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.
Keywords: Genotyping; elimination; measles; molecular epidemiology.