Mumps clinical diagnostic uncertainty

Mumps clinical diagnostic uncertainty Abstract Background During recent years, various mumps outbreaks have occurred among populations vaccinated for mumps worldwide. In Italy, improving routine coverage with two doses of measles, mumps and rubella (MMR) vaccine is one of the key strategies to eliminate measles and rubella. To monitor the effect of the vaccination programme on the population, the surveillance of these vaccine-preventable diseases has been implemented. This provided the opportunity to evaluate the accuracy of the clinical diagnosis of those diseases, including mumps. In fact, vaccinated children may develop a variety of diseases caused by a series of different viruses [Epstein-Barr virus (EBV), parainfluenza virus types 1–3, adenoviruses, herpes virus and parvovirus B19] whose symptoms (i.e. swelling of parotid glands) may mimic mumps. For this reason, laboratory diagnosis is essential to confirm clinical suspicion. Methods The accuracy of clinical diagnosis of mumps was evaluated by differential diagnosis on EBV in Italy, a country at low incidence of mumps. This retrospective study investigated whether the etiology of 131 suspected mumps cases with a negative molecular/serological result for mumps virus, obtained from 2007 to 2016, were due to EBV, in order to establish a diagnosis. Results Differential diagnosis revealed a EBV positivity rate of 19.8% and all cases were caused by EBV type 1. Conclusions This study confirms the importance of a lab based differential diagnosis that can discriminate between different infectious diseases presenting with symptoms suggestive of mumps and, in particular, emphasize the importance to discriminate between mumps and EBV-related mononucleosis. Introduction Mumps is caused by a virus (MuV) belonging to the family Paramixoviridae.1,2 The disease usually occurs among children and in the pre-vaccine era the annual reported mumps incidence in Western European countries ranged between 100 and 600 per 100 000 inhabitants.3 With the availability of a live-attenuated mumps vaccine since the 1960s,4 disease incidence dramatically decreased in countries with mumps vaccination programmes.5,6 However, several outbreaks of mumps have occurred among measles, mumps and rubella (MMR) vaccinated individuals in various countries worldwide.7–10 Like measles and rubella (MR), mumps is efficiently transmitted from person to person. Before the start of extended programmes of immunization, mumps was typically a childhood disease, with the highest incidence among children between 5 and 9 years of age, presenting with a generally benign course, which is asymptomatic in about one third of the infected children. However, mumps may affect people of any age, causing more severe complications among adults. Clinical mumps is defined as the acute onset of unilateral or bilateral tender, self-limiting swelling of the parotid or other salivary glands (from which the popular name ‘mumps’), lasting two or more days without other apparent cause. Swelling of the parotid glands is the most important clinical sign and may result in pain associated with chewing and swallowing. Fever and malaise are also common. Chills, headache, and a slight rise in temperature may occur ∼24 h before the onset of parotid swelling. In total 15–20% of mumps infections can be asymptomatic and about 50% are associated with non-specific or respiratory symptoms.11–13 Children with mumps usually recover within few days after symptoms onset. The most common complications include encephalitis (0.02–0.3%), meningitis (0.5–15%), pancreatitis (4%) and hearing loss. Finally, infection during the first 12 weeks of pregnancy is associated with a high percentage of miscarriages (25%), but not the risk of fetal malformations.2 When parotitis is present during a mumps outbreak or epidemic, the clinical diagnosis of mumps is generally straightforward. However, when the incidence rate of mumps is low, other causes of parotitis, in particular viral infections such as those due to the Epstein-Barr virus (EBV), parainfluenza viruses, influenza A virus, coxsackieviruses, adenoviruses, parvovirus B19, lymphocytic choriomeningitis virus and HIV, should be considered.12,14 For this reason, an approach based on the laboratory testing is essential to confirm the clinical suspicion of mumps, especially in areas where the incidence of the disease is low. In Italy, mumps diagnosis is performed within the framework of MR surveillance. Indeed, to support case ascertainment, the National Reference Laboratory (NRL) for MR performs laboratory surveillance to confirm suspected cases of measles, rubella and mumps.15 In February 2013, the Italian Minister of Health published a document which regulates the integrated surveillance for MR in which indications about the laboratory mumps surveillance were further included.16 This retrospective study investigated whether the etiology of Italian sporadic suspected mumps cases with a negative molecular/serological result for mumps virus obtained at the NRL, in 2007–16, were due to EBV, in order to establish a diagnosis. Methods Mumps case definition The case definition and classification is that stipulated by EU Commission Decision of 8 August 2012. Clinical criteria Fever and at least two of the following: sudden onset of unilateral or bilateral tender swelling of the parotid or other salivary glands without other apparent cause or orchitis or meningitis. Laboratory criteria At least two of the following (i) isolation of mumps virus from a clinical specimen; (ii) detection of mumps virus nucleic acid; (iii) MuV specific antibody response characteristic for acute infection in serum or saliva. Epidemiological criteria An epidemiological link by human-to-human transmission. Case classification includes ‘possible case’ (any person meeting the clinical criteria), ‘probable case’ (any person meeting the clinical criteria and with an epidemiological link), ‘confirmed case’ (any person not recently vaccinated and meeting the clinical and the laboratory criteria). Study population and clinical samples During the period between June 2007 and 2016, oral and/or blood samples of 193 suspected cases of mumps were sent to the NRL from various Italian regions for laboratory confirmation of the clinical diagnosis. All these were clinically compatible and sporadic cases, not linked to outbreak settings. The vaccination status was known for 190/193 patients: 42 had never received a vaccine against mumps (41.6%), 79 were vaccinated with 1 dose (36.3%), 69 with 2 doses (22.1%). Mumps virus genome can be detected from oral fluid within the first week after symptoms onset. Oral fluid from suspected mumps cases were tested for mumps by RT Real-Time PCR, while blood samples were tested for specific IgM anti-mumps detection by Elisa. Oral fluid samples still available of those negative patients were further tested for EBV by PCR. Serological diagnosis for mumps The detection of anti-mumps IgM was performed with the Enzygnost Anti-Parotitis Virus/IgM kit (Dade/Behring, Siemens) on blood samples collected and treated as previous described in.17 Molecular detection of mumps and EBV RNA and DNA were extracted from oral fluid specimens using QIAmp Viral RNA Kit and QIAamp DNA Mini Kit (Qiagen), respectively, according to the manufacturer's instructions. A 7 µl aliquot of RNA was used for a reverse transcription PCR Real-time with the RealTime Ready RNA Virus Master kit (Roche) according to CDC’s indications.18 A portion of 169 bp of the BXLF1 gene of EBV was amplified by PCR. The reaction was performed with PCR Supermix (Invitrogen), 10 pmol of each forward (EBV1 5′-GGGGCAAAATACTGTGTTAG-3′, position 143 411) and reverse primers (EBV2 5′-CGGGGGACACCATAGT-3′, position 143 579), and 3 µl of extracted DNA.19 The cycling conditions consisted of an initial denaturation of 10 s at 95°C, followed by 45 cycles of 40 s at 95°C, 1 min at 58°C and 40 s at 72°C and a final extension of 5 min at 72°C. Genetic analysis Samples positive for MuV were further amplified for genotyping by PCR followed by a Nested PCR on the MuV SH gene,20 using the SuperScript One-Step RT-PCR with PlatinumR Taq System and PCR SuperMix kits (Invitrogen), respectively. Before sequencing, PCR products were purified with the QIAquick PCR Purification Kit (QIAGEN) and sequencing reactions performed by Macrogen Inc. (Seoul, South Korea). Nucleotide sequences were aligned with sequences of the reference strains and with those that showed a high percentage of identity after Blast analysis, using CLUSTAL W (BioEdit) software.21 The Bayesian Information Criterion was used to determine the model of nucleotide substitution that best fit the data using the selection tool available in MEGA6.22 Evolutionary analyses were conducted using the maximum likelihood method based on the Tamura 3-parameter (T92) model and evolutionary rates among sites were modelled by a discrete Gamma distribution (+G). Samples positive for EBV were tested by PCR to amplify a portion of the gene EBNA3C in order to discriminate between EBV genotype type 1 or type 2.23 PCR was performed with PCR Supermix (Invitrogen) with 5 min at 95°C, followed by 35 cycles of 45 s at 95°C, 45 s at 56°C and 1 min at 72°C and 10 min at 72°C. Amplicons were analysed by electrophoresis on 1.5% agarose gel and gel-red staining. Results From June 2007 to 2016, 148 oral fluid and 169 blood samples from a total of 193 patients with suspect mumps were collected and tested at the NRL. As reported in table 1, 11/193 (5.7%) patients were found positive for MuV infection either by serological or molecular assay, and 182 were negative. Three cases were positive by IgM serology but negative by PCR probably due a bad sampling. Detailed results and vaccination status for each positive patient are reported in table 2. Vaccination status was available for 9 out of 11 positive cases: 5 mumps infected patients had received one dose of MMR vaccine, 2 patients had received two doses and 2 were not vaccinated. For five patients, it was possible to calculate the time elapsed after vaccination (ranging from 2.5 to 13 years). For those negative cases, 66.5% (121/182) had received at least one dose of vaccine against mumps while 20.9% (38/182) were not vaccinated. Table 1 Results obtained for mumps diagnosis by molecular and serological tests and for EBV by molecular tests     2007    2008  2009  2010  2011  2012  2013  2014  2015  2016  Total  Mumps  pos  4  1  1  0  1  0  0  3  0  1  11  neg  45  41  13  5  14  25  17  10  9  3  182  tested  49  42  14  5  15  25  17  13  9  4  193  EBV  pos  12  10  4  0  0  0  0  0  0  0  26  neg  33  30  3  0  0  13  9  6  8  3  105  tested  45  40  7  0  0  13  9  6  8  3  131      2007    2008  2009  2010  2011  2012  2013  2014  2015  2016  Total  Mumps  pos  4  1  1  0  1  0  0  3  0  1  11  neg  45  41  13  5  14  25  17  10  9  3  182  tested  49  42  14  5  15  25  17  13  9  4  193  EBV  pos  12  10  4  0  0  0  0  0  0  0  26  neg  33  30  3  0  0  13  9  6  8  3  105  tested  45  40  7  0  0  13  9  6  8  3  131  Table 2 Patients positive for mumps tests and their vaccination status   Year  PCR  IgM  Genotype  Age  Vaccination status  Pt 229  2007  –  POS  –  17 months  1 dose  Pt 256  2007  NEG  POS  –  4 years  1 dose  Pt 419  2007  POS  POS  H  13 years  1 dose  Pt 427  2007  –  POS  –  13 years  NA  Pt 1368  2008  –  POS  –  46 years  Not vaccinated  Pt 1806  2009  NEG  POS  –  6 years  1 dose  Pt 2322  2011  POS  –  G  15 years  1 dose  Pt 3060  2014  POS  BL  G  18 years  2 doses  Pt 3143  2014  POS  POS  G  19 years  NA  Pt 3196  2014  NEG  POS  –  6 years  2 DOSES  Pt 3398  2016  POS  POS  G  29 years  Not vaccinated    Year  PCR  IgM  Genotype  Age  Vaccination status  Pt 229  2007  –  POS  –  17 months  1 dose  Pt 256  2007  NEG  POS  –  4 years  1 dose  Pt 419  2007  POS  POS  H  13 years  1 dose  Pt 427  2007  –  POS  –  13 years  NA  Pt 1368  2008  –  POS  –  46 years  Not vaccinated  Pt 1806  2009  NEG  POS  –  6 years  1 dose  Pt 2322  2011  POS  –  G  15 years  1 dose  Pt 3060  2014  POS  BL  G  18 years  2 doses  Pt 3143  2014  POS  POS  G  19 years  NA  Pt 3196  2014  NEG  POS  –  6 years  2 DOSES  Pt 3398  2016  POS  POS  G  29 years  Not vaccinated  NA, not applicable. Phylogenetic analysis was performed on 5 MuV sequences obtained from samples positive in PCR. As shown in figure 1, four strains belonged to genotype G (MuVs/Salerno.ITA/4.14/, MuVs/Bolzano.ITA/18.11/, MuVs/Livorno.ITA/24.14/, MuVs/Livorno.ITA/20.16/) and one to genotype H (MuVs/Livorno.ITA/41.07/). Sequences were deposited in GenBank database under accession numbers KX518652, KX518653, KX518654, KX518655, KX518656. Figure 1 View largeDownload slide Neighbour-joining tree for nucleotide sequences of mumps strains identified from 2007 to 2016 Figure 1 View largeDownload slide Neighbour-joining tree for nucleotide sequences of mumps strains identified from 2007 to 2016 WHO data show that genotype G has been reported in Europe, North America, South-East Asia, while genotype H has been reported also from South America and Africa. BLAST analysis showed that the same strain identified in Salerno in 2014 circulated in Europe in 2012 and 2013 and in USA in 2016. The strain that circulated in Bolzano in 2011 was also identified in Germany in the same year. No strains identical to those identified in Livorno in 2014 and 2016 have been ever reported. In addition, the unique strain belonging to the genotype H, identified in Livorno in 2007, did not show identity with any other strains after BLAST analysis. Oral fluid samples available for 131 out of 182 mumps negative cases were further tested for EBV by PCR; of them, 26 were found positive (positivity rate of 19.8%) for viral DNA (table 1). Beside genetic analysis on mumps strains, EBV positive samples were tested by PCR to distinguish between genotypes type 1 or 2, and all of them belonged to genotype 1. The incidence trend of new cases of mumps in Italy from 1996 to 2014 shows a series of oscillations, with a maximum of almost 65 000 cases reported in 1996 (figure 2). Since 1999, the incidence of mumps declined to a minimum number of 191 cases reported in 2014. This decline was probably due to MMR vaccination campaigns. Studies established that the effectiveness of any MMR vaccination in patients with a history at least one MMR vaccination adjusted for age, sex and general practice was 69% (95% CI: 41–84%)24,25 and because of the low effectiveness of the mumps MMR vaccine component, several outbreaks occurred in Europe.26,27 Figure 2 View largeDownload slide Trend of mumps cases in Italy from 1996 to 2014 (Data from: Ministry of Health and European Center for Disease Prevention and Control) Figure 2 View largeDownload slide Trend of mumps cases in Italy from 1996 to 2014 (Data from: Ministry of Health and European Center for Disease Prevention and Control) Also, the decreased efficiency of the surveillance system, leading to a low notification rate, was likely to contribute to the low number of cases reported in Italy in the last years. The standard clinical case definition of mumps used for surveillance activities consisted in ‘acute onset of unilateral or bilateral swelling of the parotid or other salivary glands lasting two or more days without any other apparent cause’.2 However, although parotitis is indeed the hallmark of mumps, there are cases in which salivary-gland swelling is not apparent, especially in individuals with mumps meningitis, many of whom do not present detectable salivary-gland enlargement.28,29 Moreover, other infectious agents may also cause salivary-gland swelling. The effect of such alternative aetiologies greatly reduces the positive predictive value of a clinical diagnosis when the disease incidence is low.30 This study reports results from the differential diagnosis of mumps with EBV-related mononucleosis provides information on the specificity of the clinical diagnosis of mumps, suggesting the importance of laboratory confirmation. Our findings show that the specificity of the case-definition of mumps is low. Studies conducted in other areas of the world provided similar results. In a study conducted in Victoria, Australia, only 7 (9%) of 74 cases clinically diagnosed as mumps parotitis could be confirmed by serology; 7 (16%) of 43 laboratory-rejected cases were positive for EBV using serological testing.30 In a study conducted in Finland, on 601 acutely ill children presenting mumps-like symptoms but seronegative for mumps, the most commonly identified viral agents were the EBV (7%), parainfluenza virus (4%) and adenovirus (3%).31 These studies highlight the importance of laboratory confirmation in diagnosing mumps, especially under non-outbreak conditions. Discussion According to WHO, introduction of routine mumps vaccination, such as other prophylactic options,31 should be a high priority. Most European health systems provide mumps vaccine in combination with MMR, with a two-dose vaccination schedule, free of charge, and some 120 countries have introduced vaccination against mumps in their national immunization programmes. To date, countries such as Finland or Sweden have completely eradicated mumps from their national territory.32 Actions should be implemented to encourage practitioners to collect oral and blood samples from mumps suspected cases and to submit these samples to the NRL or other reference labs that performs mumps virus PCR and serology. This is of special importance when the patient is vaccinated and a primary or secondary vaccination failure is suspected, being important both for individual patients and for monitoring the outcome of vaccination programmes. About that, our study revealed that three patients positive for mumps had been vaccinated before the introduction (in 2001) of the more efficient component Urabe AM 9 in the MMR vaccine in spite of the Rubini strain, responsible for some vaccine failure.33 In conclusion, the results of this study confirm the importance of a lab-based differential diagnosis that can discriminate between different infectious diseases presenting with symptoms suggestive of mumps and emphasize the importance to discriminate between mumps and EBV-related mononucleosis. Finally, the large proportion of negative results suggests that other viral infections are involved in the genesis of mumps-like syndromes. Acknowledgements We wish to thank Dr C. Fortuna and Mrs E. Benedetti for technical support, and the staff of Italian Regional and Local Health Authorities for providing clinical specimens. Funding This work was partially funded by the Italian Ministry of Health grant CCM 2015-6M21. Conflicts of interest: None declared. Key points The specificity of the case-definition of mumps is low and a large number of viral infections are involved in the genesis of mumps-like syndromes such as Epstein-Barr virus. A lab-based differential diagnosis is essential to discriminate between different infectious diseases, especially for the large proportion of mumps negative cases. Low efficiency of the surveillance system for mumps, leading to a low notification rate, contribute to the low number of cases reported in Italy in the last years. References 1 King AMQ, Adams MJ, Carstens EB, Lefkowitz EJ. Virus Taxonomy: Classification and Nomenclature of Viruses: Ninth Report of the International Committee on Taxonomy of Viruses . San Diego: Elsevier Academic Press, 2012. 2 Hviid A, Rubin S, Mühlemann K. Mumps. Lancet  2008; 371: 932– 44. Google Scholar CrossRef Search ADS PubMed  3 Levy-Bruhl D, Pebody R, Veldhuijzen I, Valenciano M, Osborne K. ESEN: a comparison of vaccination programmes - part three: measles mumps and rubella. Euro Surveill  1998; 3: 115– 119. Google Scholar CrossRef Search ADS PubMed  4 Galazka AM, Robertson SE, Kraigher A. Mumps and mumps vaccine: a global review. Bull World Health Organ  1999; 77: 3– 14. Google Scholar PubMed  5 Savage E, Ramsay M, White J, et al.   Mumps outbreaks across England and Wales in 2004: observational study. BMJ  2005; 330: 1119– 20. Google Scholar CrossRef Search ADS PubMed  6 Lievano F, Galea SA, Thornton M, et al.   Measles, mumps, and rubella virus vaccine (M-M-RII): A review of 32 years of clinical and postmarketing experience. Vaccine  2012; 30: 6918– 26. Google Scholar CrossRef Search ADS PubMed  7 Vygen S, Fischer A, Meurice L, et al.   Waning immunity against mumps in vaccinated young adults, France 2013. Euro Surveill  2016; 21: 8 Gee S, O’Flanagan D, Fitzgerald M, Cotter S. Mumps in Ireland, 2004-2008. Euro Surveill  2008; 13: 18857. Google Scholar CrossRef Search ADS PubMed  9 Mossong J, Bonert C, Weicherding P, Opp M, Reichert P, Even J, et al.   Mumps outbreak among the military in Luxembourg in 2008: epidemiology and evaluation of control measures. Euro Surveill  2009; 14:pii 19121. 10 Yung CF, Andrews N, Bukasa A, et al.   Mumps complications and effects of mumps vaccination, England and Wales, 2002-2006. Emerg Infect Dis  2011; 17: 661– 7. Google Scholar CrossRef Search ADS PubMed  11 WHO. Global Status of mumps immunization and surveillance. Weekly Epidemiol Record  2005; 48: 418– 24. 12 Hatchette TF, Mahony JB, Chong S, LeBlanc JJ. Difficulty with mumps diagnosis: What is the contribution of mumps mimickers. J Clin Virol  2009; 46: 381– 3. Google Scholar CrossRef Search ADS PubMed  13 Barrabeig I, Costa J, Rovira A, et al.   Viral etiology of mumps-like illnesses in suspected mumps cases reported in Catalonia, Spain. Hum Vaccin Immunother  2015 11: 282– 7. Google Scholar CrossRef Search ADS PubMed  14 Davidkin I, Jokinen S, Paananen A, et al.   Etiology of mumps-like illnesses in children and adolescents vaccinated for measles, mumps, and rubella. J Infect Dis  2005; 191: 719– 23. Google Scholar CrossRef Search ADS PubMed  15 Italian Ministry of Health. Piano Nazionale per l'Eliminazione del Morbillo edella Rosolia congenita (PNEMoRc). Rome: Ministero della Salute. Italian. Available from: http://www.salute.gov.it/imgs/C 17 pubblicazioni 1519 allegato.pdf 16 Italia Circolare del Ministero della Salute DGPRE n. 4460-P-20/02/2013.Istituzione di un sistema di sorveglianza integrato per il morbillo e la rosolia alla luce del nuovo Piano Nazionale di Eliminazione del morbillo e della rosolia congenita 2010–2015. http://www.trovanorme.salute.gov.it/norme/renderNormsanPdf?anno=0&codLeg= 48172&parte=1%20&serie 17 Magurano F, Baggieri M, Fortuna C, et al.   Measles elimination in Italy: data from laboratory activity, 2011-2013. J Clin Virol  2015; 64: 34– 9. Google Scholar CrossRef Search ADS PubMed  18 Centers for Disease Control and Prevention (CDC). Real-time (TaqMan®) RT-PCR Assay for the Detection of Mumps Virus RNA in Clinical Samples. Available at: https://www.cdc.gov/mumps/downloads/lab-rt-pcr-assay-detect.pdf 19 Brengel-Pesce K, Morand P, Schmuck A, et al.   Routine use of real-time quantitative PCR for laboratory diagnosis of Epstein-Barr virus infections. J Med Virol  2002; 66: 360– 9. Google Scholar CrossRef Search ADS PubMed  20 Jin L, Beard S, Brown DW. Genetic heterogeneity of mumps virus in the United Kingdom: identification of two new genotypes. J Infect Dis  1999; 180: 829– 33. Google Scholar CrossRef Search ADS PubMed  21 Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT/. Nucl Acids Symp  1999; 41: 95– 8. 22 Tamura K, Stecher G, Peterson D, et al.   MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol  2013; 30: 2725– 9. Google Scholar CrossRef Search ADS PubMed  23 Khanim F, Yao QY, Niedobitek G, et al.   Analysis of Epstein-Barr virus gene polymorphisms in normal donors and in virus-associated tumors from different geographic locations. Blood  88: 3491– 501. PubMed  24 Cohen C, White JM, Savage EJ, et al.   Vaccine effectiveness estimates, 2004-2005 Mumps outbreak, England. Emerging Infect Dis  2007; 13: 12– 7. Google Scholar PubMed  25 Harling R, White JM, Ramsay ME, et al.   The effectiveness of the mumps component of the MMR vaccine: a case control study. Vaccine  2005; 23: 4070– 4. Google Scholar CrossRef Search ADS PubMed  26 Brockhoff HJ, Mollema L, Sonder GJ, et al.   Mumps outbreak in a highly vaccinated student population, The Netherlands, 2004. Vaccine  2009; 28: 2932– 6. Google Scholar CrossRef Search ADS   27 Hukic M, Hajdarpasic A, Ravlija J, et al.   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. Euro Surveill  2014; 19:pii: 20879. 28 Johnstone JA, Ross CA, Dunn M. Meningitis and encephalitis associated with mumps infection. A 10-year survey. Arch Dis Child  1972; 47: 647– 51. Google Scholar CrossRef Search ADS PubMed  29 Ritter BS. Mumps meningoencephalitis in children. J Pediatr  1958; 52: 424– 33. Google Scholar CrossRef Search ADS PubMed  30 Guy RJ, Andrews RM, Kelly HA, et al.   Mumps and rubella: a year of enhanced surveillance and laboratory testing. Epidemiol Infect  2004; 132: 391– 8. Google Scholar CrossRef Search ADS PubMed  31 WHO. Mumps virus vaccines. Weekly Epidemiol Record  2007; 82: 49– 60. 32 Peltola H, Jokinen S, Paunio M, et al.   Measles, mumps, and rubella in Finland: 25 years of a nationwide elimination programme. Lancet Infect Dis  2008; 8: 796– 803. Google Scholar CrossRef Search ADS PubMed  33 Dias JA, Cordeiro M, Afzal MA, et al.   Mumps epidemic in Portugal despite high vaccine coverage—preliminary report. Eurosurveillance  1996; 1: 25– 28. Google Scholar CrossRef Search ADS PubMed  © The Author 2017. Published by Oxford University Press on behalf of the European Public Health Association. All rights reserved. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The European Journal of Public Health Oxford University Press

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Abstract

Abstract Background During recent years, various mumps outbreaks have occurred among populations vaccinated for mumps worldwide. In Italy, improving routine coverage with two doses of measles, mumps and rubella (MMR) vaccine is one of the key strategies to eliminate measles and rubella. To monitor the effect of the vaccination programme on the population, the surveillance of these vaccine-preventable diseases has been implemented. This provided the opportunity to evaluate the accuracy of the clinical diagnosis of those diseases, including mumps. In fact, vaccinated children may develop a variety of diseases caused by a series of different viruses [Epstein-Barr virus (EBV), parainfluenza virus types 1–3, adenoviruses, herpes virus and parvovirus B19] whose symptoms (i.e. swelling of parotid glands) may mimic mumps. For this reason, laboratory diagnosis is essential to confirm clinical suspicion. Methods The accuracy of clinical diagnosis of mumps was evaluated by differential diagnosis on EBV in Italy, a country at low incidence of mumps. This retrospective study investigated whether the etiology of 131 suspected mumps cases with a negative molecular/serological result for mumps virus, obtained from 2007 to 2016, were due to EBV, in order to establish a diagnosis. Results Differential diagnosis revealed a EBV positivity rate of 19.8% and all cases were caused by EBV type 1. Conclusions This study confirms the importance of a lab based differential diagnosis that can discriminate between different infectious diseases presenting with symptoms suggestive of mumps and, in particular, emphasize the importance to discriminate between mumps and EBV-related mononucleosis. Introduction Mumps is caused by a virus (MuV) belonging to the family Paramixoviridae.1,2 The disease usually occurs among children and in the pre-vaccine era the annual reported mumps incidence in Western European countries ranged between 100 and 600 per 100 000 inhabitants.3 With the availability of a live-attenuated mumps vaccine since the 1960s,4 disease incidence dramatically decreased in countries with mumps vaccination programmes.5,6 However, several outbreaks of mumps have occurred among measles, mumps and rubella (MMR) vaccinated individuals in various countries worldwide.7–10 Like measles and rubella (MR), mumps is efficiently transmitted from person to person. Before the start of extended programmes of immunization, mumps was typically a childhood disease, with the highest incidence among children between 5 and 9 years of age, presenting with a generally benign course, which is asymptomatic in about one third of the infected children. However, mumps may affect people of any age, causing more severe complications among adults. Clinical mumps is defined as the acute onset of unilateral or bilateral tender, self-limiting swelling of the parotid or other salivary glands (from which the popular name ‘mumps’), lasting two or more days without other apparent cause. Swelling of the parotid glands is the most important clinical sign and may result in pain associated with chewing and swallowing. Fever and malaise are also common. Chills, headache, and a slight rise in temperature may occur ∼24 h before the onset of parotid swelling. In total 15–20% of mumps infections can be asymptomatic and about 50% are associated with non-specific or respiratory symptoms.11–13 Children with mumps usually recover within few days after symptoms onset. The most common complications include encephalitis (0.02–0.3%), meningitis (0.5–15%), pancreatitis (4%) and hearing loss. Finally, infection during the first 12 weeks of pregnancy is associated with a high percentage of miscarriages (25%), but not the risk of fetal malformations.2 When parotitis is present during a mumps outbreak or epidemic, the clinical diagnosis of mumps is generally straightforward. However, when the incidence rate of mumps is low, other causes of parotitis, in particular viral infections such as those due to the Epstein-Barr virus (EBV), parainfluenza viruses, influenza A virus, coxsackieviruses, adenoviruses, parvovirus B19, lymphocytic choriomeningitis virus and HIV, should be considered.12,14 For this reason, an approach based on the laboratory testing is essential to confirm the clinical suspicion of mumps, especially in areas where the incidence of the disease is low. In Italy, mumps diagnosis is performed within the framework of MR surveillance. Indeed, to support case ascertainment, the National Reference Laboratory (NRL) for MR performs laboratory surveillance to confirm suspected cases of measles, rubella and mumps.15 In February 2013, the Italian Minister of Health published a document which regulates the integrated surveillance for MR in which indications about the laboratory mumps surveillance were further included.16 This retrospective study investigated whether the etiology of Italian sporadic suspected mumps cases with a negative molecular/serological result for mumps virus obtained at the NRL, in 2007–16, were due to EBV, in order to establish a diagnosis. Methods Mumps case definition The case definition and classification is that stipulated by EU Commission Decision of 8 August 2012. Clinical criteria Fever and at least two of the following: sudden onset of unilateral or bilateral tender swelling of the parotid or other salivary glands without other apparent cause or orchitis or meningitis. Laboratory criteria At least two of the following (i) isolation of mumps virus from a clinical specimen; (ii) detection of mumps virus nucleic acid; (iii) MuV specific antibody response characteristic for acute infection in serum or saliva. Epidemiological criteria An epidemiological link by human-to-human transmission. Case classification includes ‘possible case’ (any person meeting the clinical criteria), ‘probable case’ (any person meeting the clinical criteria and with an epidemiological link), ‘confirmed case’ (any person not recently vaccinated and meeting the clinical and the laboratory criteria). Study population and clinical samples During the period between June 2007 and 2016, oral and/or blood samples of 193 suspected cases of mumps were sent to the NRL from various Italian regions for laboratory confirmation of the clinical diagnosis. All these were clinically compatible and sporadic cases, not linked to outbreak settings. The vaccination status was known for 190/193 patients: 42 had never received a vaccine against mumps (41.6%), 79 were vaccinated with 1 dose (36.3%), 69 with 2 doses (22.1%). Mumps virus genome can be detected from oral fluid within the first week after symptoms onset. Oral fluid from suspected mumps cases were tested for mumps by RT Real-Time PCR, while blood samples were tested for specific IgM anti-mumps detection by Elisa. Oral fluid samples still available of those negative patients were further tested for EBV by PCR. Serological diagnosis for mumps The detection of anti-mumps IgM was performed with the Enzygnost Anti-Parotitis Virus/IgM kit (Dade/Behring, Siemens) on blood samples collected and treated as previous described in.17 Molecular detection of mumps and EBV RNA and DNA were extracted from oral fluid specimens using QIAmp Viral RNA Kit and QIAamp DNA Mini Kit (Qiagen), respectively, according to the manufacturer's instructions. A 7 µl aliquot of RNA was used for a reverse transcription PCR Real-time with the RealTime Ready RNA Virus Master kit (Roche) according to CDC’s indications.18 A portion of 169 bp of the BXLF1 gene of EBV was amplified by PCR. The reaction was performed with PCR Supermix (Invitrogen), 10 pmol of each forward (EBV1 5′-GGGGCAAAATACTGTGTTAG-3′, position 143 411) and reverse primers (EBV2 5′-CGGGGGACACCATAGT-3′, position 143 579), and 3 µl of extracted DNA.19 The cycling conditions consisted of an initial denaturation of 10 s at 95°C, followed by 45 cycles of 40 s at 95°C, 1 min at 58°C and 40 s at 72°C and a final extension of 5 min at 72°C. Genetic analysis Samples positive for MuV were further amplified for genotyping by PCR followed by a Nested PCR on the MuV SH gene,20 using the SuperScript One-Step RT-PCR with PlatinumR Taq System and PCR SuperMix kits (Invitrogen), respectively. Before sequencing, PCR products were purified with the QIAquick PCR Purification Kit (QIAGEN) and sequencing reactions performed by Macrogen Inc. (Seoul, South Korea). Nucleotide sequences were aligned with sequences of the reference strains and with those that showed a high percentage of identity after Blast analysis, using CLUSTAL W (BioEdit) software.21 The Bayesian Information Criterion was used to determine the model of nucleotide substitution that best fit the data using the selection tool available in MEGA6.22 Evolutionary analyses were conducted using the maximum likelihood method based on the Tamura 3-parameter (T92) model and evolutionary rates among sites were modelled by a discrete Gamma distribution (+G). Samples positive for EBV were tested by PCR to amplify a portion of the gene EBNA3C in order to discriminate between EBV genotype type 1 or type 2.23 PCR was performed with PCR Supermix (Invitrogen) with 5 min at 95°C, followed by 35 cycles of 45 s at 95°C, 45 s at 56°C and 1 min at 72°C and 10 min at 72°C. Amplicons were analysed by electrophoresis on 1.5% agarose gel and gel-red staining. Results From June 2007 to 2016, 148 oral fluid and 169 blood samples from a total of 193 patients with suspect mumps were collected and tested at the NRL. As reported in table 1, 11/193 (5.7%) patients were found positive for MuV infection either by serological or molecular assay, and 182 were negative. Three cases were positive by IgM serology but negative by PCR probably due a bad sampling. Detailed results and vaccination status for each positive patient are reported in table 2. Vaccination status was available for 9 out of 11 positive cases: 5 mumps infected patients had received one dose of MMR vaccine, 2 patients had received two doses and 2 were not vaccinated. For five patients, it was possible to calculate the time elapsed after vaccination (ranging from 2.5 to 13 years). For those negative cases, 66.5% (121/182) had received at least one dose of vaccine against mumps while 20.9% (38/182) were not vaccinated. Table 1 Results obtained for mumps diagnosis by molecular and serological tests and for EBV by molecular tests     2007    2008  2009  2010  2011  2012  2013  2014  2015  2016  Total  Mumps  pos  4  1  1  0  1  0  0  3  0  1  11  neg  45  41  13  5  14  25  17  10  9  3  182  tested  49  42  14  5  15  25  17  13  9  4  193  EBV  pos  12  10  4  0  0  0  0  0  0  0  26  neg  33  30  3  0  0  13  9  6  8  3  105  tested  45  40  7  0  0  13  9  6  8  3  131      2007    2008  2009  2010  2011  2012  2013  2014  2015  2016  Total  Mumps  pos  4  1  1  0  1  0  0  3  0  1  11  neg  45  41  13  5  14  25  17  10  9  3  182  tested  49  42  14  5  15  25  17  13  9  4  193  EBV  pos  12  10  4  0  0  0  0  0  0  0  26  neg  33  30  3  0  0  13  9  6  8  3  105  tested  45  40  7  0  0  13  9  6  8  3  131  Table 2 Patients positive for mumps tests and their vaccination status   Year  PCR  IgM  Genotype  Age  Vaccination status  Pt 229  2007  –  POS  –  17 months  1 dose  Pt 256  2007  NEG  POS  –  4 years  1 dose  Pt 419  2007  POS  POS  H  13 years  1 dose  Pt 427  2007  –  POS  –  13 years  NA  Pt 1368  2008  –  POS  –  46 years  Not vaccinated  Pt 1806  2009  NEG  POS  –  6 years  1 dose  Pt 2322  2011  POS  –  G  15 years  1 dose  Pt 3060  2014  POS  BL  G  18 years  2 doses  Pt 3143  2014  POS  POS  G  19 years  NA  Pt 3196  2014  NEG  POS  –  6 years  2 DOSES  Pt 3398  2016  POS  POS  G  29 years  Not vaccinated    Year  PCR  IgM  Genotype  Age  Vaccination status  Pt 229  2007  –  POS  –  17 months  1 dose  Pt 256  2007  NEG  POS  –  4 years  1 dose  Pt 419  2007  POS  POS  H  13 years  1 dose  Pt 427  2007  –  POS  –  13 years  NA  Pt 1368  2008  –  POS  –  46 years  Not vaccinated  Pt 1806  2009  NEG  POS  –  6 years  1 dose  Pt 2322  2011  POS  –  G  15 years  1 dose  Pt 3060  2014  POS  BL  G  18 years  2 doses  Pt 3143  2014  POS  POS  G  19 years  NA  Pt 3196  2014  NEG  POS  –  6 years  2 DOSES  Pt 3398  2016  POS  POS  G  29 years  Not vaccinated  NA, not applicable. Phylogenetic analysis was performed on 5 MuV sequences obtained from samples positive in PCR. As shown in figure 1, four strains belonged to genotype G (MuVs/Salerno.ITA/4.14/, MuVs/Bolzano.ITA/18.11/, MuVs/Livorno.ITA/24.14/, MuVs/Livorno.ITA/20.16/) and one to genotype H (MuVs/Livorno.ITA/41.07/). Sequences were deposited in GenBank database under accession numbers KX518652, KX518653, KX518654, KX518655, KX518656. Figure 1 View largeDownload slide Neighbour-joining tree for nucleotide sequences of mumps strains identified from 2007 to 2016 Figure 1 View largeDownload slide Neighbour-joining tree for nucleotide sequences of mumps strains identified from 2007 to 2016 WHO data show that genotype G has been reported in Europe, North America, South-East Asia, while genotype H has been reported also from South America and Africa. BLAST analysis showed that the same strain identified in Salerno in 2014 circulated in Europe in 2012 and 2013 and in USA in 2016. The strain that circulated in Bolzano in 2011 was also identified in Germany in the same year. No strains identical to those identified in Livorno in 2014 and 2016 have been ever reported. In addition, the unique strain belonging to the genotype H, identified in Livorno in 2007, did not show identity with any other strains after BLAST analysis. Oral fluid samples available for 131 out of 182 mumps negative cases were further tested for EBV by PCR; of them, 26 were found positive (positivity rate of 19.8%) for viral DNA (table 1). Beside genetic analysis on mumps strains, EBV positive samples were tested by PCR to distinguish between genotypes type 1 or 2, and all of them belonged to genotype 1. The incidence trend of new cases of mumps in Italy from 1996 to 2014 shows a series of oscillations, with a maximum of almost 65 000 cases reported in 1996 (figure 2). Since 1999, the incidence of mumps declined to a minimum number of 191 cases reported in 2014. This decline was probably due to MMR vaccination campaigns. Studies established that the effectiveness of any MMR vaccination in patients with a history at least one MMR vaccination adjusted for age, sex and general practice was 69% (95% CI: 41–84%)24,25 and because of the low effectiveness of the mumps MMR vaccine component, several outbreaks occurred in Europe.26,27 Figure 2 View largeDownload slide Trend of mumps cases in Italy from 1996 to 2014 (Data from: Ministry of Health and European Center for Disease Prevention and Control) Figure 2 View largeDownload slide Trend of mumps cases in Italy from 1996 to 2014 (Data from: Ministry of Health and European Center for Disease Prevention and Control) Also, the decreased efficiency of the surveillance system, leading to a low notification rate, was likely to contribute to the low number of cases reported in Italy in the last years. The standard clinical case definition of mumps used for surveillance activities consisted in ‘acute onset of unilateral or bilateral swelling of the parotid or other salivary glands lasting two or more days without any other apparent cause’.2 However, although parotitis is indeed the hallmark of mumps, there are cases in which salivary-gland swelling is not apparent, especially in individuals with mumps meningitis, many of whom do not present detectable salivary-gland enlargement.28,29 Moreover, other infectious agents may also cause salivary-gland swelling. The effect of such alternative aetiologies greatly reduces the positive predictive value of a clinical diagnosis when the disease incidence is low.30 This study reports results from the differential diagnosis of mumps with EBV-related mononucleosis provides information on the specificity of the clinical diagnosis of mumps, suggesting the importance of laboratory confirmation. Our findings show that the specificity of the case-definition of mumps is low. Studies conducted in other areas of the world provided similar results. In a study conducted in Victoria, Australia, only 7 (9%) of 74 cases clinically diagnosed as mumps parotitis could be confirmed by serology; 7 (16%) of 43 laboratory-rejected cases were positive for EBV using serological testing.30 In a study conducted in Finland, on 601 acutely ill children presenting mumps-like symptoms but seronegative for mumps, the most commonly identified viral agents were the EBV (7%), parainfluenza virus (4%) and adenovirus (3%).31 These studies highlight the importance of laboratory confirmation in diagnosing mumps, especially under non-outbreak conditions. Discussion According to WHO, introduction of routine mumps vaccination, such as other prophylactic options,31 should be a high priority. Most European health systems provide mumps vaccine in combination with MMR, with a two-dose vaccination schedule, free of charge, and some 120 countries have introduced vaccination against mumps in their national immunization programmes. To date, countries such as Finland or Sweden have completely eradicated mumps from their national territory.32 Actions should be implemented to encourage practitioners to collect oral and blood samples from mumps suspected cases and to submit these samples to the NRL or other reference labs that performs mumps virus PCR and serology. This is of special importance when the patient is vaccinated and a primary or secondary vaccination failure is suspected, being important both for individual patients and for monitoring the outcome of vaccination programmes. About that, our study revealed that three patients positive for mumps had been vaccinated before the introduction (in 2001) of the more efficient component Urabe AM 9 in the MMR vaccine in spite of the Rubini strain, responsible for some vaccine failure.33 In conclusion, the results of this study confirm the importance of a lab-based differential diagnosis that can discriminate between different infectious diseases presenting with symptoms suggestive of mumps and emphasize the importance to discriminate between mumps and EBV-related mononucleosis. Finally, the large proportion of negative results suggests that other viral infections are involved in the genesis of mumps-like syndromes. Acknowledgements We wish to thank Dr C. Fortuna and Mrs E. Benedetti for technical support, and the staff of Italian Regional and Local Health Authorities for providing clinical specimens. Funding This work was partially funded by the Italian Ministry of Health grant CCM 2015-6M21. Conflicts of interest: None declared. Key points The specificity of the case-definition of mumps is low and a large number of viral infections are involved in the genesis of mumps-like syndromes such as Epstein-Barr virus. A lab-based differential diagnosis is essential to discriminate between different infectious diseases, especially for the large proportion of mumps negative cases. Low efficiency of the surveillance system for mumps, leading to a low notification rate, contribute to the low number of cases reported in Italy in the last years. References 1 King AMQ, Adams MJ, Carstens EB, Lefkowitz EJ. Virus Taxonomy: Classification and Nomenclature of Viruses: Ninth Report of the International Committee on Taxonomy of Viruses . San Diego: Elsevier Academic Press, 2012. 2 Hviid A, Rubin S, Mühlemann K. Mumps. Lancet  2008; 371: 932– 44. Google Scholar CrossRef Search ADS PubMed  3 Levy-Bruhl D, Pebody R, Veldhuijzen I, Valenciano M, Osborne K. ESEN: a comparison of vaccination programmes - part three: measles mumps and rubella. Euro Surveill  1998; 3: 115– 119. Google Scholar CrossRef Search ADS PubMed  4 Galazka AM, Robertson SE, Kraigher A. Mumps and mumps vaccine: a global review. Bull World Health Organ  1999; 77: 3– 14. Google Scholar PubMed  5 Savage E, Ramsay M, White J, et al.   Mumps outbreaks across England and Wales in 2004: observational study. BMJ  2005; 330: 1119– 20. Google Scholar CrossRef Search ADS PubMed  6 Lievano F, Galea SA, Thornton M, et al.   Measles, mumps, and rubella virus vaccine (M-M-RII): A review of 32 years of clinical and postmarketing experience. Vaccine  2012; 30: 6918– 26. Google Scholar CrossRef Search ADS PubMed  7 Vygen S, Fischer A, Meurice L, et al.   Waning immunity against mumps in vaccinated young adults, France 2013. Euro Surveill  2016; 21: 8 Gee S, O’Flanagan D, Fitzgerald M, Cotter S. Mumps in Ireland, 2004-2008. Euro Surveill  2008; 13: 18857. Google Scholar CrossRef Search ADS PubMed  9 Mossong J, Bonert C, Weicherding P, Opp M, Reichert P, Even J, et al.   Mumps outbreak among the military in Luxembourg in 2008: epidemiology and evaluation of control measures. Euro Surveill  2009; 14:pii 19121. 10 Yung CF, Andrews N, Bukasa A, et al.   Mumps complications and effects of mumps vaccination, England and Wales, 2002-2006. Emerg Infect Dis  2011; 17: 661– 7. Google Scholar CrossRef Search ADS PubMed  11 WHO. Global Status of mumps immunization and surveillance. Weekly Epidemiol Record  2005; 48: 418– 24. 12 Hatchette TF, Mahony JB, Chong S, LeBlanc JJ. Difficulty with mumps diagnosis: What is the contribution of mumps mimickers. J Clin Virol  2009; 46: 381– 3. Google Scholar CrossRef Search ADS PubMed  13 Barrabeig I, Costa J, Rovira A, et al.   Viral etiology of mumps-like illnesses in suspected mumps cases reported in Catalonia, Spain. Hum Vaccin Immunother  2015 11: 282– 7. Google Scholar CrossRef Search ADS PubMed  14 Davidkin I, Jokinen S, Paananen A, et al.   Etiology of mumps-like illnesses in children and adolescents vaccinated for measles, mumps, and rubella. J Infect Dis  2005; 191: 719– 23. Google Scholar CrossRef Search ADS PubMed  15 Italian Ministry of Health. Piano Nazionale per l'Eliminazione del Morbillo edella Rosolia congenita (PNEMoRc). Rome: Ministero della Salute. Italian. Available from: http://www.salute.gov.it/imgs/C 17 pubblicazioni 1519 allegato.pdf 16 Italia Circolare del Ministero della Salute DGPRE n. 4460-P-20/02/2013.Istituzione di un sistema di sorveglianza integrato per il morbillo e la rosolia alla luce del nuovo Piano Nazionale di Eliminazione del morbillo e della rosolia congenita 2010–2015. http://www.trovanorme.salute.gov.it/norme/renderNormsanPdf?anno=0&codLeg= 48172&parte=1%20&serie 17 Magurano F, Baggieri M, Fortuna C, et al.   Measles elimination in Italy: data from laboratory activity, 2011-2013. J Clin Virol  2015; 64: 34– 9. Google Scholar CrossRef Search ADS PubMed  18 Centers for Disease Control and Prevention (CDC). Real-time (TaqMan®) RT-PCR Assay for the Detection of Mumps Virus RNA in Clinical Samples. Available at: https://www.cdc.gov/mumps/downloads/lab-rt-pcr-assay-detect.pdf 19 Brengel-Pesce K, Morand P, Schmuck A, et al.   Routine use of real-time quantitative PCR for laboratory diagnosis of Epstein-Barr virus infections. J Med Virol  2002; 66: 360– 9. Google Scholar CrossRef Search ADS PubMed  20 Jin L, Beard S, Brown DW. Genetic heterogeneity of mumps virus in the United Kingdom: identification of two new genotypes. J Infect Dis  1999; 180: 829– 33. Google Scholar CrossRef Search ADS PubMed  21 Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT/. Nucl Acids Symp  1999; 41: 95– 8. 22 Tamura K, Stecher G, Peterson D, et al.   MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol  2013; 30: 2725– 9. Google Scholar CrossRef Search ADS PubMed  23 Khanim F, Yao QY, Niedobitek G, et al.   Analysis of Epstein-Barr virus gene polymorphisms in normal donors and in virus-associated tumors from different geographic locations. Blood  88: 3491– 501. PubMed  24 Cohen C, White JM, Savage EJ, et al.   Vaccine effectiveness estimates, 2004-2005 Mumps outbreak, England. Emerging Infect Dis  2007; 13: 12– 7. Google Scholar PubMed  25 Harling R, White JM, Ramsay ME, et al.   The effectiveness of the mumps component of the MMR vaccine: a case control study. Vaccine  2005; 23: 4070– 4. Google Scholar CrossRef Search ADS PubMed  26 Brockhoff HJ, Mollema L, Sonder GJ, et al.   Mumps outbreak in a highly vaccinated student population, The Netherlands, 2004. Vaccine  2009; 28: 2932– 6. Google Scholar CrossRef Search ADS   27 Hukic M, Hajdarpasic A, Ravlija J, et al.   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. Euro Surveill  2014; 19:pii: 20879. 28 Johnstone JA, Ross CA, Dunn M. Meningitis and encephalitis associated with mumps infection. A 10-year survey. Arch Dis Child  1972; 47: 647– 51. Google Scholar CrossRef Search ADS PubMed  29 Ritter BS. Mumps meningoencephalitis in children. J Pediatr  1958; 52: 424– 33. Google Scholar CrossRef Search ADS PubMed  30 Guy RJ, Andrews RM, Kelly HA, et al.   Mumps and rubella: a year of enhanced surveillance and laboratory testing. Epidemiol Infect  2004; 132: 391– 8. Google Scholar CrossRef Search ADS PubMed  31 WHO. Mumps virus vaccines. 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The European Journal of Public HealthOxford University Press

Published: Feb 1, 2018

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