Brauer M.(1), Wolfaardt M.(2), Taylor M.B.(2), Webber L.M.(1)

1. Molecular detection and characterisation of mumps virus in cerebrospinal fluid in a Gauteng laboratory Brauer M.(1), Wolfaardt M.(2), Taylor M.B.(2), Webber L.M.(1) Department of Medical Virology, University of Pretoria/National Health Laboratory Service Department of Medical Virology, Faculty of Health Sciences, University of Pretoria

2. Introduction • Acute mumps virus (MuV) infection is commonly associated with central nervous system (CNS) manifestations • Up to 50% of mumps meningitis cases may be clinically indistinguishable from other types of meningitis due to the absence of parotid swelling • May be underdiagnosed unless specifically tested for

3. Introduction • Prior to the introduction of mumps vaccination, MuV was a common cause of viral meningitis • Currently the mumps vaccine is not included in the South African Extended National Immunization Schedule and is only administered in the private health sector • MuV surveillance is not prioritised in South Africa • Epidemiologic data regarding MuV and its complications is very limited • The last epidemiologic data regarding MuV meningitis in the mostly unvaccinated population of Gauteng dates back 20 years

4. Aim The aim of this study was to determine the presence of MuV in cerebrospinal fluid (CSF) specimens of patients who presented with acute CNS infection, and to do molecular characterisation of detected MuV strains

5. Methods • A total of 260 CSF specimens were selected from specimens received in the diagnostic laboratory of National Health Laboratory Service Tshwane Academic Division • Tested negative for other pathogens • Following manual RNA extraction, an in-house one-step real-time RT-PCR was used to amplify and detect the MuV F gene and the endogenous internal control Homo sapiens RNase gene • F gene: highly conserved

6. Methods • A conventional nested RT-PCR was performed to amplify the entire SH gene for nucleotide sequence analysis • SH: most variable region of the genome • Genotypes are assigned based on sequence analysis of the SH gene • Phylogenetic relationships were determined using reference strains from GenBank

7. Results • Mumps virus was detected in 1.5% (3/260) of CSF specimens • Invalid results were obtained for 28 (10.8%) specimens on account of internal control failure

8. Results • The nucleotide sequence of one MuV positive specimen (CSF074) could be determined • Phylogenetic analysis revealed that this strain clustered with the Jeryl-Lynn and RIT4385 vaccine strains with 99% nucleotide sequence similarity

9. Results • Clinical case report • CSF074 was collected from 17 month old male • Presented with a history of vomiting and diarrhoea • HIV exposed but tested HIV negative at the referring hospital • Severe failure to thrive, appeared chronically ill • On examination: palpable axillary lymph nodes & generalised muscle wasting • Immunisations were up to date acc. to mother

10. Results • Clinical case report • No stool passed since admission to the referring hospital • Presumptive diagnosis of bowel obstruction was made • Transferred to Steve Biko Academic Hospital • Laparotomy was performed • Unremarkable except for the presence of ascites and mesenteric lymphadenopathy • Working diagnosis: functional bowel obstruction

11. Results • Clinical case report • Day 2 post-operation: developed neck stiffness • Positive Babinski sign • Hyperreflexia & increased tone L > R • CSF was collected and submitted for viral studies • Intravenous acyclovir was administered empirically

12. Results

13. Results • Clinical case report

14. Results • Clinical case report • Laboratory results on CSF specimen for case CSF074 • Gram stain, bacterial antigen tests and culture negative for bacteria • Culture and PCR for M tuberculosis were negative • PCRs for herpes simplex virus and enteroviruses were negative • Neurological signs resolved spontaneously during hospital stay

15. Results • Clinical case report • Other laboratory results on CSF specimen for case CSF074 • Gram stain, bacterial antigen tests and culture were all negative for bacteria • Culture and PCR for M tuberculosis were negative • PCRs for herpes simplex virus and enteroviruses were negative • Neurological signs resolved spontaneously during hospital stay

16. Conclusion • A low detection rate of MuV RNA in CSF specimens was shown in the study population • To the authors’ knowledge this is the first study to provide virological proof that the Jeryl-Lynn or RIT4385 vaccine strain may be associated with aseptic meningitis • While aseptic meningitis has been well documented following vaccination with other mumps vaccine strains, the Jeryl-Lynn and RIT4385 strains are considered to be the least neurovirulent

17. Conclusion • The clinical history of the case was in keeping with the CSF findings • Although this finding highlights the potential adverse events that may be associated with MuV vaccination, the risk of aseptic meningitis due to natural MuV infection remains significantly greater • No reports of long-term sequelae following vaccine-associated aseptic meningitis • Continued efforts to detect and characterise MuV in this population is recommended

18. Acknowledgement • Supervisor: Prof LM Webber • Co-supervisor: Dr M Wolfaardt • Prof MB Taylor • University of Pretoria – Conference funding Discovery Foundation - Academic Fellowship Award NHLS Research Trust funding