Journal of Infection (I989) x9, 219-228

Enterovirus-specific IgM in the diagnosis of meningitis

Carol Day, Hamish Cumming and Jacqueline Walker

Public Health Laboratory, West Park Hospital, Horton Lane, Epsom, Surrey KTI9 8PB, U.K.

Accepted for publication 6 April I989

Summary Samples of serum from 557 patients with a clinical diagnosis of meningitis or encephalitis and referred to the Epsom Public Health Laboratory during a period of 3 years were tested for enterovirus-specific IgM in a /~ capture enzyme-linked immunosorbent assay (ELISA).

Enterovirus-specific IgM was detected in 45 ~o samples from all age groups. In the 3-5-year age group, 67 % specimens were positive. A notable male predominance (73 ~o) was seen in the age group o-I5 years.

As predicted, a seasonal increase in incidence was found in the summer and autumn months. Data from a questionnaire sent to the referring laboratories showed only a 5 ~o enterovirus isolation rate from cerebrospinal fluids when isolation of a virus was attempted.

The enterovirus IgM ELISA is a sensitive economical and rapid method for use in the diagnosis of viral meningitis.

Introduction

Aseptic meningitis is a common manifestat ion of enterovirus infection. Al though such meningitis is generally regarded as benign compared to the bacterial form, serious sequelae may arise in the first I2 months of life. 1 The non-polio group of enteroviruses has been estimated to account for 8o-9o % viral meningit is for which an aetiology can be determined. 2 Encephalitis is a rarer clinical manifestat ion of enterovirus infection. I t is most common in infants with generalised neonatal infection 3 al though severe cases in adults with focal temporal lobe necrosis have been recorded. 4 Encephalit is leading to coma and death has been reported in agammaglobulinaemic children who are at particular risk of enterovirus infection. 5-9

Techniques such as virus isolation and neutralisation tests provide diagnostic evidence of infection but often after the patient has been discharged f rom hospital. At tent ion was therefore directed to the development of methods of detecting IgM as an indication of current or recent infection.

Several groups have developed tests for detecting IgM to Coxsackie B viruses 1°-18'2° many of which detect I g M to other enteroviruses predominant ly echoviruses and Coxsackie A Viruses. In one s tudy of 45 patients with suspected meningitis , and in which I g M detection was compared with virus isolation, 67 ~o patients were positive in the IgM test compared with 22 % by isolation. 21

T h e method which we have developed is a /z capture double-ant ibody- sandwich enzyme immunoassay that incorporates the use of antigens prepared

oi63-4453/89/o6o219+ Io $02.00/0 © i989 The British Society for the Study of Infection

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from Coxsackie B virus I - 5. The test detects IgM to other enteroviruses also but heterotypic reactions are specific to the enterovirus group.

This study was designed to investigate the incidence of enterovirus IgM in patients presenting with a clinical diagnosis of meningitis or encephalitis and to examine any age/sex relationship and seasonal influence.

Materials and methods

Samples

Samples of serum were selected from patients with a clinical diagnosis of meningism, meningitis or encephalitis and referred to the Public Health Laboratory, Epsom from other laboratories in England and Wales during the years of 1984-1986 inclusive. Samples tested came from 557 patients of whom 28I were male and 276 female, they were representative of all ages. Samples for comparison were obtained from persons in age-matched groups, some before routine surgery, some at the time of antenatal booking while some were blood donors, all without clinical signs of infection.

Controls

Positive control samples were selected from those patients with a clinical diagnosis indicative of an enterovirus infection and tested in the enzyme- linked immunosorbent assay (ELISA) system. Those giving a net absorbance value >/0"5o were put together in order to form a pool of positive control serum. Dilutions of control samples giving a net absorbance value of I'O were used in the ELISA system.

Samples remaining after antenatal screening and that came from healthy young women were tested in the ELISA. Those giving a net absorbance value ~< o.I were used to provide a pool of negative control serum. Titration of the pool in the ELISA established the dilution giving an average net absorbance value of o.I.

Antigens

Viral antigens were prepared from confluent monolayers of Vero cells maintained on serum-free Medium 199 (Gibco) buffered with o'o5 M N-Tris (hydroxymethyl) methylglycine 19 and inoculated with Coxsackie B viruses, types 1-5. Cultures were harvested when showing 8o-9o ~o cytopathic effect, freeze-thawed twice and clarified by centrifugation at IOOO g for IO min. The optimal dilution for use was obtained in the ELISA system by 'chessboard' titration and was usually I in 5. A negative control antigen was prepared from uninfected cells in a likewise manner and was used at the same dilution.

Rabbit antiserum

Immunising antigens were prepared from test antigens. The virus was precipitated with an equal volume of saturated ammonium sulphate at 4 °C and the precipitate washed twice with 5o % saturated ammonium sulphate at 4 °C. After centrifugation, the pellet was resuspended in I /3oth of the original volume of phosphate buffered saline A solution (PBSA) (Dulbecco) and the virus deposited in an ultracentrifuge at IOO ooo g for 9o rain. The antigen was

Enterovirus-specific IgM in meningitis 22 I

fur ther purified on a density gradient of caesium chloride centr i fuged at I65OOO g for I8 h. Fract ions containing infectious virus were pooled and the volume made up to 6 ml.

N e w Zealand white rabbits were inoculated initially with I ml antigen in each thigh and I ml intravenously. A further I ml was inoculated intravenously on days I4, 28 and 56, and the rabbits bled on days 42 and 70. T he specificity of each ant iserum was confirmed by neutralisation of the corresponding virus type in Vero cells. T h e op t imum dilution for use in the E L I S A system was de termined by ' ches sboa rd ' t i tration and was I in 6000 for each rabbi t antiserum.

ELISA m e t h o d

T h e capture ant ibody [affinity-purified goat ant i -human I g M (Tago)] was di luted I in 200 with carbonate buffer, p H 9"0, before Ioo #1 of it was adsorbed on to each well of 96-well microassay plates (Falcon) by incubation at 37 °C in a humidif ied a tmosphere for 2 h. T h e plates were washed three times in P B S A - T w e e n 20, p H 7"2, in a 'T i t r e t ek i 2 o ' microplate washer. Excess washing buffer was tapped out on to absorbent paper. T h e plates were washed by this me thod after each per iod of incubation.

T h e samples of serum were di luted I in IOO in dilution buffer (PBSA- T w e e n 20 plus 0"05 % bovine albumin) and tested separately against each Coxsackie B virus antigen as follows. T o four consecutive vertical wells on each specific antigen plate were added IOO #1 each sample while ioo #1 of positive and negative control sera were added in duplicate on every plate. T h e plates were incubated at 37 °C in a humidif ied a tmosphere for 3 h.

Viral and control antigens were di luted I in 5 in dilution buffer. T o the top two wells allotted to each serum on the designated antigen plate were added Ioo #1 viral antigen while Ioo/~1 control antigen were added to the remaining wells on every plate. T h e plates were covered and incubated at room tempera ture overnight.

The specific rabbi t anti-virus antisera were di luted I in 6000 in dilution buffer and IOO #1 added to every well on the corresponding antigen plate. T h e plates were incubated at 37 °C in a humidif ied a tmosphere for 2 h.

Peroxidase-conjugated goat ant i - rabbi t ant iserum (Tago) was di luted I in 20000 (as de termined by ' ches sboa rd ' t i tration in the E L I S A ) in dilution buffer and IOO #1 added to all wells. T h e plates were incubated at 37 °C in a humidif ied a tmosphere for 2 h.

T o every well on each plate were added ioo #1 freshly prepared substrate [40 mg o-phenylenediamine dihydrochlor ide (OPD) dissolved in Ioo ml citrate buffer, p H 5"0 plus 40 #1 hydrogen peroxide 30 volume]. T he reaction was s topped after I2 minutes by the addit ion of 50 #1 2"5 M sulphuric acid.

The plates were read at a wavelength of 492 nm on a Dyna tech Microelisa Auto Reader M R 5 8 o which was linked to an Apple I Ie microcomputer . This was p rogrammed to calculate the ratio of the test to negative average net absorbances, as well as s tandard deviations f rom the mean of the negative samples, for each sample tested against each antigen.

T h e working dilution of each reagent was de termined by ' chessboard t i t ra t ion ' in order to achieve max imum sensitivity with the max imum dilution

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Enterovirus-specific IgM in meningitis 223

of reagent while absorbance values within the range of the plate reader (I'5oo) were achieved.

A positive enterovirus-specific IgM result was recorded when the absorance value of the test-sample was /> three times that of the mean of the negative control samples. This corresponded to ~> 5 standard deviations from the mean of the negative control samples.

Statistical analysis of the results employed the X 2 formula with Yate's correction.

R e s u l t s

Table I shows the proportions of patients with IgM-positive serum according to age and clinical diagnosis. The age groups correspond approximately to neonates, infants, pre-school, school age and three adult groups. The results for those patients with a clinical diagnosis of meningitis were all found to be significant when P = o-ooI or P = o.oI. The group of patients with encephalitis is much smaller than that of those with meningitis and results for the 6 to > 45 years age groups were significant when compared to the normal group. The relatively large proportion (I 8 %) of healthy controls in the 3-5- year age group with enterovirus IgM reflects the high incidence of inapparent enterovirus infection in pre-school children.

Table II contains data from the meningitis patients included in Table I classified by age and sex and who had IgM-positive samples. The results reveal that in children < 16 years of age with enterovirus meningitis, 69 % (43 of 62) were male. This is statisitically significant (P < 0"05). In some groups the difference was greater although the numbers are very small. The overall proportion of patients classified by sex was approximately even. In adults (> / I6 years of age) there was no significant difference between the sexes. Figure I contains the data from the meningitis and encephalitis patients in the Epsom enterovirus-specific IgM-study I984-I986 while fig. 2 shows the seasonal distribution of enterovirus isolates from patients with meningitis and encephalitis reported to the Public Health Laboratory Service Communicable Disease Surveillance Centre. Both sets of data show seasonal peaks from July to November which reflect the known summer /au tumn incidence of enterovirus activity in the population.

Many of the samples tested for this survey were referred to us from laboratories all over England and Wales. We were therefore largely unaware of any other virological or bacterial investigations that had been performed on the patients concerned. For each positive patient a questionnaire was devised in order to provide this information and was sent to each referring laboratory. Table III summarises the data obtained. It includes those patients who had lymphocytic or pleocytic cerebro-spinal fluid (CSF) with sterile bacterial culture and for whom no other viral aetiology had been demonstrated.

Only 5 % samples of CSF yielded an enterovirus, either an ECHO 9 virus or an ECHO i I virus. More viruses were isolated from faeces and throat swabs but the isolation rate overall appeared to be very low although isolation may not have been attempted in some cases. Only two of the viruses isolated were Coxsackie B viruses.

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Discuss ion

T h e Coxsackie B I g M E L I S A employed in this survey has been in routine use in the E p s o m laboratory for several years. Heterotypic responses i.e. a positive response to two or more antigens has often been observed. In a previous study, we included Coxsackie Ag, A I 6 and Echo 6, 7, 9 and II viral antigens in the

Enterovirus-specific IgM in meningitis 225

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ELISA. Some of the heterotypic responses recorded were, to a greater or lesser extent, to all I I antigens. Such results reflect those obtained by Minor et al . 11 and Dorries and ter Meulen. 16 Both type and group determinants are located on the VPI polypeptide. 1~'13 These may be exposed to varying extents according to the method of antigen production.

The incidence of enterovirus-specific IgM presented here probably underestimates the true incidence in cases of aseptic meningitis since only Coxsackie B I-5 viral antigens were included. Indeed, in the previous study, some samples reacted to only the non-Coxsackie B viral antigens. In this study we have employed a cut-offvalue based on a ratio of 3: I (5 standard deviations from the mean of that obtaining with control samples). The testing of several thousand samples since then has led us to lower the cut-off value to one based on a ratio of 2"5 : ~ or 3 standard deviations. Although we realised that our cut- off value of 3 was high, we decided to err on the side of caution due to the ubiquitous nature of enteroviruses. Lowering the value to 2"5 would include only another 15 of a total of 557 patients but would also increase the percentage of positive persons in the normal population. Significance values, however, would remain unaltered (unpublished data). The age and sex distribution of such additional patients make no appreciable difference to the results of this study.

Initial standardisation of the E L I S A showed it to be enterovirus-specific. We suspect that the heterotypic response in the E L I S A reflects an anamnestic response by the host, i.e. that infection by an enterovirus of any serotype stimulates production of IgM related to any enterovirus to which the host has been previously exposed. In support of this proposition, we have observed that older patients, having experienced more enterovirus infections, exhibit more heterotypic reactions.

Enterovirus-specific IgM was detected in 45 % samples from persons in all age groups. The o-I5-year age groups show that the greatest proportion of patients with enterovirus-specific IgM-positive meningitis, notably 67 % in the 3-5-year age group, 73 % of whom were males. This group is composed of many susceptible persons. The predominance of patients with enterovirus IgM-positive meningitis in this age group reflects the known fact that enterovirus infections are common in children.

A male predominance in enterovirus infections has been noted by others. Studies have shown that males outnumber females by 2 : I among children with aseptic meningitis but the sexes are more equally distributed among adult patients. 22 Lake et al . noted a 72 % predominance of male infants in a study of enterovirus infection in neonates 23 and in a study of fatal echovirus I i infection in children over a period of IO years Berry and Nagington noted a 66 % male predominance. 3

Other illnesses associated with enterovirus infection and that have a male preponderance are Bornholm disease and myocarditis but no explanation for this distribution has yet been offered. 22

Encephalitis was seen mainly among the older children and young adults. This is probably due to intrafamilial spread of virus from children to adults and the fact that adults often experience more severe illness due to many viral infections, e.g. polio, mumps. It is also known that susceptibility of the central nervous system increases with age in poliovirus infection. 22

Enterovirus-specific I g M in meningitis 227

It is well known that enteroviruses circulate in the communi ty mostly during the summer and autumn. Our results correlate well with reports of enterovirus infections made to the U.K. Public Heal th Labora tory Service Communicable Disease Surveillance Centre (CDSC). It is worth noting, however, that the I g M results indicate some activity all through the year. I t may be that, in the winter months , specimens are not cultured for enteroviruses whereas otherwise negative samples of serum are referred for Coxsackie B I g M tests.

I g M may persist for several months in some patients. This may account for the greater evidence of enteroviral activity reflected by the IgM results compared with that derived f rom C D S C Communicable Disease Reports. Enteroviruses were isolated in the au tumn and winter months th roughout our s tudy so it is reasonable to assume that a proport ion of positive IgM tests related to infection at that t ime of year. Serial samples from patients were not tested in this s tudy but previous work has shown that I g M may be detected for at least 3 months after an acute enteroviral infection. Persistence of IgM has been reported in certain conditions such as recurrent pericarditis, 24 myalgic encephalomyelitis and chronic fatigue syndrome. 25 Work in this laboratory on the role of enteroviruses in juvenile-onset diabetes involved the testing of serial samples from patients and immediate family-members . Persistent homotypic IgM responses lasting up to 5 years were seen in a few otherwise healthy family members of diabetic patients (unpublished data). A genetic influence on the response to enterovirus infection in such a group, however, may be important and it is unwise to apply these results to the normal population.

T h e overall enterovirus-specific I g M detection rate was 45 % compared to a rate of 5 % for viral isolation f rom cerebral spinal fluid. T h e E L I S A takes 2 days to complete and is relatively cheap to perform. We therefore conclude that the Coxsackie B IgM E L I S A is a rapid, economical diagnostic test for investigating an enteroviral aetiology in patients with aseptic meningitis.

(We thank the staff of other laboratories for completing our questionnaire and the Director of the U.K. Communicable Disease Surveillance Centre for permission to reproduce data from the I984 Communicable Disease Reports.)

R e f e r e n c e s

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2. Centers for Disease Control. Aseptic Meningitis Surveillance Annual Summary I976. United States Department of Health, Education and Science I979. U.S.A.

3. Berry PJ, Nagington J. Fatal infection with Echovirus I I. Arch Dis Child I982; 57: 22-29. 4. Heathfield KWG, Pilsworth R, Wall BJ, Corsellis JAN. Coxsackie B5 infections in Essex,

I965, with particular reference to the nervous system. Q J Med I967; 36: 579-595. 5. Johnson PR, Edwards KM, Wright PF. Failure of intraventricular gammaglobulin to

eradicate Echovirus encephalitis in a patient with cross-linked agammaglobulinaemia. N Engl J Med I985; 3x3:I546-I547 •

6. Dayan AD. Chronic encephalitis in children with severe immunodeficiency. Acta Neuropathol (Berl) I97I ; I9: 234-24I.

7- Webster ADB, Tripp JH, Hayward Aet al. Echovirus encephalitis and myositis in primary immunoglobulin deficiency. Arch Dis Child I978 ; 53: 33-37.

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8. Wilfert CM, Buckley RH, Mohanakumar T et al. Persistent and fatal central nervous system Echovirus infections in patients with agammaglobulinaemia. N Engl J Med 1977; 296: 1485-I489.

9. Weiner LS, Howell JT, Langford et al. Effect of specific antibodies on chronic Echovirus type 5 encephalitis in a patient with hypogammaglobulinaemia. J Infect Dis 1979; I4O: 858-863.

IO. Schmidt NJ, Lennette EH. Gel double diffusion studies with Group B and Group A type 9 Coxsackieviruses. II . Serological diagnosis of Coxsackievirus infections by gel double diffusion technique. J Immunol 1962 ; 89 : 96-1o5 •

I I. Minor TE, Helstrom PB, Nelson DB, D'Alession DJ. Counter immunoelectrophoresis test for immunoglobulin M antibodies to Group B Coxsackieviruses. J Clin Microbiol 1979; 9: 5o3-5o6.

12. Katz MG, Crowell RL. Indirect enzyme-linked immunosorbent assay (ELISA), for the detection of Coxsackievirus Group B antibodies. J Gen Virol 198o; 48: 225-229.

I3. Katz MG, Crowell RL. Immunological studies of Group B Coxsackieviruses by the sandwich enzyme-linked immunosorbent assay (ELISA), and immunoprecipitation. J Gen Virol 198o; 5o: 357-367.

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15. Dorries R, ter Meulen V. Detection of Enterovirus-specific IgG and IgM antibodies in humans by an indirect solid-phase radioimmunoassay. Med Microbiol Immunol (Bed) I98O; I68: I59- I7 I .

I6. Dorries R, ter Meulen V. Specificity of IgM antibodies in acute human Coxsachievirus B infections analysed by indirect solid phase enzyme immunoassay and immunoblot technique. 37 Gen Virol 1983; 64: 159-167.

17. Morgan-Capner P, McSorley C. Antibody capture radioimmunoassay (MACRIA), for Coxsackievirus B4- and B5-specific IgM. ] Hyg (Lond) 1983; 90: 333-349.

i8. Chan D, Hammond GW. Comparison of serodiagnosis of Group B Coxsackievirus infections by an immunoglobulin M capture enzyme immunoassay versus micro- neutralisation. J Clin Microbiol i985; 2I (5): 83o-834 •

19. Gardner RS. The use of tricine buffer in animal tissue cultures. J Cell Biol 1969; 42: 320-321.

20. Torfason EG, Frisk G, Diderholm H. Indirect and reverse radioimmunoassays and their apparent specificities in the detection of antibodies to enteroviruses in human sera. 37 Med Virol I984; I3: I3-3I .

2i . Bell EJ, McCarmey RA, Basquill D, Chaudhum AKR. #-Antibody Capture ELI SA for the rapid diagnosis of Enterovirus infections in patients with aseptic meningitis. 37 34ed Virol 1986; 19: 213-217.

22. Gamble DR. Enteroviruses: P o l i o - E c h o - and Coxsackie viruses. In: Principles of bacteriology, virology and immunity, Vol. 4 Chapter 99, 1984; 4o5-4o6.

23. Lake AM, Lauer BA, Clark JC et al. Enterovirus infections in neonates 37 Paediatr 1976; 89 : 787-791.

24. Tilzey A, Signy M, Banatvala JE. Persistent Coxsackie B virus specific IgM response in patients with recurrent pericarditis. Lancet 1986; i : 1491-1492.

25. McCarmey RA, Banatvala JE, Bell EJ. The routine use of # antibody capture E L I S A in the serological diagnosis of Coxsackie B infection. 37 Med Virol 1986; I9: 2o5-212.