Detection of the first appearance of SARS-CoV-2 virus in Hungary based on retrospective testing of respiratory samples / A SARS-CoV-2 vírus magyarországi megjelenésének kimutatása légúti minták retrospektív vizsgálata alapján.

INTRODUCTION: In Hungary, SARS-CoV-2 was first detected in the swab samples of two Iranian patients on March 4, 2020. After finding the first positive cases, the question arose whether the virus had entered Hungary and caused infections before this date. Before March 4, 2020, except for the two above-mentioned samples, none of the 224 swab samples received specifically for SARS-CoV-2 tested positive. AIM: The National Reference Laboratory for Respiratory Viruses of the National Public Health Center aimed to carry out a retrospective study of the swab and other samples taken for testing respiratory virus infections between January 1, and April 19, 2020 sent by sentinel physicians within the influenza surveillance for diagnostic purposes. METHOD: For the study, we used swab samples taken weekly by sentinel physicians of the influenza surveillance service, and other samples received for diagnostic purposes. Tests were performed using real-time PCR. RESULTS: All the 465 swab samples sent by sentinel physicians were found to be SARS-CoV-2 negative. Also, of the 551 samples collected for diagnostic reasons of other respiratory viruses, no SARS-CoV-2 positive was found among those taken before March 4. CONCLUSION: Based on our data, it is very likely that prior to the first cases diagnosed on March 4, 2020, SARS-CoV-2 did not cause clinically symptomatic infections in Hungary. Orv Hetil. 2020; 161(38): 1619-1622.

Extraordinary increase in West Nile virus cases and first confirmed human Usutu virus infection in Hungary, 2018.

BackgroundDuring the 2018 WNV transmission season, similarly to other endemic areas in Europe, a large number of human West Nile virus (WNV) infections were reported in Hungary.AimsWe summarise the epidemiological and laboratory findings of the 2018 transmission season and expand experiences in flavivirus differential diagnostics.MethodsEvery patient with clinical suspicion of acute WNV infection was in parallel tested for WNV, tick-borne encephalitis virus and Usutu virus (USUV) by serological methods. Sera, whole blood and urine samples were also tested for the presence of viral nucleic acid.ResultsUntil the end of December 2018, 215 locally acquired and 10 imported human WNV infections were notified in Hungary. All reported cases were symptomatic; most of them exhibited neurological symptoms. In a large proportion of tested individuals, whole blood was the most appropriate sample type for viral nucleic acid detection, but because whole blood samples were not always available, testing of urine samples also extended diagnostic possibilities. In addition, the first human USUV infection was confirmed in 2018 in a patient with aseptic meningitis. Serological cross-reactions with WNV in different serological assays were experienced, but subsequent molecular biological testing and sequence analysis identified Europe lineage 2 USUV infection.ConclusionCareful interpretation and simultaneous application of different laboratory methods are necessary to avoid misdiagnosis of human USUV cases. Expansion of the laboratory-confirmed case definition criteria for detection of viral RNA in any clinical specimens to include urine samples could increase diagnostic sensitivity.

Comparative study of antibody levels developed by vaccination against polio virus in population after vaccine type alteration.

During clinical trials, samples from Hungarian patients of different age groups were tested for antibodies against all 3 serotypes of poliovirus, a member of Picornaviridae family. During the virus neutralization serological test, blood samples were titrated using permanent virus concentration. Based on the cythopathic effect observed under a light microscope, the antibody level of the patient was assessed. The 100 people examined were classified into 5 groups based on age and type of original vaccine: I. Newborns, no vaccination given; II. Immunosuppressed patients; III. Born before 1986, received only OPV vaccine; IV. Born between 1992-2005, received a combination of OPV and IPV vaccines; V. Born after 2006, received only IPV vaccine. Results show that vaccination coverage meets all the criteria. None of the immunized persons was seronegative to all three polioviruses. Both IPV and OPV vaccines are effective against poliovirus. Blood samples from newborn babies with no immunization were also examined. Results show that most newborns have maternal antibodies in their blood. Results of group II show that immunosuppression does not have a negative influence on blood antibody levels against polioviruses. In spite of the low number of samples, our results show that seroconversion after immunization in the Hungarian population is adequate. For more accurate results about vaccination coverage in the population, further trials would be necessary.

Detection of enteric viruses in Hungarian surface waters: first steps towards environmental surveillance.

Waterborne viruses infect the human population through the consumption of contaminated drinking water and by direct contact with polluted surface water during recreational activity. Although water related viral outbreaks are a major public health concern, virus detection is not a part of the water quality monitoring scheme, mainly due to the absence of routine analysis methods. In the present study, we implemented various approaches for water concentration and virus detection, and tested on Hungarian surface water samples. Eighty samples were collected from 16 sites in Hungary. Samples were concentrated by glass wool and membrane filtration. Human adenoviruses were detected by conventional and quantitative real-time polymerase chain reaction (PCR) methods in 56% (45/80) of the samples; viral titers ranged from 8.60 × 10(1) to 3.91 × 10(4) genome copies per liter. Noroviruses and enteroviruses were detected in 30% (24/80) and 13% (10/80) of samples, respectively, by reverse transcription-PCR assays. Results indicate a high prevalence of viral human pathogens in surface waters, suggesting the necessity of a detailed survey focusing on the quality of natural bathing waters and drinking water sources.

Hand hygiene perception among health care workers in Hungarian hospitals: prior to a nationwide microbiological survey.

Transmission of pathogens via healthcare workers' (HCWs) hands is one of the most frequent means of spreading multi-resistant organisms and occurring healthcare-associated infections (HAIs) in hospitals. The role of contaminated hands in pathogen transmission was recognized by Hungarian physician, Ignác Semmelweis. Hand hygiene prevents cross-infections in hospitals, but numerous epidemiological and microbiology-based studies have documented low compliance of HCWs with this simple procedure. Furthermore, hand hygiene perception of HCWs plays an important role in determining hand hygiene compliance. Our aim was to describe the opinion of HCWs about their perception regarding hand hygiene practice. Our further goal was to strengthen a laboratory basis for bacterial backup control of nosocomial pathogens. A cross-sectional descriptive study was conducted between December 2010 and February 2011 in 13 participating hospitals in Hungary. HCWs know that there is correlation between contaminated hands and HAIs (83%), but neither the frequency (62%) nor the implementation (73%) of their hand hygiene performance are satisfying.We recommend that multimodal interventions - highlighted active microbiological surveillance of HCWs' hands - are the most suitable strategies to reduce the occurrence of HAIs and to determine their impact on cross-transmission of microorganisms and to overcome barriers of HCWs.

Surveillance of human rotaviruses in 2007-2011, Hungary: exploring the genetic relatedness between vaccine and field strains.

BACKGROUND: The availability of rotavirus vaccines has resulted in an intensification of post vaccine strain surveillance efforts worldwide to gain information on the impact of vaccines on prevalence of circulating rotavirus strains. OBJECTIVES: In this study, the distribution of human rotavirus G and P types in Hungary is reported. In addition, the VP4 and VP7 genes of G1P[8] strains were sequenced to monitor if vaccine-derived strains were introduced and/or some strains/lineages were selected against. STUDY DESIGN: The study was conducted in 8 geographic areas of Hungary between 2007 and 2011. Rotavirus positive stool samples were collected from diarrheic patients mostly <5 years of age. Viral RNA was amplified by multiplex genotyping RT-PCR assay, targeting the medically most important G and P types. When needed, sequencing of the VP7 and VP4 genes was performed. RESULTS: In total, 2380 strains were genotyped. During the 5-year surveillance we observed the dominating prevalence of genotype G1P[8] (44.87%) strains, followed by G4P[8] (23.4%), G2P[4] (14.75%) and G9P[8] (6.81%) genotypes. Uncommon strains were identified in a low percentage of samples (4.12%). Phylogenetic analysis of 318 G1P[8] strains identified 55 strains similar to the Rotarix strain (nt sequence identities; VP7, up to 97.9%; VP4, up to 98.5%) although their vaccine origin was unlikely. CONCLUSIONS: Current vaccines would have protected against the majority of identified rotavirus genotypes. A better understanding of the potential long-term effect of vaccine use on epidemiology and evolutionary dynamics of co-circulating wild type strains requires continuous strain surveillance.

Are we protected? Imported measles - on the way to eradication.

In accordance with the 2015 regional goal for measles and rubella elimination of the WHO European Region, only a few imported cases have been documented of both diseases in Hungary for years.This paper presents a case of a Hungarian woman, born in 1975, who received measles vaccination at age of 12 months and later at age of 11 years, according to her certificate of vaccination. In 2009, after arriving home from a vacation in Ireland, she developed acute measles infection with clinical symptoms. It was confirmed by the detection of measles specific IgM, IgA and IgG antibodies, and by detection of viral nucleic acid from throat swab in virus transport medium.Additionally, an outbreak occurred in December of 2011 among a family emigrated from Romania to Hungary. No new measles cases were diagnosed among the contact persons of neither the young Hungarian woman returning from Ireland, nor the family emigrated from Romania. This observation refers to the effectiveness of the Hungarian vaccination program.

Experimental infection of goats with tick-borne encephalitis virus and the possibilities to prevent virus transmission by raw goat milk.

OBJECTIVES: The aim of this work was to study the tick-borne encephalitis virus (TBEV) infection of goats and the possibilities to prevent human milk-borne infections either by immunizing animals or the heat treatment of milk. METHODS: An experiment was conducted with 20 milking goats. Ten goats (half of them immunized) were challenged with live TBEV and 10 were left uninfected. Clinical signs and body temperatures of the animals were recorded and milk samples were collected daily. The presence of viral RNA and infectious virions in milk were detected by RT-PCR and intracerebral inoculation of suckling mice, respectively. Milk samples containing infectious virions were subjected to various heat treatment conditions and retested afterwards to assess the effect on infectivity. RESULTS: The infected goats did not show any clinical signs or fever compared to uninfected ones. Infectious virions were detected for 8-19 days from the milk samples (genome for 3-18 days by PCR) of infected goats. Immunized goats did not shed the virus. After heat treatment of the milk, the inoculated mice survived. CONCLUSIONS: Goats shed the virus with their milk without showing any symptoms. Human milk-borne infections can be avoided both by immunizing goats and boiling/pasteurizing infected milk.

Serologic evidence of West Nile virus infection in patients with exanthema in Hungary.

The presence of WNV in Europe has been well known for decades, although the first human infections and avian outbreaks were diagnosed in Hungary only in 2003. An annual average of 6-8 cases of the neuroinvasive form of WNV infection has been detected in the region since then, but a higher number (17) of WNV associated neuroinvasive disease occurred in 2008. In 2004, a surveillance system was established for monitoring WNV-associated meningo-encephalitis cases in Hungary, but a milder type of illness (with fever, rash and/or influenza like symptoms) is not followed. Fifty-two sera of 45 patients with mild clinical symptoms (fever, exanthema) were tested for anti-WNV antibodies in 2008 in a retrospective study by immunofluorescence test and ELISA. Seven patients had antibodies against WNV, serologic evidence of recent WNV infection was found in 4 out of the 7 patients. Infections could be acquired predominantly in August and in September, which seems to be a risk period for WNV in Hungary. The possibility of a recent WNV infection should be taken into consideration in the occurrence of fever and rush at late summer. Differential diagnosis of exanthematous patients should include WNV serology tests and should be done routinely.

Interference among viruses circulating and administered in Hungary from 1931 to 2008.

Viral interference was discovered about 60 years ago. Molecular epidemiology revealed that this phenomenon possesses important biological implications, it can reduce the epidemic spread of certain viruses from time to time (influenza and enteroviruses) and the efficiency of live vaccination can be impaired, too. Phenomena observed during the last 80 years in Hungary are analyzed. It is suggested to concentrate the distribution of MMR vaccines to seasons of limited influenza and enterovirus circulation. Interference seems to impair the progress of wild poliovirus eradication in the endemic tropical countries. It is recommended to enhance enterovirus surveillance in the region of European countries, since the exchange of the oral poliovirus vaccine to the enhanced inactivated polio vaccine might result in enhanced circulation of non-polio enteroviruses leading to the increase in the number of type I (juvenile) diabetes patients.

Detection of non-polio enteroviruses in Hungary 2000-2008 and molecular epidemiology of enterovirus 71, coxsackievirus A16, and echovirus 30.

Human enteroviruses are associated with various clinical syndromes from minor febrile illness to severe, potentially fatal conditions like aseptic meningitis, paralysis, myocarditis, and neonatal enteroviral sepsis. Between June 2000 and August 2008 echovirus (E) type 2, 4, 6, 7, 9, 11, 13, 25, 30, coxsackievirus (CV) -A16, -A19, -B5, and enterovirus 71 (EV71) were reported in Hungary. In this study, 29 previously enterovirus positive samples from 28 patients diagnosed with hand, foot and mouth disease, meningitis and encephalitis, were molecularly typed. The genetic relationships of identified serotypes CV-A16, EV71, and E30 were assessed by direct sequencing of genomic region encoding the capsid protein VP1. The sequences were compared to each other and sequences from other geographical regions possessed in Genbank. The phylogenetic analysis of CV-A16 revealed that the viruses were mostly of Far-Eastern or Asia-Pacific origin. Typing of EV71 showed that one virus from 2000 belonged to genotype C1 and five viruses observed in 2004 and 2005 were identified as genotype C4. The 11 echovirus 30 strains showed homology with those of neighbor European countries. The molecular examination of E30 revealed that three separate lineages circulated in 2000, 2001, and 2004-2006 in Hungary.

Molecular characterization of poliovirus isolates from children who contracted vaccine-associated paralytic poliomyelitis (VAPP) following administration of monovalent type 3 oral poliovirus vaccine in the 1960s in Hungary.

Hungarian children were immunized with monovalent oral poliovaccine (mOPV) delivered at 6-week intervals in the order Sabin 1, Sabin 3, Sabin 2, from 1959 until 1992. During that period, 90 cases of vaccine-associated paralytic poliomyelitis (VAPP) were reported, 52 of which were associated with Sabin 3-related virus (76% of VAPP cases with virologic data). Because of renewed interest in type 3 mOPV (mOPV3), molecular methods were used to reanalyze 18 of the Sabin 3-related isolates from 15 VAPP patients, confirming the original identification. All isolates had the U472C 5'-untranslated region (5'-UTR) substitution associated with reversion to neurovirulence, and from zero to seven nucleotide substitutions in the virus protein 1 (VP1) region. No evidence was found for prolonged mOPV3 replication in the VAPP patients or for spread of Sabin 3-related viruses beyond close vaccinee contacts. The VAPP diseases were prevented by a single dose of inactivated poliovirus vaccine from 1992 to 2006 in Hungary, as proved by continuous surveillance of acute flaccid paralysis.

Tick-borne encephalitis outbreak in Hungary due to consumption of raw goat milk.

A tick-borne encephalitis outbreak involving 25 patients of 154 exposed persons occurred in Hungary in August 2007. None of the patients had a history of tick-bite, however all of them drank unpasteurized raw goat milk from the same farm. The aim of this study was to identify the goats on the farm which could have spread the infection through their milk. Blood samples were taken from 75 goats on the farm and were examined by various serological methods, namely indirect immunofluorescent assay, hemagglutination inhibition, microneutralization and an ELISA adapted to testing material from goats, to determine antibody levels in the serum. The four methods have proved different levels of specificity. The least specific was the indirect immunofluorescent assay, which showed a low titre in all sera. Comparison of the results of the other three methods indicates that two sera were positive for anti-TBEV IgG and one for anti-TBEV IgM. The goat with the IgM positive serum sample could have been a source of the infected milk. It has been concluded that serological results for goats by the different methods should be compared before final diagnosis because the specificity of methods in use can differ significantly.

Heterogeneous pathways of maternal-fetal transmission of human viruses (review).

Several viruses can pass the maternal-fetal barrier, and cause diseases of the fetus or the newborn. Recently, however, it became obvious, that viruses may invade fetal cells and organs through different routes without acute consequences. Spermatozoa, seminal fluid and lymphocytes in the sperm may transfer viruses into the human zygotes. Viruses were shown to be integrated into human chromosomes and transferred into fetal tissues. The regular maternal-fetal transport of maternal cells has also been discovered. This transport might implicate that lymphotropic viruses can be released into the fetal organs following cellular invasion. It has been shown that many viruses may replicate in human trophoblasts and syncytiotrophoblast cells thus passing the barrier of the maternal-fetal interface. The transport of viral immunocomplexes had also been suggested, and the possibility has been put forward that even anti-idiotypes mimicking viral epitopes might be transferred by natural mechanisms into the fetal plasma, in spite of the selective mechanisms of apical to basolateral transcytosis in syncytiotrophoblast and basolateral to apical transcytosis in fetal capillary endothelium. The mechanisms of maternal-fetal transcytosis seem to be different of those observed in differentiated cells and tissue cultures. Membrane fusion and lipid rafts of high cholesterol content are probably the main requirements of fetal transcytosis. The long term presence of viruses in fetal tissues and their interactions with the fetal immune system might result in post partum consequences as far as increased risk of the development of malignancies and chronic pathologic conditions are discussed.

Detection of human bocavirus from fecal samples of Hungarian children with acute gastroenteritis.

OBJECTIVES: Human bocavirus (HBoV), a newly identified member of the Parvoviridae family is associated with respiratory tract and gastroenteric infections, mostly of young children. HBoV infections show a seasonal distribution with the peak in temperate areas being in the winter months. METHODS: In our study, 35 throat swabs from children under 5 years with acute respiratory symptoms and 61 stool samples from children (<5 years) with acute gastroenteritis were collected in the period of October 2007-March 2008. A HBoV-specific polymerase chain reaction for detection of the virus, and sequence analysis for identification of virus variants were performed. RESULTS: Although respiratory samples were all negative, 3.3% of stool samples (2/61) proved to be positive for HBoV. The virus carrier children were 3 and 5 years old. The ratio of HBoV positive samples is similar to international results (2.1-5.5%). CONCLUSIONS: According to the result of sequence analysis of HBoV, the occurrence of genotype 2 of HBoV in Hungary is confirmed.

Mutation spectra of the surface-protein-coding region of the HBV genome in HBV-vaccinated and non-vaccinated individuals in Hungary.

Hepatitis B virus (HBV) infection has a major effect on health care systems, with about one-third of the world's population currently infected with the virus. There is an effective vaccine against HBV, which contains a recombinant "surface antigen" produced in an expression vector. Vaccination has proved to be successful in Hungary: the number of acute HBV cases has decreased in the past 10 years. Although an increasing number of publications report on "vaccine-escape" HBV variants which can infect HBV-vaccinated individuals, such mutant HBV strains have not yet been detected in Hungary. We therefore surveyed two risk groups for vaccine-escape or immunoglobulin-escape HBV mutations in Hungary: 28 actively and/or passively HBV-immunized children of HBV carrier mothers who proved to be HBsAg and/or anti-HBc positive and 40 symptomless HBV carrier pregnant women (presumably carrying genotype B or C). We focused on the coding sequences of the "a" immundominant region of the surface protein. We could not detect the G145R amino acid substitution associated with vaccine escape mutant virus. However, we could map other mutations potentially affecting the immunodominant "a" region of the HBV surface protein.

First description of swine Torque teno virus (TTV) and detection of a new genogroup in Hungary: short communication.

Torque teno virus (TTV) belongs to the floating genus of Anellovirus. It was discovered in a human patient, and later it was also found in animals including pigs. The aim of this study was to investigate the presence and estimate the prevalence of swine TTV in Hungarian pig herds for the first time, and to characterise the viruses found. Serum samples of 82 adult swine from 13 piggeries and 44 weaned pigs from one large herd were tested by PCR for the presence of TTV DNA. Viral DNA was found in 30% of the adult swine and 73% of the weaned pigs tested. Liver and intestine of weaned pigs were also tested and found to be infected at a lower rate. The TTV sequences found in sera and intestines were similar and could be clustered as swine genogroup 1. However, the sequences derived from one liver were remarkably different from all other known genogroups and seemed to represent a new genogroup.

TT virus in Hungary: sequence heterogeneity and mixed infections.

The majority of the viral hepatitis cases is caused by five hepatitis viruses (A,B,C,D,E). In 1997, TT virus was discovered. It was supposed that a number of the unknown hepatitis cases was caused by the TT virus. The aim of this study was to characterize TT viruses carried by healthy individuals and patients suffering from hepatitis of unknown origin in Hungary. TTV DNA was detected by seminested PCR with the commonly used N22 primers. Twenty of the 108 sera (18.5%) taken from healthy persons and 115 of the 228 sera (50.4%) of patients with hepatitis of unknown origin were found to be positive. The nucleotide sequences of 26 clones derived from 17 hepatitis patients and 15 clones from nine healthy persons were determined and a phylogenetic tree was constructed. Genotype 2 (group 1) was found to be the most frequent, but other group 1 genotypes (1, 6) and genotypes 8 and 17 of group 2 were also detected. Mixed TTV infections were found in eight cases (two healthy persons and six hepatitis patients). Variants belonging to the same group were carried in seven cases, and the presence of group 1 (genotype 2) and group 2 (genotype 8) TTV sequences were found in one single hepatitis patient.

Prevalence of GB virus C/hepatitis G virus in Hungary.

In 1995 a new flavivirus, GB virus C/hepatitis G virus (GBV-C/HGV), was discovered. The aim of this study was to determine the prevalence of the virus in healthy persons and hepatitis patients in Hungary. The sera of 408 healthy persons older than 60 years were tested for the presence of GBV-C/HGV antibodies, and 113 were positive (28%). Eight of the 71 healthy persons younger than 60 years and twenty of the 51 sera (39%) taken from patients suffering from hepatitis of unknown origin proved to be positive for GBV-C/HGV antibodies. Ten of the 124 sera (8%) of healthy persons and 36 of the 247 sera (14.6%) of hepatitis patients proved to be positive for GBV-C/HGV RNA. Eleven PCR products were sequenced, and the sequences were found to be different from each other and from the previously published ones. However, three sequences taken from the same patient at different times were identical. These results show that GBV-C/HGV is present in Hungary and cannot be considered rare.