Safety and immunogenicity of a seasonal trivalent inactivated split influenza vaccine: a double blind, phase III randomized clinical trial in healthy Serbian adults.

This study was a phase III, multicenter, double-blind, randomized, placebo-controlled trial to evaluate the safety and immunogenicity of a seasonal trivalent split, inactivated influenza vaccine (TIV) in healthy Serbian adults between the ages of 18 and 65 years. This egg-based vaccine was manufactured by the Institute of Virology, Vaccines and Sera, Torlak, Belgrade, Serbia. A total of 480 participants were assigned randomly in a ratio of 2:1 to receive a single intramuscular dose (0.5 ml) of the vaccine (15 µg of hemagglutinin per strain) or placebo (phosphate-buffered saline). Participants were monitored for safety, including solicited and unsolicited adverse events (AEs) and serious adverse events (SAEs). No SAEs related to vaccination were reported. Injection site pain (51.3%), injection site tenderness (40.4%), tiredness (17.0%), and headache (15.1%) were the most commonly reported solicited events in the vaccine group. Incidence of related unsolicited AEs was low (1.3%) among vaccinees. Hemagglutinin inhibition (HAI) titers were measured before and 21 days after vaccination in 151 participants. Overall, HAI seroconversion rates to H1 and H3 were observed in 90.1% and 76.2% of vaccinees, respectively. For B antigen, it was 51.5%, likely due to high pre-vaccination titers. Post-vaccination seroprotection rates were in the range of 78.2-95.0% for the three antigens. Post-vaccination geometric mean titers (GMT) were at least 3.8 times higher than baseline levels for all the three strains among vaccinees. Overall, the study showed that the vaccine was safe and well tolerated, and induced a robust immune response against all three vaccine strains. ClinicalTrials.gov identifier: NCT02935192, October 17, 2016.

Safety and immunogenicity of a seasonal trivalent inactivated split influenza vaccine: a phase I randomized clinical trial in healthy Serbian adults.

This study was a phase I double-blind, randomized, placebo-controlled trial to evaluate the safety and immunogenicity of a Serbian-produced seasonal trivalent split, inactivated influenza vaccine in healthy adults. The vaccine was manufactured in eggs by the Torlak Institute of Virology, Vaccines and Sera, Belgrade, Serbia and contained A/H1N1, A/H3N2 and B viruses. The clinical trial took place at the Clinical Center of Serbia in Belgrade. Sixty healthy volunteers, aged 18-45 years, were enrolled in the trial. On the day of immunization, volunteers were randomly assigned to receive either a single dose of the trivalent seasonal influenza vaccine (15 µg of hemagglutinin per strain) or placebo (phosphate-buffered saline). Subjects were monitored for adverse events through a clinical history and physical examination, and blood was taken for testing at screening and on day 8 to assess vaccine safety. Serum samples obtained before and 21 days after immunization were tested for influenza antibody titers using hemagglutination-inhibition (HAI) and microneutralization (MN) tests. No serious adverse events were reported. Pain and tenderness at the injection site were the most commonly reported symptoms in both vaccine and placebo groups. Overall, serum HAI responses of fourfold or greater magnitude were observed to H1, H3, and B antigen in 80%, 75%, and 70% of subjects, respectively. Seroprotection rates as measured by HAI were also high (100%, 100% and 86.67%, respectively, for H1, H3 and B). Thus, Torlak's seasonal trivalent influenza vaccine was not associated with adverse events, was well-tolerated and immunogenic. It should be further evaluated in clinical trials to provide sufficient safety and immunogenicity data for licensing in Serbia.

Influenza A H1N1 virus infection among pregnant women in a tertiary hospital in Belgrade, Serbia.

INTRODUCTION: Pregnant women are at higher risk of developing severe influenza. Our aim was to analyze clinical course and risk factors for fatal outcome in pregnant women with influenza (H1N1) infection. METHODOLOGY: This retrospective study enrolled eleven pregnant women with confirmed Influenza A (H1N1) infection treated in the Clinic for Infectious and Tropical Diseases, Belgrade, Serbia in a 6-years period. RESULTS: The mean age of pregnant women was 28.9 ± 5.2 years, and mean gestational age was 23.1 ± 7.0 weeks. Nine (81.8%) pregnant women had pneumonia (six had interstitial and three had bacterial pneumonia). Pregnant women developed pneumonia more often than other women in the reproductive period, but without statistical significance (81.8% vs. 65.7%, p = 0.330, OR (95% CI) 2.35 (0.47-11.80)). Nine (81.8%) pregnant women recovered. None of them experienced preterm delivery or abortion. Two women (18.2%) died due to acute respiratory distress syndrome. In one of them fetal death occurred one day before she died. The other one was performed caesarean section three days before death. Her newborn and children of all recovered women were healthy at birth. Prolonged time to initiation of oseltamivir and higher body mass index were statistically significantly associated with fatal outcome (p = 0.002, and p = 0.007, respectively). Gestational week of pregnancy, the etiology of pneumonia and comorbidity were not found to be risk factors for death (p = 0.128, p = 0.499 and p = 1.000, respectively). CONCLUSION: Pregnant women with H1N1 infection are at higher risk of pneumonia and death than other women in the reproductive period. Early antiviral therapy reduces the risk of unfavorable outcome.

Tenofovir disoproxil fumarate (TDF) vs. emtricitabine (FTC)/TDF in lamivudine resistant hepatitis B: A 5-year randomised study.

BACKGROUND & AIMS: Long-term treatment with tenofovir disoproxil fumarate (TDF) alone, or in combination with emtricitabine (FTC) is associated with sustained viral suppression in patients with lamivudine resistant (LAM-R) chronic hepatitis B (CHB). METHODS: LAM-R CHB patients were randomised 1:1 to receive TDF 300mg or FTC 200mg and TDF 300mg once daily in a prospective, double blind, study. The proportion of patients with plasma hepatitis B virus (HBV) DNA<69IU/ml (<400copies/ml) at week 96 (primary efficacy endpoint) was reported previously. Here we present week 240 follow-up data. RESULTS: Overall, 280 patients were randomised to receive TDF (n=141) or FTC/TDF (n=139), and 85.4% completed 240weeks of treatment. At week 240, 83.0% of patients in the TDF arm, and 82.7% of patients in the FTC/TDF treatment arm had HBV DNA<69IU/ml (p=0.96). Rates of normal alanine aminotransferase (ALT) and normalised ALT were similar between groups (p=0.41 and p=0.97 respectively). Hepatitis B e antigen loss and seroconversion at week 240 were similar between groups, (p=0.41 and p=0.67 respectively). Overall, six patients achieved hepatitis B surface antigen (HBsAg) loss and one patient (FTC/TDF arm) had HBsAg seroconversion by week 240. No TDF resistance was observed up to week 240. Treatment was generally well tolerated, and renal events were mild and infrequent (∼8.6%). The mean change in bone mineral density at week 240 was -0.98% and -2.54% at the spine and hip, respectively. CONCLUSIONS: TDF monotherapy was effective and well tolerated in LAM-R CHB patients for up to 240weeks. LAY SUMMARY: The goal of oral antiviral treatment for chronic hepatitis B (CHB) is to achieve and maintain undetectable HBV DNA levels. Treatment options with enhanced potency, and low risk of resistance development for patients infected with lamivudine resistant (LAM-R) HBV are required. Tenofovir disoproxil fumarate (TDF) monotherapy was effective and well tolerated without TDF resistance development in CHB patients with LAM-R, for up to 240weeks. Clinical trial number: NCT00737568.

Predictors of Hospitalization and Admission to Intensive Care Units of Influenza Patients in Serbia through Four Influenza Seasons from 2010/2011 to 2013/2014.

A retrospective analysis of the surveillance data on laboratory confirmed cases of influenza in 4 post pandemic seasons in Serbia was performed to evaluate predictors of hospitalization and admission to intensive care units (ICU). The specimens, including nasal and throat swabs were tested for influenza. Univariate and multivariate logistic regression analyses were performed. Data of a total of 777 confirmed influenza cases were analyzed. Age > 65 years, the presence of any co-morbidity or the presence of ≥ 2 comorbidities, infection with influenza virus subtype A (H1) pdm09, and an interval greater than 3 days between symptom onset and the first physician visit, were independently associated with hospital admission. These variables, as well as infection with non-subtype influenza virus A, were predictors for ICU admission. Obesity and chronic neurological disease were independent predictors for ICU admission but not hospitalization. Overall, 41.7% of patients with influenza had at least one co-morbidity, but only 3% of all patients were vaccinated against influenza. Identification of high risk groups and education of these groups regarding their increased susceptibility to severe forms of influenza, and in particular regarding the importance of influenza vaccination, is essential.

Antimicrobial resistance in patients with urinary tract infections and the impact on empiric therapy in Serbia.

INTRODUCTION: Surveillance of antimicrobial resistance is essential in establishing treatment guidelines for urinary tract infections. The aim of this pilot study was to analyse resistance rates of pathogens, across different demographics and determine whether adjustments in empiric therapy should be considered for different age and gender groups. METHODOLOGY: A 5-year retrospective study included 256 patients hospitalised, under the initial diagnosis of Fever of Unknown Origin who were then subsequently diagnosed with a urinary tract infection at the Clinic for Infectious and Tropical Diseases, Clinical Centre of Serbia. Patients were evaluated using demographic, clinical, and antimicrobial resistance data with appropriate statistical analysis including ANOVA significance testing, univariate, and multivariate analysis. RESULTS: Resistance rates were above the threshold of 20% for the majority of the antimicrobials tested, the only exception being carbapenems. Amikacin, cefepime, and norfloxacin were agents that could be effectively used as empiric therapy in younger adults with resistance rates of 4.2, 8.0, and 10.0%, respectively. Moderate resistance rates of 17.4% for amikacin and 19.1% for cefepime were observed in the age group 35-64 years. High resistance rates were observed for all antimicrobials among patients 65 years and over. Among male patients, resistance rates to most antimicrobials were high. In female patients, amikacin and cefepime had resistance rates less than 20%. Younger age presented as a negative risk factor for infection by a multi-drug resistant pathogen. CONCLUSION: Age and gender demonstrated to be significant factors for determining proper empiric therapy; large-scale studies from Serbia are needed to solidify these findings.

Biofilm formation on tympanostomy tubes depends on methicillin-resistant Staphylococcus aureus genetic lineage.

Bacterial biofilm formation has been implicated in the high incidence of persistent otorrhoea after tympanostomy tube insertion. The aim of the study was to investigate whether biofilm formation on tympanostomy tubes depends on the genetic profile of methicillin-resistant Staphylococcus aureus (MRSA) strains. Capacity of biofilm formation on fluoroplastic tympanostomy tubes (TTs) was tested on 30 MRSA strains. Identification and methicillin resistance were confirmed by PCR for nuc and mecA genes. Strains were genotypically characterised (SCCmec, agr and spa typing). Biofilm formation was tested in microtiter plate and on TTs. Tested MRSA strains were classified into SCCmec type I (36.7 %), III (23.3 %), IV (26.7 %) and V (13.3 %), agr type I (50 %), II (36.7 %) and III (13.3 %), and 5 clonal complexes (CCs). All tested MRSA strains showed ability to form biofilm on microtiter plate. Capacity of biofilm formation on TTs was as following: 13.3 % of strains belonged to the category of no biofilm producers, 50 % to the category of weak biofilm producers and 36.7 % to moderate biofilm producers. There was a statistically significant difference between CC, SCCmec and agr types and the category of biofilm production on TTs tubes (p < 0.001): CC5, SCCmecI type and agrII type with a moderate amount of biofilm, and CC8 and agrI type with a low amount of biofilm. Biofilm formation by MRSA on TTs is highly dependent on genetic characteristics of the strains. Therefore, MRSA genotyping may aid the determination of the possibility of biofilm-related post-tympanostomy tube otorrhea.

Retrospective PCR-based species identification of Leishmania in two patients with visceral leishmaniasis in Serbia.

INTRODUCTION: Retrospective molecular identification of Leishmania parasites in two patients with visceral leishmaniasis (VL) previously treated in Serbia was carried out. DNA was isolated from unstained bone marrow smears (BMSs) kept for 11 and 8 years. Genus-specific real-time PCR was combined with conventional PCR and sequencing for detection and species identification. CASE PRESENTATION: In 2003, a 40-year-old Serbian male was admitted to the Clinical Centre of Serbia (CCS) with fever, sweating, fatigue and splenomegaly, which developed over a period of 7 weeks. He had frequently travelled around Europe. VL was confirmed by microscopy of Giemsa-stained BMS. Treatment by pentavalent antimonials was successfully completed. Two years later, the patient developed post-kala-azar dermal leishmaniasis. Treatment resulted in symptom resolution. Later on, Leishmania infantum was identified as the causative agent of the VL by sequencing of the ITS (internal transcribed spacer) region; mixed Leishmania spp. infection could not be excluded. In 2006, a 33-year-old female from Vojvodina, Serbia, with pre-existing diabetes mellitus and chronic meningoencephalitis and a history of frequent visits to the Montenegrin seacoast, was admitted to the CCS with fever, pancytopenia and moderate hepatosplenomegaly. A stained BMS revealed abundant Leishmania amastigotes. Indirect haemagglutination analysis was positive with a titre of 1 : 2048, and a rapid dipstick rK39 test was also positive. Treatment by liposomal amphotericin B was successful; however, shortly after, the patient developed neural infection and pneumonia and died. The causative agent was identified as L. infantum. CONCLUSION: Molecular diagnosis of VL and species delineation using DNA from unstained BMSs stored for several years is possible.

HUMAN SUBCUTANEOUS DIROFILARIASIS - CASE REPORT.

INTRODUCTION: Human dirofilariasis is a zoonotic disease caused by Dirofilaria repens and Dirofilaria immitis. It usually presents as a nodular lesion in the lung, subcutaneous tissues or eyes. In animals, dirofilariasis is a very common disease with serious cardiovascular and respiratory manifestations. If adequate therapy is not given at the beginning ofthe disease, dirofilariasis can lead to animal death. On the contrary, human dirofilariasis is frequently mild, sporadic and asymptomatic disease. Complications in humans are very rare. In Europe, human dirofilariasis is a very rare zoonotic disease even in endemic areas such as Italy, Spain and the Mediterranean. CASE REPORT: The authors reported the case of a 43-year-old male with a subcutaneous nodule caused by Dirofilaria repens. The patient who lives in Budva, Montenegro, had a nodule in the right-hand side of the anterior abdominal walljust below the sternum with maximum diameter of 3 cm. His health condition was good and all laboratory analyses were normal. The lesion was surgically removed and the histopathological examination confirmed the parasitic infection by Dirofilaria repens. After surgical excision, the patient was treated with dual antimicrobial therapy (100 mg doxycycline per os twice a day for 28 days and 200 mg albendazole per os twice a day for 10 days). CONCLUSION: It is very difficult to make the diagnosis of a subcutaneous nodule. The difficulties arise in the differential diagnosis because subcutaneous nodules are suspected to be malignant neoplasm or other pathologies such as tuberculosis, fungal infections, sebaceous cysts, hamartomas, abscesses, and so on. Although human dirofilariasis is a rare disease, the number of reported cases has recently been increasing worldwide.

Oral teicoplanin for successful treatment of severe refractory Clostridium difficile infection.

INTRODUCTION: Clostridium difficile is the leading cause of hospital-acquired diarrhoea. There is no defined protocol for treating severe Clostridium difficile infection (CDI) refractory to vancomycin or vancomycin and metronidazole combination therapy. The aim of this study was to evaluate the rate of clinical cure, time to resolution of diarrhoea and recurrence rate in patients with severe refractory CDI treated with oral teicoplanin. METHODOLOGY: A one-year prospective study was carried out in the Clinic for Infectious and Tropical Diseases, Clinical Center Serbia. Patients with severe and complicated CDI who failed to respond to oral vancomycin and intravenous metronidazole combination therapy were enrolled. They were given oral teicoplanin 100 mg bi-daily. Patients were followed for recurrence for eight weeks. RESULTS: Nine patients with a mean age of 70.8±9.4 years were analyzed. All patients had pseudomembranous colitis, and five had complicated disease. In four patients intracolonic delivery of vancomycin was also performed in addition to oral vancomycin and intravenous metronidazole prior to initiating teicoplanin, but without improvement. After teicoplanin initiation all patients achieved clinical cure. The mean time to resolution of diarrhoea after teicoplanin introduction was 6.3±4.5 days. There was no statistically significant difference in time to resolution of diarrhoea according to initial leucocyte count, age over 65 years, the presence of ileus, complicated disease and the use of concomitant antibiotic therapy (p = 0.652, 0,652, 0.374, 0.374, and 0,548, respectively). None of the patients experienced recurrence. CONCLUSIONS: Oral teicoplanin might be a potential treatment for severe and complicated refractory CDI, but further studies are required.