|Year : 2015 | Volume
| Issue : 3 | Page : 378-382
Mumps disease outbreak in Davangere district of Karnataka, India
CG Raut, DP Sinha, H Jayaprakash, H Hanumiah, MJ Manjunatha
National Institute of Virology, Bangalore Unit, Bangalore Near NIMHANS, Karnataka - 560 029, India
|Date of Submission||27-May-2014|
|Date of Acceptance||05-Feb-2015|
|Date of Web Publication||12-Jun-2015|
C G Raut
National Institute of Virology, Bangalore Unit, Bangalore Near NIMHANS, Karnataka - 560 029
Source of Support: None, Conflict of Interest: None
Background: Mumps is a vaccine-preventable disease that usually occurs as a parotitis, but it can also lead to several life- threatening complications, including pancreatitis, meningitis and encephalitis. Objective: To determine and diagnosis of mumps disease, which is communicable disease usually affects childrens. Although it is seen worldwide, but outbreaks not common in India. Materials and Methods: Thirty one suspected mumps cases, who presented to the unimmunized population of Chikkahallivana village in Davangere district of Karnataka, India in January 2014, with clinical evidence of fever, cervical lymphadenitis and ear pain, manifest with self-limited uni-or bilateral parotitis. A total of 31 cases consisting of 31 blood and 31 throat swabs were tested for diagnosis of mumps disease. Results: Of the 31 suspected cases, laboratory results showed 18 positive for mumps IgM antibodies and 7 cases showed presence of mumps virus RNA by RT-PCR using MV specific nested primers. From 31 cases, 5 were positive with both the methods. Conclusion: We confirmed the cases by serological as well as a sensitive RT-nested PCR-based method and sequencing results for the molecular identiﬁcation of mumps infection. Sequencing results of the SH gene identified outbreak strain as genotype C, which was consistent with other outbreaks in India.
Keywords: IgM and RT-PCR, mumps disease, unimmunized
|How to cite this article:|
Raut C G, Sinha D P, Jayaprakash H, Hanumiah H, Manjunatha M J. Mumps disease outbreak in Davangere district of Karnataka, India. Indian J Med Microbiol 2015;33:378-82
|How to cite this URL:|
Raut C G, Sinha D P, Jayaprakash H, Hanumiah H, Manjunatha M J. Mumps disease outbreak in Davangere district of Karnataka, India. Indian J Med Microbiol [serial online] 2015 [cited 2019 Nov 19];33:378-82. Available from: http://www.ijmm.org/text.asp?2015/33/3/378/158558
| ~ Introduction|| |
Mumps disease is caused by Mumps virus (MV), which belongs to the genus Rubulavirus of the subfamily Paramyxovirinae in the family Paramyxoviridae. MV is antigenically monotypic and spreads by respiratory droplets. Mumps is a vaccine-preventable disease that is endemic in most parts of the world. 
Mumps disease outbreak occurred in an unvaccinated population of Chikkahallivana village in Davangere district of Karnataka, India in January 2014. Most of the affected unimmunized population belonged to a worker community who opted out of scheduled vaccination. The disease was managed using provincial mumps control guidelines, including programmes for enhanced surveillance and public awareness, in the specific geographic affected region. After the outbreak resolved, we utilized clinical and laboratory data to investigate the period of mumps virus detection, the onset of parotitis and to diagnose laboratory findings with mumps status.
Following natural infection, 10% of infected individuals remain asymptomatic: 40-50% develops a nonspecific respiratory prodrome and ear pain, and the remaining 30-40% manifest with self-limited uni-or bilateral parotitis and cervical lymphadenitis. However, the virus can cause severe complication including meningitis, deafness, pancreatitis and orchitis. 
The epidemic curve is presented in [Figure 1], with the beginning of the outbreak in the second week of January, the peak in the last week of January, and the last cases in the first date of February.
|Figure 1: Number of cases of mumps reported during the period from 2nd week in January 2014 till the first week of February, 2014|
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Mumps vaccination has been incorporated into the regular immunization schedule of many countries, usually along with measles and rubella vaccines in a triple formulation. These vaccines have enabled the WHO to establish global strategies for the advanced control of measles and rubella leading to an elimination target in some regions. However, in contrast to rubella and measles, secondary vaccine failure occurs frequently in the case of mumps and circulation of MV within highly vaccinated populations has been frequently reported. ,,,,,
Twelve different genotypes of MV based on genetic variation in the SH gene are currently recognized by the WHO. ,,
MV shows a distinct geographic distribution where more than one genotype may circulate simultaneously in a region.  The magnitude and epidemiology of mumps infection in India are poorly understood. There are few literatures available in PubMed related to mumps virus circulation in India. ,,,
However, detailed investigations on sporadic cases or outbreak (s) of mumps are necessary to understand the circulation of wild type viruses in the context of the global epidemiology of mumps. , This study describes diagnostic tests performed for lab confirmation of mumps cases from a village-Chikkahallivana of Karnataka state, India.
| ~ Materials and Methods|| |
Chikkahallivana is a small village in Davangare district, Karnataka. The district headquarters of Davangare is located at National Highway-4 (Chennai to Mumbai National Highway), Chikkahallivana village is 40 kms away from the district of Davangare and has an estimated population of 364 being rural. The village dwelling setting has two lanes namely, Lane-1 and Lane-2.
A cross-sectional survey was carried out by three members from the National Institute of Virology, Bangalore Unit with technical support from state Government district medical officer and it was decided that any identified case will be provided necessary treatment at the earliest possible time.
The Chikkahallivana village was covered under this study. The contact was made with the village members to seek their cooperation for the investigation. A total village was surveyed by an investigating team in village who reported having mumps cases. The standard case definition was used for diagnosis of mumps. A disease symptoms criteria was used to clinically identify the mumps cases. A study subject was considered to have mumps if presented with one of the symptoms criteria and any of the minor criteria. 
Only the clinical definition of mumps was considered as the index case was already evaluated by experts.  The household was a sampling unit and the villagers were extensively covered by house-to-house visits made by the teams. With regard to the uni-or bilateral parotitis or fever cases in the household in the last 3 weeks, if any such case was found, extensive information was collected, for that, study subject enquiries about mumps cases were made from the mothers or responsible persons. Information was collected regarding age, gender, Table-1 history of mumps in the last 3 weeks.
Samples were collected from a total of 31 patients. Distribution of the samples was 31 blood and 31 throat swabs. Throat swabs were collected in virus transport medium. All the samples were carried on cold chain to the laboratory for diagnosis, according to WHO guidelines for specimen collection of mumps virus detection. 
The blood samples were processed to have the best quality serum and further tested for Mumps IgM antibodies. The test was done with standard positive control using IgM ELISA kit (Calbiotech, CA) as per the protocol. The kit is used for qualitative detection and quantitative determination of specific IgM antibodies to Mumps virus in human serum.
Total RNA was extracted from 140 μl of throat swab and blood samples using a QIAmp viral RNA kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. RNA was eluted in 50 μl of nuclease free water. For the conventional PCR gel-based platform, a minimum of one standard positive control RNA and one negative control along with samples of target RNA included.
We used diagnostic RNA virus based nested reverse-transcriptase PCR (RT-PCR) to detect mumps virus from clinical samples.  A precautions were considered to prevent cross-contamination.
First round of RT-PCR was done using a standard primer pairs (PRV1 forward primer 5'- CGCGTGGTCTACGGGGACACGGA-3' and PRV1 reverse primer 5'- ATGACGCC GATGTACTTCTTCTT-3') to amplify a 600 bp fragment in the SH gene which included the 456 bp amplicon recommended for genotyping.  PCR conditions for RT-PCR were reverse transcription step at 42°C for 45 min; followed by 94°C for 2 min for pre-denaturation and denaturation at 94°C for 1 min, annealing at 47°C for 1 min and extension 68°C for 2 min, and an extension at 72°C for 7 min for 35 cycles. Thermocycling was done in a PCR system (Applied Biosystems, Foster City, CA) without an oil overlay.
The second PCR round was performed including 1 μl of undiluted ﬁrst PCR product as template in a reaction containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 200 μM each dNTP, 2.5 mM MgCl 2 , 2.5 units of Taq polymerase (Qiagen, Germany), 10 pmol each of internal control primers PRV2-(5'- GGGACACGGACTCGGTCTTC-3') and PRV2-(5'- CCGGAAGG TCTTCTCGCACTC-3') and 10 pmol each of forward and reverse primers. Nested PCR assays were subjected to an initial cycle of 94°C for 2 min and 30 cycles of 94°C for 1 min, 50°C for 1 min and 72°C for 1 min, followed by ﬁnal incubation at 72°C for 5 min. After amplification, 10 μl portion of each product was analyzed by agarose gel electrophoresis utilizing 2.0% agarose gel (Sigma Aldrich) and gels were stained with ethidium bromide at 5 μg/ml and visualized by Gel documentation system (Bio-Rad, USA) using the software Quantity One version 4.6.3 to determine the band size. A 1 kb DNA ladder (Invitrogen, Life Technologies) was used as a molecular ladder.
Nucleotide sequencing for genotyping and phylogenetic analysis
The positive mumps viruses of second round PCR products of representative samples were sequenced by dideoxy terminator sequencing method and comparison of the nucleotide sequence was performed using the NCBI BLAST programme with the GenBank database. Mumps SH-gene sequence obtained in this study confirmed with both strands and aligned with WHO reference SH-gene sequence  using software ClustalW version 1.83. The viral-DNA sequence was also deposited in Genbank (http://www.ncbi.nlm.nih.gov).
| ~ Results|| |
Serological investigation of the clinical samples showed 18 children (58%) positive for mumps IgM antibodies, from the total 31 samples subjected to serological analysis. In majority, children below the age of 10 years were found positive for mumps IgM antibodies. Total of six children were found positive between the age of 1-6 years, 11 children between the age of 7-13 years and one positive case of 30 age. Out of total 18 children, five boys and 13 girls were found positive for the mumps IgM antibodies [Table 1]. The results revealed girls were predominantly affected with the mumps disease as compare to boys [Figure 2].
|Figure 2: Mumps samples age and sex- wise distribution in Chikkahallivana village|
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Molecular assay results
Mumps virus was detected from throat swab, using nested RT-PCR and PCR product size were 448bp with primers targeting the SH genes [Figure 3].
|Figure 3: Detection of SH gene by Nested PCR. Lane M: DNA Ladder (1 kb); Lane 1 to 7: Positive Samples; Lane 8: Negative Control; Lane 9: Positive Control|
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Out of 31 cases, 7 (22%) were confirmed by RT-PCR. All acute phase specimens are positive by RT-PCR. The age group involved was 1-15 yrs. It included two boys and five girls positive with RT-PCR results.
Sequence information revealed that the sequence from infected mumps virus share 99% homology with Mumps virus strain MuVi/Chennai.IND/45.12 small hydrophobic protein (SH) gene, complete cds having Genbank accession no. KC429765 from Chennai. Sequencing of the SH gene identified outbreak strain as genotype C, was named as MuVi/Davangare. IND and deposited in Genbank (Acc. No. KJ468460, KJ468461) which was consistent with other outbreaks in India. ,,
The phylogeny obtained by analysis of representative strains of all genotypes and our sample sequences allowed quick and easy differentiation of the corresponding genotype [Figure 4]. Phylogenetic tree of mumps virus genotypes based on the 316 nucleotides of the entire small hydrophobic gene: Our isolated virus sequences were matched with WHO reference strains. Neighbour-joining methods of MEGA 5 programme were used.  The parameter employed was Kimura 2-parameter model and the robustness of the internal branches was determined by 1000 bootstrap replications. The horizontal length of the bar denotes percentage difference between sequences (see scale at bottom) and the bootstrap numbers (%) are given at each node.
|Figure 4: Phylogenetic relationships of the SH sequences of the reference strains and NIV BU sequenced strain for mumps genotyping|
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The clustering pattern clearly divides two groups, each representing a genotype group included in this study. The bootstra P values ranges from 62-99 for genotype A, F, J and 11-99 for C, E, D, K, M, H, L, I, B, N and G representing of diversity in accession of these mumps strain. The sequence identity of C genotypes with the WHO reference genotype C was ranged from 39 to 99. MuVi/Davangare isolate is close to MuVi/Chennai stain. Furthermore, articles reporting sets of data show temporal changes in the dominant genotype, as well as co-circulation of genotypes in the same or in neighbouring states. According to our findings, these different lineages showed within genotype 'C' that the virus responsible for an outbreaks in South India was similar as earlier reported in India. ,,
| ~ Discussion|| |
The use of vaccine in developed countries successfully reduced mumps virus infection, though outbreaks are often reported. Due to absence of effective vaccination programmes, mumps is still a major health problem in several developing countries. Mumps disease burden in India is still not completely understood. Few published reports indicate more occurrence of the disease in children of the age-group 5-9 years. , In the present study it was shown that mumps virus exhibited a positive laboratory result is defined as either: IgM positive or virus detection by RT-PCR. Suspected mumps cases were confirmed by systematic laboratory testing which included IgM test, RT-PCR and sequencing analysis.
A positive IgM test result indicates current infection or reinfection. The detection of mumps virus by RT-PCR method confirms the outbreak due to mumps virus and sequencing analysis further revealed circulation of 'C' genotype as reported earlier in South India.
| ~ Acknowledgement|| |
We are thankful to Dr D. T. Mourya, director NIV for providing constant support and valuable suggestions during progression of this study.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]