Diagnostic value of urine qRT-PCR for the diagnosis of West Nile virus neuroinvasive disease.

Short and low-level viremia and virorachia, antibody cross-reactivity, IgM persistence, and inaccessibility of neutralization test, make laboratory diagnosis of West Nile virus (WNV) infection difficult. Recent investigations imply that WNV is excreted in urine longer and at higher concentrations compared to blood. The detection of WNV nucleic acid in cerebrospinal fluid (CSF), serum, and urine samples collected from 41 patients with suspected WNV neuroinvasive disease, was done by real-time RT-PCR assay. CSF and serum samples were also serologically tested using anti-WNV IgM/IgG ELISA kits. WNV infection was confirmed in 46.3% of patients by positive WNV RNA results in serum and/or CSF samples. The WNV RNA testing of urine allowed confirmation of 31.7% more cases. No association between WNV RNA urine positivity and age, gender, or the day of sample collection was found. The urine qRT-PCR can be a valuable diagnostic test for confirmation of probable cases of WNV neuroinvasive disease.

Human papillomavirus-specific antibody status among unvaccinated subjects in the region of Vojvodina, Serbia.

OBJECTIVES: The aim of the study was to evaluate the immune status of young people from the Vojvodina province, Serbia, through the detection of IgG antibodies specific for the L1 protein of HPV types 6, 11, 16, and 18 contained in quadrivalent vaccine. METHODS: The study enrolled 514 healthy persons of both genders, aged between 18 and 30 years. All potential participants were informed about the project's aims by trained interviewers before venous blood collection. Also, participants completed a specially designed anonymous questionnaire to identify socio-demographic characteristics and individual behaviours associated with HPV seroprevalence. VPL HPV L1-specific IgG antibodies were measured using a semi-quantitative HPV IgG ELISA kit (Dia.Pro, Italy). RESULTS: A total of 472 (91.8%) young subjects had no detectable antibodies against high- and low-risk HPV types covered by the quadrivalent vaccine. A slightly higher number of seropositive individuals were detected in the age group of 26-30 years compared to younger than 25. Multivariate analysis showed that the number of lifetime sexual partners was the most powerful predictor of HPV seropositivity (OR = 3.483, 95% CI: 1.294-9.379). CONCLUSIONS: Obtained data point out low levels of naturally induced HPV-specific serum antibodies among the target population in the Vojvodina province. The present work highlights the significance and potential benefits of HPV vaccination. Routine HPV vaccination should be the public health priority in our country and should be included in the national immunization programme as soon as possible.

Negative trend in seroprevalence of anti-toxoplasma gondii IgG antibodies among the general population of the province of Vojvodina, Serbia, 2008-2021.

This study aimed to estimate dynamic changes in seroprevalence of Toxoplasma gondii within the general population living in the northern part of the Republic of Serbia (Province of Vojvodina) during a 14-year period. The differences in prevalence of anti-toxoplasma antibodies were analyzed in correlation with age, gender, residential area (rural/urban) and meteorological factors. In this cohort retrospective study, 24,440 subjects between 1 and 88 years old were enrolled. To determine the presence of T. gondii-specific IgM and IgG antibodies in serum samples, commercially available ELISA kits were used (Euroimmun, Luebeck, Germany). During the study period, the overall T. gondii seroprevalence was 23.5%. The seroprevalence continuously decreased over time from 31.7% in 2008 to 20.4% in 2021 (0.81% per year, p < 0.001). Approximately 2% of patients had a serologic profile positive for both anti-Toxoplasma IgG and IgM antibodies. The seroprevalence was higher (28.87%) among men compared to women (24.28%), while urban residents (24.94%) had lower seroprevalence than the rural population (28.17%). A statistically significant negative correlation (r = -0.559) was found between serologic profile of patients positive for both T. gondii IgG and IgM antibodies and the annual mean air temperature. No significant association was observed between seropositivity to T. gondii infection and examined meteorological factors. These data could be useful to national and regional health authorities to create an optimal health policy to reduce rate of T. gondii infections.

The prevalence of 30 HPV genotypes detected by EUROArray HPV in cervical samples among unvaccinated women from Vojvodina province, Serbia.

This study evaluates the pre-vaccination prevalence of HPV infection in women from Vojvodina, Serbia, according to age and cytological status. A total of 1,495 women, ranging from 18 to 65 years of age, with different cytological results were enrolled. The HPV genotyping assay was performed using the EUROArray HPV test in order to detect thirty genitally relevant HPV subtypes. In our study, the most prevalent genotypeswere HPV 16, 31, 51, and 53. Among these, HPV 16 was consistently present in all cytological subgroups. Twelve HPV genotypes classified as carcinogenic to humans (Group 1) were detected in 77.8.0% of HSIL/ASCH and 55.0% of NILM with abnormal colposcopy findings. Six possible carcinogens-HRs (group 2B) were often found in women with normal cytology (14.8%) and mild abnormalities (ASCUS and LSIL), but with lower frequence in HSIL/ASCH lesions (7.1%). HPVs 6 and 11(Group 3) were not found in the cases of HSIL/ASCH. Unclassified HPV types were equally distributed in all cytology groups: 20.7%, 19.1%, 16.3% and 13% of NILM, ASCUS, LSIL and HSIL/ASCH, respectively. Our findings highlight that majority of abnormal Pap test results are caused by Group 1 HPVs among women from our region. Low frequency HPVs of group 2A/2B, especially HSIL/ASCH, supports the conclusion that individual genotypes require consideration of each type as an individual agent. We expect a positive impact of HPV vaccine in reducing HPV-associated cervical lesions among women from Vojvodina province, after establishing vaccination programs in our country.

Prevalence of oncogenic Human papillomavirus and genetic diversity in the L1 gene of HPV16 HPV 18 HPV31 and HPV33 found in women from Vojvodina Province Serbia.

The main purpose of this paper is to estimate the pre-vaccination prevalence of 12 hrHPV types among 564 women from Vojvodina province (Serbia). The corrected contingency coefficient (Ccorr) was used to estimate the importance of association of examined HPV types and cytological diagnosis. The highest association with the abnormal cytology was observed for HPV 16 (Ccorr = 0.493) in all age groups of participants. The effect of HPV 16 was especially clear within the group of women older than 35 years (Ccorr = 0.691), compared with women younger than 35 (Ccorr = 0.333). The molecular characterization at the level of L1 gene of HPV 16, 18, 31 and 33 variants was for the first time assessed in our region. Nearly all HPV 16 isolates cluster with variant lineage A (96.4%) the remaining isolates clustering with variant lineage D. All of HPV 18 and HPV 33 isolates are clustering within the lineage A while isolates of HPV 31 group with lineages A and C. This contributes to understanding of intrinsic geographical and biological differences of examined HPV types and could be useful for development of cervical cancer screening strategies in Vojvodina (Serbia) and diagnosis of HPV related cervical cancer in general.

West Nile Virus Lineage 2 Associated with Human Case in Republic of Serbia.

A suspicion on West Nile virus (WNV) in Serbia was first reported in 1972 by a seroprevalence study, after which no data were available for four decades. We report full sequence of the isolate obtained for the first time from a human sample in Serbia. The closest clustering was obtained with lineage 2 WNV identified in Greece in 2010. Since WNV lineage 2 emerged in Europe in 2004, a cocirculation of lineages 1 and 2-as observed in Hungary and Italy-cannot be excluded. The reinforcement of surveillance will be required to investigate the possible cocirculation of the two lineages and the burden of WNV in the local population.

West Nile virus 'circulation' in Vojvodina, Serbia: Mosquito, bird, horse and human surveillance.

Efforts to detect West Nile virus (WNV) in the Vojvodina province, northern Serbia, commenced with human and mosquito surveillance in 2005, followed by horse (2009) and wild bird (2012) surveillance. The knowledge obtained regarding WNV circulation, combined with the need for timely detection of virus activity and risk assessment resulted in the implementation of a national surveillance programme integrating mosquito, horse and bird surveillance in 2014. From 2013, the system showed highly satisfactory results in terms of area specificity (the capacity to indicate the spatial distribution of the risk for human cases of West Nile neuroinvasive disease - WNND) and sensitivity to detect virus circulation even at the enzootic level. A small number (n = 50) of Culex pipiens (pipiens and molestus biotypes, and their hybrids) females analysed per trap/night, combined with a high number of specimens in the sample, provided variable results in the early detection capacity at different administrative levels (NUTS2 versus NUTS3). The clustering of infected mosquitoes, horses, birds and human cases of WNND in 2014-2015 was highly significant, following the south-west to north-east direction in Vojvodina (NUTS2 administrative level). Human WNND cases grouped closest with infected mosquitoes in 2014, and with wild birds/mosquitoes in 2015. In 2014, sentinel horses showed better spatial correspondence with human WNND cases than sentinel chickens. Strong correlations were observed between the vector index values and the incidence of human WNND cases recorded at the NUTS2 and NUTS3 levels. From 2010, West Nile virus was detected in mosquitoes sampled at 43 different trap stations across Vojvodina. At 14 stations (32.56%), WNV was detected in two different (consecutive or alternate) years, at 2 stations in 3 different years, and in 1 station during 5 different years. Based on these results, integrated surveillance will be progressively improved to allow evidence-based adoption of preventive public health and mosquito control measures.

TICK-BORNE ENCEPHALITIS VIRUS INFECTION IN HUMANS.

INTRODUCTION: Tick-borne meningoencephalitis virus is a flavivirus that causes the most important vector-borne central nervous system infection in many countries of Europe and Asia. There are three subtypes of tick-borne encephalitis virus: European, Siberian and the Far-Eastern subtype. TRANSMISSION: In endemic areas, the virus remains in transmissive cycles between Ixodes ticks and small rodents. CLINICAL PICTURE: In most cases (70-98%) infection goes asymptomatically. In about one-third of meningitis cases, meningoencephalitis or meningomyelitis is developed. Postencephalytic syndrome may be the complication of the infection, presenting with neurological symptoms. DIAGNOSIS: Etiologic diagnosis of tick-borne meningoencephalitis is only made on basis of laboratory analyses. Reverse transcription-polymerase chain reaction is used for determining the presence of virus in the blood and cerebrospinal fluid. Antibodies in blood and cerebrospinal fluid can be detected by serological tests. PREVENTION: The most efficient way to control this potentially severe disease with possible serious long-term consequences is vaccination. It should be recommended to persons who live or travel to endemic areas. CONCLUSION: In Serbia, tick-borne encephalitis virus infection belongs to the list of reportable diseases; however, there are no reported cases because the diagnostics is not performed routinely. We believe that the significance of this zoonosis must be examined in our country and some of its parts because of preliminary positive serological findings found out in Vojvodina as well as because of reported cases in neighboring countries such as Hungary and Croatia and its worldwide distribution.

Frequency of twelve carcinogenic human papilloma virus types among women from the South Backa region, Vojvodina, Serbia.

BACKGROUND/AIM: The aim of this study was to determine the presence and age distribution of different oncogenic human papilloma virus (HPV) types in women in the South Backa region and its relationship to Pap results. MATERIALS AND METHODS: In a group of 1087 women with normal and abnormal cytology, the commercial HR HPV Real-TM kit (Sacace Biotechnologies, Italy) was used. RESULTS: Overall, 50.5% of the women were HPV positive. The presence of HPV types 18, 31, 51, and 58 was significantly influenced by age, while the presence of HPV types 16 and 45 was significantly influenced by cervical cytology. Results of the LSD test show a wide spectrum of high risk HPV among women with normal cytology and women with a low grade cervical lesion rate (atypical squamous cell of undetermined significance (ASCUS) and low grade squamous intraepithelial lesions (LSIL). The most prevalent HPV types found were 16, 31, 51, 18, and 52. In the HSIL group the most prevalent HPV types were 16 and 45. CONCLUSION: The reported results provide new data on the circulation of oncogenic HPV genotypes and frequency of multiple infections among women in Vojvodina and suggest that a prophylactic vaccine against HPV 16 and 18 has the potential to prevent approximately half of the high-grade lesions.

Testing for viral material in water of public bathing areas of the Danube during summer, Vojvodina, Serbia, 2014.

From August to September 2014 a water quality study was conducted on five popular public Danube beaches in Vojvodina, Serbia. To assess the safety of Danube water for bathing, physical, chemical, bacteriological tests were performed. While many parameters for monitoring the quality of water are regulated by law, there are neither national nor international legislations addressing the presence of viruses in recreational waters. In this study, we performed analyses that surpassed national requirements, and investigated if adenovirus, enterovirus or rotavirus genetic material was present in samples of recreational water collected for quality monitoring. Of 90 water samples obtained during the study, enterovirus material was not found in any sample, but adenovirus and rotavirus genetic materials were respectively detected in 60 and 31 samples. Statistical analyses showed a significant correlation between adenovirus DNA and total coliforms in the water. Even when water samples were adequate for recreational use, adenoviruses were detected in 75% (57/76) of such samples. Our results indicate that implementation of viral indicators in recreational water might be helpful to better assess public health safety. This might be particularly relevant in areas where urban wastewater treatment is insufficient and surface waters affected by wastewater are used for recreation.

Risk factors for severe influenza A virus infections in post-2009 pandemic period.

Introduction: Literature data concerning risk factors for severe influenza in post-2009 pandemic period, from low- and middle-income Central and Eastern European countries are very limited. Objective: The aim of this study was to investigate the risk factors for severe A(H1N1)pdm09 and A(H3N2) influenza during the post-2009 pandemic period. Methods: During four consecutive seasons of 2010/2011­2013/2014, nasopharyngeal or nasal and pharyngeal swab samples from 153 patients with mild and 147 patients with severe influenza were tested using real-time reverse transcription polymerase chain reaction (real-time RT PCR) assays. Results: The study indicated three statistically significant risk factors of influenza severity, including presence of chronic underlying illness/condition [odds ratio (OR) of 15.2, 95% confidence interval (CI) of 1.8­125.4, p = 0.001), age ≥15 years (OR 9.2, 95% CI 3.5­24.1, p < 0.001), and delay in medical care of more than two days after the symptoms onset (OR 3.2, 95% CI 1.6­6.4, p = 0.001). Conclusion: Obtained results confirmed that patients with chronic underlying illness/condition and older than 15 years had the highest risk for serious complications from influenza and highlighted the importance of start of antiviral therapy within the first two days of illness in order to reduce the risk for the most severe outcomes of influenza, such as acute respiratory distress syndrome and lethal outcome.

Influenza B Viruses in the Population of Province of Vojvodina during the 2012/2013 Season: Differentiation of B/Yamagata and B/Victoria Lineages by Real-time RT-PCR, Antigenic and Phylogenetic Characterization.

INTRODUCTION: At present, two phylogenetically distinct influenza B virus lineages, B/Yamagata and B/ Victoria, co-circulate worldwide and can cause significant morbidity and mortality. OBJECTIVE: The aim of this study was to determine the prevalences of two influenza B virus lineages in the population of Vojvodina and to identify their antigenic and phylogenetic properties. METHODS: A total of 369 and 334 nasopharyngeal, or nasal/throat swab samples, collected during the 2012/2013 and 2013/2014 seasons, respectively, were tested using specific singleplex influenza A, influenza B, influenza B/Yamagata and influenza B/Victoria real-time reverse transcription polymerase chain reaction (RT-PCR) assays. Antigenic and genetic testing were done by hemagglutination inhibition assay and hemagglutinin and neuraminidase gene sequence analysis, respectively. RESULTS: During the 2012/2013 season, influenza B viruses were present in 53.4% (101/189) of influenza positive samples.The B/Yamagata-like viruses (81.2%) significantly predominated over the B/Victoria-like viruses (18.8%). Comparing to B/Victoria-like positive patients, among B/Yamagata-like positive patients, children 5-14 years of age were significantly more represented (5.3% vs. 35.4%, respectively), as well as patients with mild form of illness (15.8% vs. 45.1%, respectively). The results of sequence analysis and antigenic testing showed that tested viruses were not closely related to B/Wisconsin/1/2010, the vaccine virus for 2012/2013. During the 2013/2014 season influenza B viruses were not detected. CONCLUSION: The results of this study confirmed the health significance of influenza B viruses and indicated that B/Yamagata-like viruses were significantly more prevalent than B/Victoria lineage viruses, during the 2012/2013 season. They also showed a sub-optimal match between the tested viruses and the vaccine virus for season 2012/2013.

Detection of enteroviruses in clinical samples of patients with aseptic meningitis by rapid antigen detection assay.

INTRODUCTION: Human enteroviruses represent the most common etiological agents of aseptic meningitis. Rapid diagnosis of aseptic meningitis affects the management of patients. OBJECTIVE: The aim of this work was to assess the efficacy of rapid antigen detection (RAD) assay for the confirmation of human enteroviruses in comparison to that of the conventional cell culture (CCC), and to identify the serotypes associated with aseptic meningitis cases in the Autonomous Province of Vojvodina. METHODS: RAD assay was carried out using centrifugation of inoculated Vero, RD and HEp-2 cell cultures and indirect immunofluorescence with Pan-Enterovirus 2E11 reagent (Millipore-Chemicon). CCC was performed using the same type of cells and the same immunofluorescence reagent for enterovirus confirmation. RESULTS: Out of 70 analyzed samples (29 cerebrospinal fluid specimens, 27 stool specimens, 9 rectal and 5 throat swabs), 36 (51.4%) were positive by immunofluorescence after CCC and 34 (48.6%) by the RAD assay. The sensitivity of RAD assay was 94.4% and specificity 100%. Detection time of enteroviruses by the RAD assay was 3 days, and by CCC varied from 3 to 13 days (mean time 6.1 days). Within 72 h from inoculation, a cytopathic effect (CPE) occurred in only 19 (47.5%) of 40 CPE positive samples. Serotyping revealed 11 types of enteroviruses: coxsackievirus A16, B3, B4, B5, and echovirus 2, 4, 6, 11, 13, 16 and 30. CONCLUSION: The RAD assay was slightly less sensitive than CCC and significantly shortened the detection time of enteroviruses, so it may be useful in rapid diagnosis of enteroviral meningitis.

[Avian influenza viruses--new causative agents of human infections].

INTRODUCTION: Influenza A viruses can infect humans, some mammals and especially birds. Subtypes of human influenza A viruses: A(H1N1), A(H2N2) and A(H3N2) have caused pandemics. Avian influenza viruses vary owing to their 15 hemagglutinins (H) and 9 neuraminidases (N). HUMAN CASES OF AVIAN INFLUENZA A: In the Netherlands in 2003, there were 83 human cases of influenza A (H7N7). In 1997, 18 cases of H5N1 influenza A, of whom 6 died, were found among residents of Hong Kong. In 2004, 34 human cases (23 deaths) were reported in Viet Nam and Thailand. H5N1 virus-infected patients presented with fever and respiratory symptoms. Complications included respiratory distress syndrome, renal failure, liver dysfunction and hematologic disorders. Since 1999, 7 cases of human influenza H9N2 infection have been identified in China and Hong Kong. THE IMPORTANCE OF HUMAN INFECTION WITH AVIAN INFLUENZA VIRUSES: H5N1 virus can directly infect humans. Genetic reassortment of human and avian influenza viruses may occur in humans co-infected with current human A(H1N1) or A(H3N2) subtypes and avian influenza viruses. The result would be a new influenza virus with pandemic potential. All genes of H5N1 viruses isolated from humans are of avian origin. PREVENTION AND CONTROL: The reassortant virus containing H and N from avian and the remaining proteins from human influenza viruses will probably be used as a vaccine strain. The most important control measures are rapid destruction of all infected or exposed birds and rigorous disinfection of farms. Individuals exposed to suspected animals should receive prophylactic treatment with antivirals and annual vaccination.