First evaluation of completeness and sensitivity of the measles surveillance system in the Czech Republic, January 1, 2018 until June 30, 2019.

AIM: The aim of study was to evaluate completeness and estimate sensitivity of the measles surveillance using the new electronic version of the national notification system of infectious diseases (ISIN) in order to assess its performance. MATERIAL AND METHODS: The completeness of measles reporting in the ISIN for demographic characteristics (week and region of reporting, age and gender), date of onset, complications, hospitalisations, vaccination status, used laboratory methods and country of import from January 2018 to June 2019 was assessed. The register from National Reference Laboratory (NRL) and the ISIN were compared using the capture-recapture method (CRM). Cases were matched using unique personal identifier. The total number of measles cases in the population was assessed using the Chapmans formula. Sensitivity of reporting was calculated by dividing the number of reported cases by the CRM estimated true number of cases. RESULTS: In the ISIN, 765 measles cases were registered within specified time period. For many variables 100% completeness was found. The data were missing mainly for vaccination status (20%), serology results (55%) and used laboratory methods (8%). The NRL confirmed 653 patient samples in respected period. Within both registries (ISIN and NRL) the total 612 cases were matched. Estimated real number of measles cases using the CRM was 816 (95% CI: 809-823) compared to 806 reported cases. The estimated surveillance system sensitivity was 98.8%. Five percent (n = 41) of cases tested positively in the NRL were not reported to the ISIN. CONCLUSIONS: We found high level of reported measles data completeness in the ISIN for most variables. Estimated real and reported number of cases was in a good correlation and calculated sensitivity of the ISIN was on very high level. Though, the data sources used in the study were not independent on each other, therefore results may not be fully accurate. The technical changes (more mandatory fields and more logical syntax to check data) in the ISIN to improve data completeness are being recommended. Data providers should report all measles cases to the ISIN with maximum precision in entering individual variables and investigating laboratories should send samples for confirmation to the NRL in required cases.

Measles outbreaks in 2017-2019 - molecular surveillance started in the Czech Republic.

OBJECTIVE: Between 2017 and 2019, measles virus spread globally, causing a large measles epidemic that suddenly ended in 2020. Measles outbreaks also occurred in the Czech Republic (CR) as part of the global public health problem. In the recent alarming epidemiological situation, molecular surveillance is becoming increasingly important as it plays a vital role in the identification of imported cases and in the monitoring of virus transmission. Molecular surveillance makes it possible to obtain evidence of the discontinuation of the endemic spread and is indispensable for the verification of measles elimination. The study aim is to find out whether any of measles virus genotypes circulated in the CR for more than 12 months in order to either confirm or refute the endemic spread of measles virus in the country in relation to the recent loss of the measles elimination status. Another aim is to assess the current laboratory diagnosis from the perspective of recent measles outbreaks and the obligation to refer samples for confirmation and genotyping. MATERIAL AND METHODS: In total, 243 positive nasopharyngeal swabs collected from outbreak patients from all over the CR in 2018 and 2019 were analysed by molecular methods. The most variable part of the measles virus genome, the nucleoprotein gene (N-450), was sequenced according to the WHO protocol. The sequence analysis was performed by Sanger method using the Applied Biosystems 3 500 sequencer, and sequence data were analysed by the bioinformatics programe Geneious. RESULTS: In the CR, only two genotypes were found in measles outbreaks in 2018-2019, eight variants of the dominant D8 and six B3 variants, while genotype A was detected in eight samples. The dominant genotype of 2017 (D8, 4283) was identified for the first time in the CR in January 2018. Four months later, it was replaced by genotype D8, 4683, occurring in the CR from March 2018 to June 2019. This genotype was identified in 170 of 243 samples (70%). There was a 3-month window between the first and the second detection of this genotype, which does not imply that in the meantime the virus did not circulate in the population. The analysis of seven samples from 2017 conducted by the collaborating Regional Reference Laboratory at the Robert Koch Institute (RRL RKI) in Berlin assigned five samples from Ostrava to genotype B3 and detected two variants of genotype D8 (Praha, Liberec). Laboratory diagnosis was facilitated by a higher proportion of clinical specimens available for direct detection of the virus, which increased from 18% in 2017 to 43% in 2019. Samples were referred to the National Reference Laboratory (NRL) in Prague for sequencing in accordance with the set legal rules. Between 2018 - 2019, laboratories sent 424 samples. Two hundred and forty-three samples (60%) were successfully sequenced, while the sequencing of the remaining samples failed due to low viral load. CONCLUSIONS: Measles virus sequencing was introduced in the Czech Republic as a necessary part of molecular surveillance, and almost 60% of positive samples were analysed. The sequencing analysis confirmed the endemic spread of measles virus, with genotype D8, 4683 MVs/GirSomnath.IND/42.16 found to circulate in the CR for 16 months between 2018 and 2019. Laboratory diagnosis is recently focusing more on direct detection of the virus, which along with genotyping extended to include another part of the genome will improve molecular surveillance.

COVID-19 reinfections.

Reports of SARS-CoV-2 reinfections are on the rise. This study focused on reinfections in patients with confirmed COVID-19 in the Czech Republic. Between 1 March 2020 and 9 November 2020, 362 084 cases with the onset of symptoms before 31 October 2020 were reported. Overall, 28 cases of symptomatic SARS-CoV-2 reinfections were identified, 11 in males and 17 in females, age range 25-80 years, median age 46 years. The interval between the first and second episodes of the disease ranged from 101 to 231 days, and the median interval was 201.5 days. During both symptomatic episodes, all patients have been tested by RT-PCR. Altogether 26 patients (92.9%) have been tested negative after recovery from the first episode of COVID-19. Symptomatic reinfections occurred in nearly 0.2% of all patients at risk. Most patients with reinfection had mild symptoms in both episodes, and only three episodes were moderate to severe. Thus, reinfections may have been underdiagnosed. In summary, COVID-19 reinfections are possible and not exceptional.

[The benefit from mumps virus IgG antibody avidity testing in the population with high vaccine coverage in the context of other serological methods for laboratory diagnosis of mumps and the current epidemiological]. / Prínos avidity IgG protilátek u príusnic ve vysoce proockované populaci v kontextu s ostatními sérologickými metodami laboratorní diagnostiky príusnic a soucasnou epidemiologickou situací.

AIM: Regular vaccination against mumps resulted in a significant reduction in epidemic mumps in the Czech Republic. However, mumps cases have recently shown an upward trend, even in the vaccinated population where a considerable proportion of cases have occurred. The aim of this study was to find out, by mumps virus IgG antibody avidity testing, whether the high incidence of mumps in the vaccinated population is a result of primary or secondary vaccine failure and whether the vaccinated differ from the naturally immunised in anamnestic antibody avidity. Given the problematic laboratory diagnosis of mumps in the population with high vaccination coverage, the informative value of the detected IgM, IgA, and IgG antibodies was also considered as well as the potential of antibody avidity testing for improving laboratory diagnosis from a single sample of blood, the most commonly analysed clinical material, in patients with suspected mumps. MATERIAL AND METHODS: Sixty-four patients laboratory confirmed with mumps, whose vaccination status was known, were included in the study (groups 1 and 2). Other study groups were 30 healthy naturally immunised subjects (group 3) and 22 vaccinated children 2-4-years of age with no etiological link to the mumps virus (group 4). The avidity index (AI) was determined using the Siemens Enzygnost Anti-Mumps/IgG kit and 6M urea, able to induce the dissociation of antigen-antibody bonds proportionally to the antibody avidity. IgM, IgG, and IgA antibodies were tested using the Siemens Enzygnost Anti-Mumps/IgM and /IgG, and Mast Diagnostica Mastazyme Mumps IgA kits. The EPIDAT system served as the data source. RESULTS: The results showed that the mumps virus induces antibodies with a low AI after both vaccination, even recent, and natural immunisation. Antibodies with a high AI were only detected in convalescent sera of the vaccinated patients or in re-infected, naturally immunised persons, as a result of recent contact with the mumps virus. The comparison of the results of acute sera testing revealed that in the vaccinated patients, 56% of cases were laboratory confirmed based on IgA positivity, i.e. 20% more cases in comparison with routine detection of IgM antibodies, while of unvaccinated cases, 87% were IgA positive and 74% IgM positive. CONCLUSION: The results of mumps virus IgG antibody avidity testing suggest that the high proportion of cases in the vaccinated patients result from secondary vaccine failure, also known as waning immunity. Diagnostic benefit from antibody avidity testing has been observed in convalescent sera and/or acute sera from both vaccinated and naturally immunised patients collected from day 6 after the onset of the disease when significant increase in AI occurs.The comparison of the serological methods for the detection of IgM, IgG, and IgA antibodies in acute sera revealed that the highest percentage of mumps infection was detected by IgA antibody testing. The addition of this serological method to mumps laboratory diagnosis made the latter considerably more effective, particularly in the vaccinated patients.

[Differential diagnosis of the viral etiology of suspected mumps in a population with high vaccine coverage]. / Diferenciální diagnostika virové etiologie suspektních príusnic ve vysoce proockované populaci.

AIM: In this study, buccal swabs from patients with the clinical picture of parotitis epidemica in whom mumps virus (MV) infection was not confirmed by direct detection or serologically were tested. The aim was to detect by molecular methods nucleic acids (NAs) of other respiratory viruses possibly involved in salivary gland swelling. At the same time, paired sera, if available, were tested. MATERIAL AND METHODS: The study group consisted of 72 buccal swabs from patients of the Clinic of infectious, tropical, and parasitic diseases, Na Bulovce Hospital. Paired sera were available from ten patients. Samples were collected in 2013 to 2015. Buccal swabs were tested by PCR for the presence of NAs of adenoviruses (AdV), bocaviruses (hBoV), parainfluenza viruses of types 1-4 (HPIV), human metapneumovirus (hMPV), coronaviruses (HCoV: NL63, OC43, HKU1, and 229E), respiratory syncytial virus (RSV), influenza A virus, influenza B virus, and Epstein-Barr virus (EBV). Paired sera were screened by the complement fixation test (AdV and influenza A and B viruses), hemagglutination inhibition test (HPIV types 2 and 3), ELISA (AdV, EBV), and immunofluorescence (EBV). RESULTS: NAs from viruses other than the mumps virus were detected in 27 of 72 patients with clinical symptoms of parotitis epidemica, and serological tests revealed etiological links with parainfluenza viruses in three more cases. Overall, 30 (41.7%) of 72 patients with suspected mumps tested positive for one or more viruses from the study panel. The most commonly detected viruses were AdV 11/72 (15.3%), EBV 9/72 (12.5%), and HPIV 3/72 (4.2%), but influenza A virus (H3N2) 1/72 (1.4%) was also found. Some patients tested positive for more than one virus: 2/72 (3%) for AdV plus hBoV and 1/72 (1.4%) for HPIV plus HCoV. In addition, examination of paired sera revealed HPIV positivity in three more patients. PCR and serology detected etiological link with HPIV in six (8.3%) of 72 patients tested. CONCLUSION: In our study group, nearly 42% of patients with the clinical picture of parotitis epidemica in whom mumps virus (MV) infection was not confirmed by direct detection or serologically tested positive for viruses other than the mumps virus. Thorough laboratory diagnosis of suspected mumps in vaccinated persons is important not only for the treatment of patients and adoption of isolation and other measures, but also for a better understanding of the epidemiology of the disease and outcomes of the immunisation programmes.

[Report on a measles epidemic in the Ústí nad Labem Region]. / Vyhodnocení epidemie spalnicek v Ústeckém kraji.

The Czech Republic is a measles free country where only isolated, mostly imported cases have been reported. A measles epidemic that occurred in the Ústí nad Labem Region is presented, with the first case diagnosed early in February 2014 and the last one reported in August 2014. The index case and source of infection to other susceptible patients was an adult male with a history of travel to India. The diagnosis of measles was difficult to make as the patient presented with feverish condition due to co-infection with dengue fever, chikungunya, and measles. The primary measles outbreak occurred in contacts and spread to health workers of the Masaryk Hospital in Ústí nad Labem. The infection further spread to the general population of adults. In total, 305 persons presented with suspected measles. One hundred and eighty-six and of them (61%) met the criteria for a confirmed case (positive clinical symptoms and laboratory test). Fifty (16.4%) patients developed typical clinical symptoms and were epidemiologically linked to confirmed cases, but turned out to be antibody negative. In 69 (22.6%) patients, measles were ruled out. Cases were confirmed by the detection of IgM and IgG antibodies against the measles virus or by RT--PCR. Nearly all cases were verified by the National Reference Laboratory for Rubella, Measles, Mumps, and Parvovirus B 19 of the National Institute of Public Health in Prague. In response to the epidemic, apart from common anti-epidemic measures, emergency vaccination was provided to health professionals of the Masaryk Hospital in Ústí nad Labem. Within two weeks after vaccination, a considerable decline in cases was seen in the vaccinated group. Measles most often occurred in persons born in 1970-1980, but were also observed in the smallest, non-vaccinated children (32 cases). Isolated cases also emerged in duly vaccinated children and adolescents. General recommendations are provided based on practical experience from the epidemic.

[Contribution of the detection of IgA antibodies to the laboratory diagnosis of mumps in the population with a high vaccination coverage]. / Prínos stanovení protilátek IgA pro laboratorní diagnostiku príusnic ve vysoce proockované populaci.

STUDY OBJECTIVE: Serological diagnosis of epidemic mumps can be difficult in vaccinated persons, particularly due to the absence of specific IgM antibodies. The aim was to find whether adding the detection of IgA antibodies to the currently used routine serological diagnosis of mumps (detection of IgM and IgG antibodies in an acute serum sample) would make the serological diagnosis of mumps more effective in a population with a high vaccination coverage. At the same time, ELISA kits for the detection of early IgA and IgM antibodies against the mumps virus were compared and statistical analysis of the results was performed. MATERIAL AND METHODS: Sixty-four acute sera from patients with laboratory confirmed diagnosis of mumps were included in the study. Clinical specimens were collected at the onset of clinical symptoms. To test the sera, the MASTAZYME ELISA Mumps IgA kit (MAST DIAGNOSTICA, Germany) with the MASTSORB sorbent (RF and IgG) and Enzygnost Anti-Parotitis-Virus/IgM kit (Siemens, Germany) were used. A panel of 121 acute sera with no epidemiological link to mumps virus served as specificity controls for the IgA assay. The epidemiological data were derived from the EPIDAT system. The level of agreement was assessed using the McNemara test and Cohen's coefficient kappa. The Stata 9.2 software (Stata Corp LP, College Station, USA) was used for statistical analysis. RESULTS: The detection of IgA and IgM antibodies against the mumps virus yielded concordant results in 50/64 acute sera, 32 positive and 18 negative, i.e. an agreement of 78.12 %. Of the remaining 14 samples, 13 were only IgA positive and one was only IgM positive. The controls showed non-specific IgA positivity in 5/121 samples which indicates a 96% specificity. CONCLUSION: The absence of specific IgM antibodies against mumps virus is relatively often seen in vaccinated indivi-duals; nevertheless, the test is routinely used in patients with suspected active infection. The test for IgA antibodies, which is not routinely performed, significantly increased the detection rate of the disease. Based on the results of the present study, it can be concluded that the combination of the anti-mumps IgM and IgA assays increased the effectiveness of the serological diagnosis at the onset of clinical symptoms from less than 52% to nearly 72%.

[Measles re-emerging in the Ústí Region]. / Návrat spalnicek do Ústeckého kraje.

A report is given of a measles outbreak in healthcare workers and the general population that occurred in February to March in Ústí nad Labem. The source of infection was a man with a history of travel to India. The outbreak was primarily observed in the patients contacts and then spread widely to healthcare workers of the Masaryk Hospital in Ústí nad Labem. The outbreak further reached the general population including family contacts of the patients. By the end of March 2014, 171 measles cases were reported, 68 (39.8%) of which in healthcare workers. Cases we confirmed by laboratory analysis of IgM antibodies and IgG antibody dynamics and, possibly, by R-PCR. In addition to standard preventive anti-epidemic measures, the healthcare workers of the Masaryk Hospital were vaccinated with an extra dose of the Priorix vaccine. Measles cases were most often reported in the population born in 1970-1980. This age group was the target population for an extra dose of the Priorix vaccine. General recommendations have been formulated based on the experience from the outbreak.

[Genotyping results, laboratory diagnosis, and epidemiology of the mumps virus circulating in the Czech Republic in 2012]. / Výsledky genotypizace, laboratorní diagnostika a epidemiologie viru príusnic cirkulujícího v Ceské republice v roce 2012.

GOAL: To extend the present routine serological diagnosis of mumps with the methods of direct detection of the pathogen and subsequent genotyping of the isolated viruses in an attempt to obtain more detailed data on recent mumps viruses circulating in the Czech Republic. Sub-goals were to point out the particularities of the laboratory examination in the population with a high vaccine coverage and to evaluate the current epidemiological situation. MATERIAL AND METHODS: Altogether 47 buccal swabs from patients with suspected mumps were included in the study. Clinical specimens collected at the onset of clinical symptoms were obtained from five administrative regions of the Czech Republic from February 2012 to December 2012. Vero cell cultures were used for virus isolation and isolates were identified using real-time quantitative polymerase chain reaction (RT-qPCR). Genotyping was performed by the WHO Regional Reference Laboratory for Measles, Mumps, and Rubella (RRL MMR), Robert Koch Institute, Berlin. The EPIDAT system was used as a source of epidemiological data. RESULTS: From 47 buccal swabs, 20 mumps viruses were isolated on Vero cells and in seven other specimens, the presence of viral RNA without positive isolation was only detected by RT-qPCR. Nineteen isolates were referred for genotyping. The phylogenetic analysis of the SH gene classified them into genotype G, as four variants. In both 2011 and 2012, most cases occurred in vaccinated patients (80%), with 15-19-year-olds being the most affected age group. The leading complication was orchitis, followed by meningitis. More complications were reported in non-vaccinated individuals. CONCLUSIONS: The increased incidence of mumps cases in the Czech Republic in 2012 was due primarily to genotype G, the leading cause of mumps in most European countries since 2005. The presence of genotype G was first reported in the Czech Republic in 2006. In the context of the unfavourable epidemiological trend, molecular epidemiological studies including genotyping of recent mumps virus strains appear to be necessary. A detailed monitoring could be helpful in elucidating the pattern of virus circulation and in designing strategies to control emerging outbreaks. The vaccination efficacy in relation to the causative genotype and possible role of waning immunity in mumps outbreaks are the issues that need to be addressed.

Increased incidence of mumps in the Czech Republic in the years 2011 and 2012.

A nation-wide vaccination against mumps that had been launched in the Czech Republic in 1987 eliminated great outbreaks (up to 100,000 cases per year) of this disease in 1955-1988, but did not prevent small outbreaks (a few thousand cases per year) in 1995-1996, 2005-2007, and 2010-2012. The extent of these small outbreaks shows an increasing trend. The article describes mumps outbreaks in the Czech Republic in 2011 and 2012 with the aim to bring additional data contributing to the clarification of repeated outbreak triggers. In the years 2011 and 2012 there have been reported 2885 and 3902 mumps cases, respectively, in the Czech Republic. Similarly to other countries, a shift in the age-specific incidence of the disease towards higher age has been found, with the highest occurrence seen in the age group of 15-19 years. Men were slightly more affected than women. Clinical complications and vaccination status of patients were also observed.

Adjuvant effect of Bacillus firmus in intranasal immunization of guinea pigs with inactivated type B infuenza virus.

Intranasal immunization of guinea pigs with inactivated type B influenza virus plus inactivated Bacillus firmus as an adjuvant compared to the virus alone yields higher titers of serum hemagglutination-inhibiting antibodies and virus-neutralizing antibodies. This phenomenon could be useful in standard serology, especially in the preparation of immune sera against highly pathogenic strains for in vitro diagnosis.