequency of HA‐specific T cells was observed 7 days after vaccination. In addition, in 12 of these donors, this increase was accompanied with fourfold increase of H1N1 antibody titre. The increase in frequency of HA‐specific CD8+/IFN‐γ+ cells was low and peaked 28 days after vaccination in three of the six donors tested. Frequencies of HA‐specific CD8+ T cells and antibody titre returned to prevaccination values 1 year after vaccination. Subunit influenza vaccines have the ability to induce HA‐specific CD8+ cells. As the immune response to this vaccine decreased significantly after 1 year, our results confirm the importance of annual immunization for adequate protection.