Early Diagnosis of Tularemia by Flow Cytometry, Czech Republic, 2003-20151.

We retrospectively assessed the utility of a flow cytometry-based test quantifying the percentage of CD3+ T cells with the CD4-/CD8- phenotype for predicting tularemia diagnoses in 64 probable and confirmed tularemia patients treated during 2003-2015 and 342 controls with tularemia-like illnesses treated during 2012-2015 in the Czech Republic. The median percentage of CD3+/CD4-/CD8- T cells in peripheral blood was higher in tularemia patients (19%, 95% CI 17%-22%) than in controls (3%, 95% CI 2%-3%). When we used 8% as the cutoff, this test's sensitivity was 0.953 and specificity 0.895 for distinguishing cases from controls. The CD3+/CD4-/CD8- T cells increased a median of 7 days before tularemia serologic test results became positive. This test supports early presumptive diagnosis of tularemia for clinically suspected cases 7-14 days before diagnosis can be confirmed by serologic testing in regions with low prevalences of tularemia-like illnesses.

The Scottish model of vancomycin dosing and therapeutic drug monitoring improves both efficacy and safety of vancomycin therapy.

OBJECTIVE: Vancomycin therapeutic drug monitoring (TDM) is recommended for better treatment efficacy and safety. The Scottish Antimicrobial Prescribing Group model recommends weight-based loading dose, next 3-4 doses based on creatinine clearance and maintenance doses according to trough concentrations. METHODS: Retrospective cohort study of all adult patients treated with vancomycin before and after introducing the guideline in a large Czech hospital in 2015 compares the success rate in achieving recommended trough concentrations (10-20 mg/l) during first measurement and efficiency of maintaining these concentrations subsequently. Assessment of vancomycin related nephrotoxicity is included. RESULTS: In 2014, 74/163 (45.4 %) patients achieved recommended concentrations in the first measurement, compared to 101/160 (63.1 %) patients in 2016 (χ2; p = 0.001). Recommended trough concentrations in more than half of subsequent measurements were detected in 51/105 patients (48.6 %) in 2014 and in 80/117 patients (68.4 %) in 2016. Not a single level in subsequent measurements within the range was detected in 28 (26.7 %) cases in 2014 and in 10 (8.5 %) cases in 2016 (Mann-Whitney; p = 0.026). There was no difference in nephropathy occurrence (χ2; p = 0.286). CONCLUSION: The adopted Scottish model of vancomycin TDM resulted in very significantly higher achievement of recommended trough concentrations during first measurements and significantly more effective maintenance of subsequent concentrations, without increased nephrotoxicity. Key words: guideline - therapeutic drug monitoring - vancomycin.

Variation of the specificity of the human antibody responses after tick-borne encephalitis virus infection and vaccination.

UNLABELLED: Tick-borne encephalitis (TBE) virus is an important human-pathogenic flavivirus endemic in large parts of Europe and Central and Eastern Asia. Neutralizing antibodies specific for the viral envelope protein E are believed to mediate long-lasting protection after natural infection and vaccination. To study the specificity and individual variation of human antibody responses, we developed immunoassays with recombinant antigens representing viral surface protein domains and domain combinations. These allowed us to dissect and quantify antibody populations of different fine specificities in sera of TBE patients and vaccinees. Postinfection and postvaccination sera both displayed strong individual variation of antibody titers as well as the relative proportions of antibodies to different domains of E, indicating that the immunodominance patterns observed were strongly influenced by individual-specific factors. The contributions of these antibody populations to virus neutralization were quantified by serum depletion analyses and revealed a significantly biased pattern. Antibodies to domain III, in contrast to what was found in mouse immunization studies with TBE and other flaviviruses, did not play any role in the human neutralizing antibody response, which was dominated by antibodies to domains I and II. Importantly, most of the neutralizing activity could be depleted from sera by a dimeric soluble form of the E protein, which is the building block of the icosahedral herringbone-like shell of flaviviruses, suggesting that antibodies to more complex quaternary epitopes involving residues from adjacent dimers play only a minor role in the total response to natural infection and vaccination in humans. IMPORTANCE: Tick-borne encephalitis (TBE) virus is a close relative of yellow fever, dengue, Japanese encephalitis, and West Nile viruses and distributed in large parts of Europe and Central and Eastern Asia. Antibodies to the viral envelope protein E prevent viral attachment and entry into cells and thus mediate virus neutralization and protection from disease. However, the fine specificity and individual variation of neutralizing antibody responses are currently not known. We have therefore developed new in vitro assays for dissecting the antibody populations present in blood serum and determining their contribution to virus neutralization. In our analysis of human postinfection and postvaccination sera, we found an extensive variation of the antibody populations present in sera, indicating substantial influences of individual-specific factors that control the specificity of the antibody response. Our study provides new insights into the immune response to an important human pathogen that is of relevance for the design of novel vaccines.

Specificities of human CD4+ T cell responses to an inactivated flavivirus vaccine and infection: correlation with structure and epitope prediction.

Tick-borne encephalitis (TBE) virus is endemic in large parts of Europe and Central and Eastern Asia and causes more than 10,000 annual cases of neurological disease in humans. It is closely related to the mosquito-borne yellow fever, dengue, Japanese encephalitis, and West Nile viruses, and vaccination with an inactivated whole-virus vaccine can effectively prevent clinical disease. Neutralizing antibodies are directed to the viral envelope protein (E) and an accepted correlate of immunity. However, data on the specificities of CD4(+) T cells that recognize epitopes in the viral structural proteins and thus can provide direct help to the B cells producing E-specific antibodies are lacking. We therefore conducted a study on the CD4(+) T cell response against the virion proteins in vaccinated people in comparison to TBE patients. The data obtained with overlapping peptides in interleukin-2 (IL-2) enzyme-linked immunosorbent spot (ELISpot) assays were analyzed in relation to the three-dimensional structures of the capsid (C) and E proteins as well as to epitope predictions based on major histocompatibility complex (MHC) class II peptide affinities. In the C protein, peptides corresponding to two out of four alpha helices dominated the response in both vaccinees and patients, whereas in the E protein concordance of immunodominance was restricted to peptides of a single domain (domain III). Epitope predictions were much better for C than for E and were especially erroneous for the transmembrane regions. Our data provide evidence for a strong impact of protein structural features that influence peptide processing, contributing to the discrepancies observed between experimentally determined and computer-predicted CD4(+) T cell epitopes. Importance: Tick-borne encephalitis virus is endemic in large parts of Europe and Asia and causes more than 10,000 annual cases of neurological disease in humans. It is closely related to yellow fever, dengue, Japanese encephalitis, and West Nile viruses, and vaccination with an inactivated vaccine can effectively prevent disease. Both vaccination and natural infection induce the formation of antibodies to a viral surface protein that neutralize the infectivity of the virus and mediate protection. B lymphocytes synthesizing these antibodies require help from other lymphocytes (helper T cells) which recognize small peptides derived from proteins contained in the viral particle. Which of these peptides dominate immune responses to vaccination and infection, however, was unknown. In our study we demonstrate which parts of the proteins contribute most strongly to the helper T cell response, highlight specific weaknesses of currently available approaches for their prediction, and demonstrate similarities and differences between vaccination and infection.

[Dengue fever cases in Czech workers returning from the Maldives]. / Prípady horecky dengue u ceských pracovníku zamestnaných na Maledivách.

AIM: The objective of this study is to present epidemiological characteristics and clinical symptoms of dengue fever cases in Czech workers who acquired the infection while working on the island of Fushivelavaru, Maldives. Furthermore, the study compares the sensitivity of novel direct detection assays, i.e. Real-Time Polymerase Chain Reaction (RT-PCR) and detection of Dengue NS1 antigen. MATERIAL AND METHODS: The retrospective study evaluated the clinical course of dengue fever cases in Czech workers returning from a job in the Maldives who were diagnosed with dengue virus infection from September 1 to October 31, 2012. The laboratory diagnosis of dengue fever was based on the serological detection of IgM and IgG antibodies, detection of dengue NS1 antigen by enzyme-linked immunosobent assay (ELISA), and detection of dengue virus DNA by RT-PCR. RESULTS: The infection with dengue virus was confirmed in 18 males with a median age of 40 years (IQR 36-47) who returned from a job in the Maldives. Only one patient required admission to the hospital while the others were treated on an outpatient basis. The most frequently observed symptoms were fever (18), headache (9), muscle and joint pain (8 and 7, respectively), and rash (9). Typical laboratory findings were leukocytopenia and thrombocytopenia, a low CRP level, and elevated aminotransferase activity. The clinical course was uncomplicated in all patients. The dengue NS1 antigen detection (positive in all 10 patients with acute dengue fever) showed significantly higher sensitivity than the detection of viral RNA using RT-PCR (positive in 4 patients), p = 0.011. CONCLUSION: Although the vast majority of dengue fever cases are diagnosed among travellers returning from the tropics, the presented study points out the risks posed by dengue fever to long-term workers in endemic areas. The infection in the serologically naïve hosts is usually uncomplicated; however, infected persons are at significant risk of developing a severe complicated clinical course if challenged by another serotype. Furthermore, sick leaves or premature departures cause a considerable economic burden to employers. In the diagnosis of acute dengue fever, preference should be given to highly sensitive and specific tests for the direct detection of dengue virus (NS1 antigen and RT-PCR assays).

[Catheter related blood stream infections--prevalence and interventions]. / Katétrové infekce krevního reciste--prevalence a intervence.

BACKGROUND: Catheter related blood stream infections are a significant complication of intensive care with worldwide prevalence rate around 5 cases per 1000 catheter-days. Only scanty Czech data have been published. Our study monitored the occurrence of catheter-related blood stream infections in a high dependency unit of regional hospital. METHODS: In 2008 we commenced to monitor the occurrence rate of catheter-related blood stream infections in short-term central venous catheters without antimicrobial coating. We organized a training session for medical staff and started to strictly adhere to published guidelines. After two years of keeping a register we analysed individual cases as proven, possible, or not proven blood stream infections. RESULTS: From March 2008 to March 2010 we inserted 142 central venous catheters for total time of 1423 catheter days (median 9 days). Ten catheters were removed after median of 17 days due to unexplained pyrexia. Blood stream infection was proven in 4 cases and possible in 2 cases. We have noted total 2.81 proven cases, and 4.22 proven and/or possible cases of blood stream infection per 1000 catheter-days. CONCLUSION: The register of catheter related blood stream infections is an inexpensive and time-efficient tool that improves the quality of intensive

Encephalitozoon cuniculi genotype I as a causative agent of brain abscess in an immunocompetent patient.

A brain abscess caused by Encephalitozoon cuniculi genotype I together with Streptococcus intermedius occurred in a patient without major immunocompromise and with diabetes. The distinguishing clinical signs were hemiparesis and epilepsy. The microsporidium was observed in the abscess aspirate, and its specific DNA was also detected in stool and urine. The patient was successfully treated with albendazole and mebendazole.

Clinical Characteristics of Patients with Tick-Borne Encephalitis (TBE): A European Multicentre Study from 2010 to 2017.

Tick-borne encephalitis (TBE) virus is a major cause of central nervous system infections in endemic countries. Here, we present clinical and laboratory characteristics of a large international cohort of patients with confirmed TBE using a uniform clinical protocol. Patients were recruited in eight centers from six European countries between 2010 and 2017. A detailed description of clinical signs and symptoms was recorded. The obtained information enabled a reliable classification in 553 of 555 patients: 207 (37.3%) had meningitis, 273 (49.2%) meningoencephalitis, 15 (2.7%) meningomyelitis, and 58 (10.5%) meningoencephalomyelitis; 41 (7.4%) patients had a peripheral paresis of extremities, 13 (2.3%) a central paresis of extremities, and 25 (4.5%) had single or multiple cranial nerve palsies. Five (0.9%) patients died during acute illness. Outcome at discharge was recorded in 298 patients. Of 176 (59.1%) patients with incomplete recovery, 80 (27%) displayed persisting symptoms or signs without recovery expectation. This study provides further evidence that TBE is a severe disease with a large proportion of patients with incomplete recovery. We suggest monitoring TBE in endemic European countries using a uniform protocol to record the full clinical spectrum of the disease.

Crushed dolutegravir/abacavir/lamivudine given via nasogastric tube in gastric outlet obstruction caused by cancer resulted in rapid viral load suppression.

Alternative modes of antiretroviral administration are sought for people with impaired intestinal passage and/or absorption. We present a case of late HIV diagnosis (CD4+ count 160 cells/µL) with gastric outlet obstruction due to stomach adenocarcinoma. Co-morbidities included oesophageal candidiasis, Helicobacter pylori-positive duodenal ulcers and cytomegalovirus duodenitis. The gastric outlet obstruction required total parenteral nutrition and parenteral medication during four weeks of diagnostic work-up leading to pyloric resection. Crushed dolutegravir, abacavir and lamivudine were administered during this time in the evening via nasogastric tube, which was kept clamped overnight. The tube was unclamped in the morning and stomach content was drained during the daytime. This mode of administration resulted in rapid and sustained viral load suppression (from 300,000 to 115 copies per mL in 28 days, 81 copies/mL after 42 days of treatment and less than 40 copies/mL thereafter). Therapeutic drug monitoring confirmed sufficient antiretroviral plasma levels during this mode of administration. The absorption of crushed dolutegravir, abacavir and lamivudine in the stomach may be considered in people with questionable gastrointestinal passage or impaired gastric emptying to achieve viral load suppression.

Hospital antibiotic management in the Czech Republic--results of the ABS maturity survey of the ABS International group.

BACKGROUND: Hospital antibiotic stewardship (ABS) programs are essential for ensuring long-lasting quality of antibiotic usage and for controlling antimicrobial resistance in the hospital setting. METHODS: A questionnaire for self-assessment of a hospital's ABS maturity was sent to 80 Czech hospitals in May 2007. The survey was focused on diagnostic issues, control of antibiotic consumption, antibiotic-related organization and tools, antibiotic-related personnel development and antibiotic-related relationships to relevant environments. RESULTS: Of 80 addressed hospitals, 45 sent back processed questionnaires (response rate 56.3%). These 80 hospitals cover about 85% of the Czech population. All Czech university hospitals were included in the replying group. The listed diagnostic tools were declared available by all hospitals; 44 of 45 hospitals have surveillance systems for antibiotic resistance rates. Control of antibiotic consumption was available only partially in 42 of 45 hospitals. Some antibiotic tools need to be improved and must be used more frequently. Official recognition, job descriptions and better funding of antibiotic personnel are needed, as well as support for further development of ABS structures and functions. DISCUSSION: The basic structure of ABS is well developed in the Czech hospitals. A network of antibiotic centers focusing their services on optimization of antibiotic usage has been in place in the Czech Republic since the 1970s. Nevertheless, the survey revealed a clear need and many opportunities for further improvement. Control of antibiotic consumption is not implemented in all Czech hospitals and some of the essential antibiotic tools should be used more widely.

Epidemiological survey of tick-borne encephalitis virus and Anaplasma phagocytophilum co-infections in patients from regions of the Czech Republic endemic for tick-borne diseases.

During the period 2000-2003, patients hospitalized for suspected tick-borne encephalitis in the Czech Republic were screened for possible A. phagocytophilum co-infection. Blood samples taken at admission were tested for the presence of A. phagocytophilum DNA by nested PCR using a modified target sequence as an internal control, and sera were tested for the presence of antibodies by indirect immunofluorescence and western blotting methods using cell-culture-derived antigens. To verify the assay specificity, a set of 45 sera of Patagonian residents served as a non-tick-exposed control group, and a set of 14 B. henselae-positive sera was used to check cross-reactivity. Of 809 patients hospitalized, 80 (9.9%) showed IgG antibodies reactive to A. phagocytophilum at > or =80 (reciprocal dilution factor) and 50 (6.2%) at > or =160; two (0.2%) patients showed elevated IgM titers of 40. No full blood obtained from 162 patients tested positive in PCR when false negativity was excluded. During hospitalization, the diagnosis of tick-borne encephalitis was confirmed in 536 patients, 57 (10.6%) of whom had anti-A. phagocytophilum IgG antibodies reactive at > or =80 and 41 (7.6%) at > or =160, which did not differ significantly from the whole set (P = 0.66/0.30), the maximum IgG titer registered was 5120, and no IgM titer reached the 40 cut-off. Available paired sera from 189 tick-borne encephalitis patients showed no significant shifts, but one case of slight seroconversion (IgG shift from < 80 to 320) was detected in one of the non-tick-borne encephalitis patients. The sex of the patient showed no significance for the prevalence of A. phagocytophilum antibodies; however, the seropositive patients were older on average than those who were seronegative (43.5 +/- 15.9 vs. 37.9 +/- 18.3 years, P = 0.05). Clinical manifestation of the disease did not differ noticeably between patients with and without A. phagocytophilumreactive antibodies, except for fever duration, which was significantly longer in patients with titers > or =1280. Overall, A. phagocytophilum co-infection did not seem to be a frequent and/or significant complication of tick-borne encephalitis acquired in the Czech Republic.

Tick-borne encephalitis: What travelers should know when visiting an endemic country.

Tick-borne encephalitis (TBE) is an acute febrile illness with neurological manifestations that is prevalent in forested areas of moderate climate in Europe and Asia. TBE virus is transmitted by ticks and rarely by unpasteurized milk and dairy products. The disease burden is attributed mainly to resulting long-term disability, especially in individuals over 50 y of age. Currently, there is no causative treatment, but a very effective vaccination is available with a good safety profile. The vaccination requires 3 basic doses to be fully effective and regular boosters afterwards. An accelerated vaccination schedule enables a patient to reach reasonably protective titres within 3 to 4 weeks from the first injection. The risk of travel-related TBE is estimated to be less than the risk of acquiring typhoid fever while visiting highly endemic regions in South Asia, but more than the risk of acquiring Japanese encephalitis, meningococcal invasive disease, or rabies. The pre-travel risk assessment of acquiring TBE should consider known risk factors which include 1) the country and regions to be visited; 2) April to November season; 3) altitude less than 1500 m above the sea level; 4) duration of stay; 5) the extent of tick-exposure associated activities including leisure and professional outdoor activities within the endemic area; and 6) age and comorbidities of the traveler. A major challenge, however, is the very low awareness of the risk of contracting TBE in those who travel to industrialized European countries.

Human CD4+ T Helper Cell Responses after Tick-Borne Encephalitis Vaccination and Infection.

Tick-borne encephalitis virus (TBEV) is a human-pathogenic flavivirus that is endemic in large parts of Europe and Asia and causes severe neuroinvasive illness. A formalin-inactivated vaccine induces strong neutralizing antibody responses and confers protection from TBE disease. CD4+ T cell responses are essential for neutralizing antibody production, but data on the functionalities of TBEV-specific CD4+ T cells in response to vaccination or infection are lacking. This study provides a comprehensive analysis of the cytokine patterns of CD4+ T cell responses in 20 humans after TBE vaccination in comparison to those in 18 patients with TBEV infection. Specifically, Th1-specific cytokines (IFN-γ, IL-2, TNF-α), CD40 ligand and the Th1 lineage-specifying transcription factor Tbet were determined upon stimulation with peptides covering the TBEV structural proteins contained in the vaccine (C-capsid, prM/M-membrane and E-envelope). We show that TBEV-specific CD4+ T cell responses are polyfunctional, but the cytokine patterns after vaccination differed from those after infection. TBE vaccine responses were characterized by lower IFN-γ responses and high proportions of TNF-α+IL-2+ cells. In vaccine-induced responses-consistent with the reduced IFN-γ expression patterns-less than 50% of TBEV peptides were detected by IFN-γ+ cells as compared to 96% detected by IL-2+ cells, indicating that the single use of IFN-γ as a read-out strongly underestimates the magnitude and breadth of such responses. The results provide important insights into the functionalities of CD4+ T cells that coordinate vaccine responses and have direct implications for future studies that address epitope specificity and breadth of these responses.

The current perspective on tick-borne encephalitis awareness and prevention in six Central and Eastern European countries: report from a meeting of experts convened to discuss TBE in their region.

Tick-borne encephalitis (TBE) is a potentially life-threatening disease in humans and is caused by a flavivirus spread by infected ticks (Ixodes ricinus and Ixodes persulcatus). TBE is endemic across much of Central and Eastern Europe and the incidence is increasing, with numbers estimated to be as many as 8755 cases per year. The reasons for this increase are multi-faceted and may involve improvements in diagnosis and reporting of TBE cases, increases in recreational activities in areas inhabited by infected ticks and changes in climatic conditions affecting tick habitats. Vaccination is the most effective method of preventing TBE; following a successful nationwide vaccination campaign in Austria, the annual number of TBE cases fell to about 10% of those reported in the pre-vaccination era. This report describes the findings of a group of leading experts from six Central and Eastern European countries who convened to discuss TBE in their region during the 28th Annual Meeting of the European Society for Paediatric Infectious Diseases (ESPID) Nice, France, 4-8 May 2010.

The Golden Agers and Tick-borne encephalitis. Conference report and position paper of the International Scientific Working Group on Tick-borne encephalitis.

The 7th meeting of the ISW TBE had the main topic "Tick-borne encephalitis in the Golden Agers". Data from 14 European countries were presented about incidence and clinical course of Tick borne encephalitis (TBE) in general and especially in the population over 50 years of age. With age immunity is impaired quantitatively and qualitatively, the reactions to vaccinations are generally slower, antibody titres reach lower values and decrease earlier. The incidence of the disease is increasing with age, also the clinical course is more severe, they suffer significantly more sequelae, need a longer rehabilitation and have a higher case fatality. Vaccination as the only efficient protection is needed in endemic areas, considering that mobility has increased very much. For the age group over 50 years regular booster vaccinations according to the recommended vaccination intervals or even shorter intervals are most important.

Tick-borne encephalitis in childhood--consensus 2004.

Tick-borne encephalitis (TBE) is a communicable disease caused by a flavi-virus, ticks being the main vectors. The nervous system is affected, four clinical features of different severity are observed: meningitis, meningoencephalitis, meningoencephalomyelitis, meningoradiculoneuritis. TBE is a preventable disease, which is rapidly becoming a growing public health problem in Europe. So far no causal treatment is possible but an efficient, safe vaccination is available. During the 6th meeting of the International Scientific Working Group on TBE with the main conference issue "Tick-borne encephalitis in childhood" an international consensus was achieved. In countries where TBE is endemic--and not prevented by immunization--both children and adults are affected. The disease in children is generally milder, although severe illness may occur and even lead to permanent impairment of the quality of life due to neuropsychological sequelae. Therefore immunization should be offered to all children living in or traveling to endemic areas.