Molecular surveillance of measles and rubella in the WHO European Region: new challenges in the elimination phase.

BACKGROUND: The WHO European Region (EUR) has adopted the goal of eliminating measles and rubella but individual countries perform differently in achieving this goal. Measles virus spread across the EUR by mobile groups has recently led to large outbreaks in the insufficiently vaccinated resident population. As an instrument for monitoring the elimination process and verifying the interruption of endemic virus transmission, molecular surveillance has to provide valid and representative data. Irrespective of the country's specific situation, it is required to ensure the functionality of the laboratory surveillance that is supported by the WHO Global Measles and Rubella Laboratory Network. AIMS: To investigate whether the molecular surveillance in the EUR is adequate for the challenges in the elimination phase, we addressed the quality assurance of molecular data, the continuity and intensity of molecular monitoring, and the analysis of transmission chains. SOURCES: Published articles, the molecular External Quality Assessment Programme of the WHO, the Centralized Information System for Infectious Diseases of the WHO EUR and the WHO Measles and Rubella Nucleotide Surveillance databases served as information sources. CONTENT: Molecular proficiency testing conducted by the WHO in 2016 has shown that the expertise for measles and rubella virus genotyping exists in all parts of the EUR. The analysis of surveillance data reported nationally to the WHO in 2013-2016 has revealed some countries with outbreaks but not sufficiently representative molecular data. Long-lasting supranational MV transmission chains were identified. IMPLICATIONS: A more systematic molecular monitoring and recording of the transmission pattern for the whole EUR could help to create a meaningful picture of the elimination process.

Challenges of measles and rubella laboratory diagnostic in the era of elimination.

The Member States of the WHO European Region adopted the goal of measles and rubella elimination more than 10 years ago, but so far only 21 of 53 countries have reached this target. Laboratory investigation of suspected cases is essential to support disease elimination efforts. Therefore, WHO maintains a network of accredited laboratories providing high-quality testing. Laboratory investigation heavily relies on specific IgM serology and increasingly on virus detection by reverse transcription (RT)-PCR, but other methods such as IgG avidity testing and genetic characterization of virus strains have gained in importance. In elimination settings, often few samples from suspected cases are available for testing, but testing proficiency must be maintained. The predictive value of an IgM-positive result decreases and other rash-fever disease aetiologies become more important. In addition, cases with a rash after measles/rubella vaccination or with mild disease after waning of vaccine-induced antibodies are seen more often. Thus, it is necessary to perform comprehensive and potentially time-consuming and costly investigations of every suspected case using quality-controlled laboratory methods. At the same time rapid feedback to public health officers is required for timely interventions. The introduction of new laboratory methods for comprehensive case investigations requires training of staff under the supervision of WHO-accredited reference laboratories and the definition of appropriate test algorithms. Clinical, laboratory, and epidemiological data are essential for final case classification and investigation of chains of transmission in the endgame of measles and rubella elimination.

A sensitive one-step TaqMan amplification approach for detection of rubella virus clade I and II genotypes in clinical samples.

Although teratogenic rubella virus (RV) causes a vaccine-preventable disease, it is still endemic in several countries worldwide. Thus, there is a constant risk of RV importation into non-endemic areas. RV monitoring, especially during measles and Zika virus outbreaks, requires reliable diagnostic tools. For this study, a TaqMan-based one-step reverse transcription-quantitative PCR (RT-qPCR) assay, with the p90 gene as a novel and so far unexplored target for detection of clade I and II genotypes, was developed and evaluated. Automated nucleic acid extraction was carried out. Performance characteristics of the TaqMan RT-qPCR assay were determined for a RV plasmid standard and RNA extracted from virus-infected cell culture supernatants representing clade I and II genotypes. Diagnostic specificity and sensitivity were validated against other RNA and DNA viruses, relevant for RV diagnostic approaches and for RV-positive clinical samples, respectively. The assay is specific and highly sensitive with a limit of detection as low as five to one copies per reaction or 200 infectious virus particles per ml. The coefficients of variation (CV) were specified as intra- (within one run) and inter- (between different runs) assay variation, and calculated based on the standard deviations for the obtained Ct values of the respective samples. Intra- and inter-assay CV values were low, with a maximum of 3.4% and 2.4%, respectively. The assay was shown to be suitable and specific for the analysis of clinical samples. With p90 as a novel target, the highly sensitive and specific TaqMan assay outlined in this study is suitable for RV diagnosis worldwide.

Epidemiologic and laboratory surveillance of the measles outbreak in the Federation of Bosnia and Herzegovina, February 2014-April 2015.

A measles outbreak with two epidemic waves involving 4649 probable and laboratory-confirmed cases was recorded in six out of ten cantons of the Federation of Bosnia and Herzegovina between February 2014 and April 2015. The majority of the patients had never received measles vaccination (3115/4649, 67.00%), and the vaccination status of another 23% was unknown (1066/4649). A total of 281 blood samples were tested serologically. Virus detection was performed using 44 nasopharyngeal swabs. About 57% (161/281) of the laboratory-investigated sera were immunoglobulin M positive, and 95% (42/44) of the swabs were reverse transcriptase-PCR positive. Phylogenetic analysis of sequences obtained from 30 swab samples showed circulation of two variants of genotype D8, but no genotype D4 strains as detected in 2007. Similar involvement of all age groups indicates a problem with vaccine refusal resulting from antivaccination activities in addition to gaps in immunization coverage during the war and postwar period (1992-1998). Differences in ethnicity, vaccine coverage, compliance with review policies of vaccination records and potentially also travel habits may partially explain why only six of ten cantons were affected by the outbreak. The second epidemic wave may in part be due to large-scale migrations due to catastrophic floods in 2014. As a result of the epidemic, 6- to 12-month-old children may now be vaccinated against measles during outbreaks, and public health recommendations for interventions have been strengthened. Additional efforts are required to implement the measures throughout the cantons.

Resurgence of measles in Serbia 2010-2011 highlights the need for supplementary immunization activities.

Between December 2010 and August 2011 an outbreak of measles occurred in Serbia with 363 reported cases. Sera and/or nose/throat swabs were collected from 193 patients and tested for measles-specific IgM antibodies by ELISA and viral RNA by RT-PCR, respectively. Epidemiological data were obtained from the surveillance database of the Institute of Public Health of Serbia. Of the 363 cases involved in the outbreak, 113 were laboratory confirmed. More than one third of the patients were hospitalized (n = 130, 35·8%) and for 15 (4·1% of the reported outbreak cases) the infection was complicated by pneumonia. Mostly pre-school children aged ⩽4 years (37·8%) and adults aged ⩾30 years (27·3%) were affected. The majority of patients belonged to the Roma population with a preponderance of female cases (57·0%). Nearly 94% of the patients were either unvaccinated or of unknown vaccination status. The main outbreak virus was the D4-Hamburg strain. The outbreak in Serbia occurred after several years of very low measles incidence despite a high routine immunization coverage in the general population, suggesting that special efforts to identify and vaccinate susceptible population groups are required even in countries with apparently good disease control.

Long-term transmission of measles virus in Central and continental Western Europe.

The World Health Organization (WHO) has adopted an elimination goal for measles and rubella, which is supposed to be met in the WHO European Region (EUR) by 2015. For verification of elimination, it is required that the genotyping data of detected measles viruses provide evidence for the interruption of endemic transmission. In order to record and assess the extent of endemic measles virus (MV) circulation in a part of the EUR, we analyzed transmission chains of the epidemiologically most relevant MV variants identified in Central and continental Western Europe (CCWE) from 2006 to 2013. Based on MV sequence data deposited in the WHO global database for molecular surveillance of measles (MeaNS), the circulation period was calculated for each MV variant at the country-level and for the entire region of CCWE. The MV variants "D5-Okinawa," "D4-Hamburg," "D4-Manchester," and "D8-Frankfurt-Main" spread widely in CCWE; they caused large and long-lasting outbreaks with secondary spread that resulted in additional outbreaks. Nation-wide outbreaks (epidemics) with thousands of measles cases occurred in four countries (Switzerland, France, Bulgaria, and Romania) and were characterized by continuous detection of the same MV variant for more than 12 months suggesting endemic transmission. In the entire region of CCWE, the circulation period of the four predominant MV variants ranged from 18 to 44 months. The long-lasting MV transmission which affected predominantly unvaccinated individuals in different hard-to-reach groups and in the general population is not consistent with the measles elimination goal. Additional efforts are necessary to meet the elimination target in the EUR.

Mumps outbreak in the Federation of Bosnia and Herzegovina with large cohorts of susceptibles and genetically diverse strains of genotype G, Bosnia and Herzegovina, December 2010 to September 2012.

A mumps outbreak reported from the Federation of Bosnia and Herzegovina involved 7,895 cases between December 2010 and September 2012. This was the largest outbreak in the country since the introduction of the measles, mumps and rubella vaccine in 1980. The highest disease incidence was found among 15 to 19 year-olds. About 39% (3,050/7,895) of cases reported to be unvaccinated; the vaccination status of 31% (2,426/7,895) was unknown. A seroprevalence study among 150 asymptomatic contacts to mumps cases showed that about one third (45/150) were susceptible to mumps. Among 105 clinically suspected mumps patients hospitalised at the Clinical Centre of the University of Sarajevo, orchitis (60% of all males: 51/85) and meningitis (9%: 9/105) were the most common complications. Among 57 outbreak sequences obtained for the small hydrophobic gene, eight different variants of genotype G viruses were identified. The outbreak affected mainly age groups comprising individuals who were not vaccinated during or after the Bosnian war, as well as cantons with single dose immunisation policies until 2001. In addition to issues related to vaccination of individuals, differential responses to vaccines and vaccine strains, waning of antibodies and potentially also the genetically diverse variants of genotype G may have compounded the size and duration of the outbreak. Our report emphasizes the need for supplementary immunisation programmes in particular for adolescents and young adults.

High prevalence of mumps in Lao People's Democratic Republic.

In the Lao People's Democratic Republic (PDR), mumps is not a notifiable disease and mumps vaccine is currently not included in the routine childhood immunization programme. In order to assess the burden of disease, we investigated the seroprevalence of mumps-specific IgG antibodies across four provinces. In addition, we genetically characterized mumps viruses from the past 3 years from several outbreaks and single cases. Blood and/or throat swabs from suspected cases were investigated for specific IgM antibodies or viral RNA. Mumps cases occurred between March and November in 2011-2013 and 5- to 15-year-olds were most affected. Four sequences from an outbreak in the north of Lao PDR in 2011 were identical and belonged to genotype G. Eight sequences from two outbreaks and two individual cases from 2012 and 2013 belonged to genotype J. In addition, sera collected from 2379 healthy infants and school pupils aged between 9 months and 19 years and from pregnant women aged between 16 and 46 years were investigated for mumps-specific IgG. Overall, 58.2% were positive, 39.5% were negative and the remaining 2.3% were equivocal. The seropositivity increased with age, with the lowest percentage found in <1-year-old infants (9.1%) and the highest in the cohort of pregnant women (69.2%). More female subjects than male subjects were seropositive (60.4 vs. 54.9%). There were some differences between the locations. Mumps should be a notifiable disease in Lao PDR in order to get more accurate case numbers and cost estimates for public health-care, and vaccination of children and high-risk groups should be considered.

An outbreak of rubella in the Federation of Bosnia and Herzegovina between December 2009 and May 2010 indicates failure to vaccinate during wartime (1992-1995).

A rubella outbreak involving 1900 cases was recorded in the Federation of Bosnia and Herzegovina between mid-December 2009 and the end of May 2010. Sera from 389 suspected rubella cases were examined for the presence of rubella-specific IgM and IgG antibodies. A total of 32 throat swabs from suspected rubella cases were tested by RT-PCR and were used to attempt virus isolation. Most patients (945/1900, 49·73%) had never received rubella vaccination or had an unknown vaccination status (563/1900, 29·63%). About 45% (178/389) of suspected rubella patients were IgM positive. From 13 of the throat swabs a virus isolate and E1 gene sequences attributed to genotype 2B were obtained. The rubella outbreak was due to failure to vaccinate during the war period (1992-1995) and emphasizes the need for additional vaccination opportunities.

High genetic diversity including potential new subtypes of hepatitis C virus genotype 6 in Lao People's Democratic Republic.

Sera from 105 anti-HCV-positive first-time blood donors collected in 2004, 2005 and 2008 in different provinces in Laos were investigated by PCR. Forty-five samples were positive for HCV (42.86%); two belonged to subtype 1b (2/45, 4.4%) and all others to genotype 6 (43/45, 95.6%), including subtypes 6b, 6h, 6k, 6l, 6n and 6q. Three groups of sequences were not clearly attributable to any genotype 6 subtype, two of which may be regarded as candidates for new subtypes of genotype 6. Two samples were mixed infected with different subtypes or clusters of genotype 6 viruses.

Ongoing large mumps outbreak in the Federation of Bosnia and Herzegovina, Bosnia and Herzegovina, December 2010 to July 2011.

From December 2010 until the end of July 2011, 5,261 mumps cases were recorded in the Federation of Bosnia and Herzegovina, Bosnia and Herzegovina, leading to an incidence of 225.8 per 100,000. Fifteen to 19 year-olds (43%) were most affected and 62% of cases were male. Mumps-specific IgM antibodies were found in about 70% of sera investigated, complications were reported in 41% of 81 hospitalised patients. The outbreak affected mainly those unvaccinated or unaware of their vaccination status and is probably due to vaccination failures during the war and postwar period (1992­1998).

Detection of a new subgenotype of hepatitis B virus genotype A in Cameroon but not in neighbouring Nigeria.

To further investigate the genetic diversity of hepatitis B virus (HBV) genotype A in Africa, we analysed 263 HBV strains from Nigeria (n=163) and Cameroon (n=100). Phylogenetic analysis of S fragment sequences attributed 175 strains (66.5%) to genotype E and 88 (33.5%) to genotype A. In Cameroon, genotype A strains were the most prevalent (79/100, 79.0%), whereas, in Nigeria, genotype E was highly dominant (154/163, 94.5%). The genotype A strains grouped with reference strains of subgenotype A3 (n=8), the provisional subgenotype A5 (n=43), a recently reported new variant from Rwanda (n=35), or as outliers (n=2). Ten complete genome sequences obtained from strains that clustered with the new variant from Rwanda formed a separate group supported by a bootstrap value of 96. The between-group distance to other potential or recognized subgenotypes of genotype A was at least 3.81%. Thus, the new group of strains could be considered as a new subgenotype of HBV genotype A, tentatively named 'A7'. Interestingly, the 'A7' strains from Rwanda and Cameroon showed an interspersed clustering, but essentially no other (sub)genotypes were shared between the two countries, suggesting that 'A7' may have evolved in a yet unknown place and may have only relatively recently spread to Rwanda and Western Cameroon. Strains attributed to provisional subgenotype A5 were found for the first time in Cameroon (n=36) and Central Nigeria (n=2), indicating that A5 is more widespread than previously thought.

[Molecular epidemiology of parvovirus infection in Belarus].

The paper analyzes a 994 nucleotide fragment of the NS1/VP1u region junction of 84 parvovirus B19 samples obtained from the sera of erythema infectiosum patients in Belarus in 2006. All the strains belong to genotype 1 as defined by Servant et al. (2002) and form two major clusters within this genotype (1A and 1B) with a clearly distinct geographic distribution. Cluster 1A mainly included B19 strains from Minsk where an outbreak of erythema infectiosum was observed during sample collection. Cluster 1B comprises parvovirus B19 strains obtained from sporadic cases in different parts of the country. The nucleotide variability within cluster 1B (1.1%) was almost two times higher than that within cluster 1A (0.6%). The comparison of the Belarus strains with all parvovirus B19 sequences from the GenBank revealed 22 unique nucleotide substitutions in the new strains, 18 (81.8%) of which were nonsynonymous. A high percentage of parvovirus B19 IgM positive sera were also PCR positive (94.0%; n = 63/67) indicating that both methods are suitable for diagnosis of the infection.

Avian metapneumovirus subtype A in China and subtypes A and B in Nigeria.

In order to detect and characterize avian metapneumovirus, organs or swabs were collected from 697 chicken and 110 turkeys from commercial farms in Southwestern Nigeria and from 107 chickens from live bird markets in Southeastern China. In Nigeria, 15% and 6% of the chicken and turkey samples, respectively, and 39% of the chicken samples from China, were positive for aMPV genome by PCR. The sequence of a 400 nt fragment of the attachment protein gene (G gene) revealed the presence of aMPV subtype A in both Nigeria and Southeastern China. Essentially identical subtype A viruses were found in both countries and were also previously reported from Brazil and the United Kingdom, suggesting a link between these countries or a common source of this subtype. In Nigeria, subtype B was also found, which may be a reflection of chicken importations from most major poultry-producing countries in Europe and Asia. In order to justify countermeasures, further studies are warranted to better understand the metapneumoviruses and their impact on poultry production.