Transmission and molecular characterisation of wild measles virus in Romania, 2008 to 2012

G Necula (gnecula@cantacuzino.ro)1,2, M Lazar1,2, A Stanescu3, A Pistol3, S Santibanez4, A Mankertz4, E Lupulescu1 1. National Reference Laboratory for Measles and Rubella and National Influenza Center, Cantacuzino Institute, Bucharest, Romania 2. These authors contributed equally to this article 3. National Centre for Communicable Diseases Surveillance and Control, National Institute of Public Health, Bucharest, Romania 4. World Health Organisation Regional Office for Europe, Regional Reference Laboratory for Measles and Rubella, Robert Koch Institute, Berlin, Germany


Introduction
Measles is a highly contagious respiratory viral disease characterised by the appearance of fever and a rash and that can be very serious or even fatal. Measles remains one of the leading causes of mortality in young children although a safe and cost-effective vaccine has been available for decades [1]. Although improvements have been made to control measles in Europe, large-scale outbreaks have recently still been observed [2][3][4][5][6][7]. The World Health Organization (WHO) was forced to postpone a number of times the target date for measles elimination from the European region, most recently to 2015 [8].
Measles has been a statutorily notifiable disease in Romania since 1978, and medical practitioners must report all clinically possible measles cases to the regional public health authorities. The first monovalent measles-containing vaccine was introduced in 1979 into the Romanian vaccination schedule for children aged nine to 11 months. The combined measlesmumps-rubella (MMR) vaccine replaced the monovalent measles vaccine in 2004 and was recommended as a first dose for children aged 12 to 15 months. The second MMR vaccine dose has been recommended since October 2005 for children aged six to seven years. In the period from 2000 to 2010, the coverage for the first dose of measles vaccine was estimated at 95-98% [9]. In 2011, measles vaccination coverage for the first dose of MMR vaccine was estimated at 84% for children aged 12 months and 93.2% for those aged 18 months [10].
Romania experienced a measles epidemic that started in December 2004 and lasted until early 2007 [11]. More than 9,000 cases were detected mostly in non-immunised patients belonging to the Roma ethnic group. The outbreak was caused by the strain MVs/Bucharest. ROM/48.04/2[D4] and variants divergent by two nucleotides or less were detected during the period 2004 to 2006 [11]. Closely related strains were detected from 2005 to 2007 in Bosnia and Herzegovina, Germany, Italy, Portugal, Serbia, Spain and Switzerland [11][12] [11].
D4 measles viruses are endemic in India, South-East Asia and in South Africa [14]. Outbreaks associated with this genotype have been reported since 2007 from all continents. In Europe, many distinct variants descend from D4-Enfield (MVs/Enfield.GBR/14/07) which became endemic in the United Kingdom (UK) in 2007 [15][16][17]. The variant D4-Hamburg initiated a transmission chain of 25,000 cases that was detected in Europe for a period of more than two years, 2009 to 2011 [18]. In 2010, the D4 genotype became predominant in Europe [2].
Genetic characterisation of measles viruses constitutes an important part of laboratory surveillance. Molecular epidemiology confirms the transmission pathway of measles virus, thereby complementing classical epidemiology. Moreover, interruption of endemic measles virus transmission (i.e. circulation of a certain variant for more than 12 months) is an important criterion for verification of measles virus elimination in Europe. Therefore, it is necessary to distinguish between endemic and imported viruses using molecular methods [16].
This study describes the genetic characterisation of measles viruses detected in Romania from 2008 to 2012, focusing on the recent outbreaks that occurred in the country between 2010 and 2012 that affected mainly groups with limited access to healthcare facilities and schools. This study underlines the importance of measles genotyping during the different phases of an outbreak.

Patients and specimens collection
According to the national strategy of measles surveillance, approved by Romanian MOH, a measles case is defined as a person with fever and maculopapular rash and at least one of the following symptoms: cough, coryza, or conjunctivitis. Possible cases are persons who met the clinical case definition with no epidemiological link to a laboratory-confirmed case. Confirmed cases are either laboratory-confirmed (by detecting measles IgM antibodies in serum samples, virus isolation, a significant rise in measles antibody levels, or measles PCR detection in all possible cases) or confirmed by the presence of measles case symptomatology and an epidemiological link to a laboratory-confirmed case.
At national level, notifications of measles cases are collected and analysed by the National Centre for Communicable Diseases Surveillance and Control in Bucharest, Romania. Specimens are sent for confirmation to the National Reference Laboratory for Measles and Rubella in Cantacuzino Institute, Bucharest, Romania.

RT-PCR and sequencing
Confirmed cases were selected for genotyping from new outbreaks (index case and two or three secondary cases). All confirmed cases with a history of travel abroad during the incubation period (7-21 days) were genotyped.

Sequence analysis
Sequences were edited manually with BioEdit (version 7.0.5; North Carolina State University) and with the use of Staden Package [20]. Partial nucleocapsid gene sequences were aligned against related sequences retrieved from GenBank and MeaNS (http:// www.who-measles.org) databases, using ClustalW [21] implemented in BioEdit (version 7.1.3.0) [22]. Starting with 2011, the programmes BioEdit and Gap4 have been replaced by the commercial programme Sequencer (Gene Codes Corporation, Ann Arbor, United States).
Phylogenetic analysis was performed using MEGA (version 5) with a neighbour-joining (NJ) algorithm inferred with Tamura-Nei parameter for sequence evolution.

Results
Romania experienced a measles epidemic with continuous virus transmission from late 2004 to early 2007. In the following years 2008 and 2009, measles activity

Figure 2
Localisation of different measles virus genotypes and D4 variants and confirmed cases detected in Romania in 2008-12 (n=73) For the outbreak dots that do not have a genotype assigned, genotyping was not performed. The genotype label is associated with outbreaks dots within the county's geographical boundary.

Measles cases in 2008
In

Measles cases in 2009
In August 2009, in Arad county, a small cluster of five measles cases was laboratory-confirmed by IgM. The index case was a child too young to be vaccinated returning from Ireland, who infected two other family members. One of them was hospitalised and passed the infection nosocomially to two additional cases in the paediatric ward. Genotyping a specimen from one of the secondary cases identified a measles virus (MVs/Arad.ROU/35.09/1[D4]) that was closely related to MVs/Limoges.FRA/17.10[D4] with a single nucleotide mismatch. As a response measure, the National Center for Surveillance and Control of Transmissible Diseases (CNSCBT) coordinated a supplementary vaccination campaign in the neighbouring Arad and Timis districts, targeting 1,054 unvaccinated or single-dose vaccinated children.

Measles cases in 2010
In 2010, measles activity increased to nine sporadic cases in five counties and 185 outbreak-related cases from in eight counties. Characteristic in that year was the occurrence of outbreaks in different geographic areas of the country, in the east (Neamt and Galati with 31 cases each) and in the north-west (Maramures with 95 cases) ( Figure 2).
The first cluster occurred in February to March, in a Roma community from Tulcea county, totalling five cases. The index case had travelled to France shortly before. Genotyping of the strain isolated from the index case revealed MVs/Tulcea.ROU/08. 10

Measles cases in 2011
In 2011, outbreaks expanded to 39 of the 42 counties of Romania, reaching 4,163 notified cases (45.9% laboratory-confirmed). Because the epidemic was so large, measles virus genotyping and phylogenetic analysis was restricted only to extended outbreaks and to imported cases. The majority of genotyped cases were associated with D4-Manchester and D4-Maramures variants (Figures 2 and 3). The cases were mainly seen in the north-western region of Romania, first in the Roma communities and subsequently spreading into the general population. The Salaj county was most affected (incidence: 141.9 per 100,000 population). Of the total laboratory-confirmed cases in 2011, 78% were not vaccinated. Of these, 16% were younger than 12 months and thus not eligible for vaccination. As a response measure to the growing number of measles cases, additional vaccination campaigns were implemented in 2011 that targeted children between the ages of seven months and seven years, leading to approximately 4,500 vaccinated children.

Measles cases in 2012
In 2012, measles activity remained at comparable intensity to the previous year, reaching 4,006 cases (79.3% laboratory-confirmed) by the end of year, but the geographical distribution shifted to the south-eastern region of Romania. Of all laboratory-confirmed cases in 2012, 84.5% were unvaccinated (26.7% too young for vaccination). The most affected age group were children younger than one year (incidence: 219/100.000) and those between one and four years of age (incidence: 78.6/100.000). The majority of these cases were associated with D4-Manchester variant (Figures 2  and 3). According to the national strategy of measles cluster control, vaccination of children aged between seven month and seven years continued in 2012, but the total number of vaccinations is not available. In response to the measles outbreaks, a wide range of control measures were implemented, including strengthened surveillance for timely identification and monitoring of cases and outbreaks, modified immunisation schedules, and supplementary immunisation activities (approximately 4,500 vaccinated) of the rural population. Despite these measures, more than 4,000 of the over 30,000 cases recorded in Europe in 2011 were from Romania [23]. In 2012, 3,843 of 8,230 total cases were reported by Romania to The European Surveillance System [24]. Frequent measles outbreaks in Romania could be the result of suboptimal vaccine coverage. In 2009, the coverage for the first dose of measles-containing vaccine in children aged 12 months was estimated at 85.1% (95% confidence interval (CI): 82. 4-87.8), but reached the target of 95% (95% CI: 93. 4-95.8) in children aged 18 months [25]. Despite this fairly high vaccination coverage in the 18 month-olds, it is of note that families not registered with a family physician are not represented in this analysis. Lately a shrinking confidence in vaccination has been observed in Romania, reflecting the progress of the anti-vaccine movement all over Europe. The ethnic group of the Roma is traditionally underserved by national health services and moreover, a part of them refuse vaccination actively. The current and previous Romanian outbreaks started in Roma communities and underline the need to develop strategies to improve their integration into the national health services. However, we want to make clear that any under-vaccinated groups or those with a low immunity rate are highly prone to measles virus infections, and represent an important factor preventing successful and sustainable elimination of measles in Romania. Our results underline the importance to strengthen immunisation programmes and to develop specific measures to address parents and their concerns regarding vaccination as well as the hard-to-reach population all over Europe.
In conclusion, a combination of epidemiological data and molecular characterisation enabled us to trace the spread of wild measles virus genotype in Romania from 2008 to 2012. Molecular surveillance of measles virus circulation in Romania will be continued to assess the effectiveness of the national measles control programme and hopefully to support the verification of measles elimination by the year 2015.
Lupulescu -writing of the manuscript and interpretation of the study.