Improved surveillance of Japanese encephalitis by detection of virus-specific IgM in desiccated blood specimens*

Bulletin of the World Health Organization, 63 (6): 1037-1042 (1985) © World Health Organization 1985 Improved surveillance of Japanese encephalitis by detection of virus-specific 1gM in desiccated blood specimens* D. S. BURKE,' K. CHATIYANONDA,2 S. ANANDRIK,3 S. NAKORNSRI,4 A. NISALAK,5 & C. H. HOKE, JR6 An IgM antibody-capture type enzyme-linked immunoassay (MAC ELISA) was compared with the haemagglutination inhibition method (HI)for establishing a laboratory diagnosis ofacute Japanese encephalitis (JE) virus infection using specimens ofdried blood elutedfrom filter paper strips. Paired samples from 243 encephalitis patients, which had been obtained by mail through a national surveillanceprogramme in Thailand, were tested. During the peak of the 1983 encephalitis epidemic, 72% of cases were diagnosed as Japanese encephalitis by MAC ELISA, compared with only 38% by HI. During non- epidemic periods, the proportions diagnosed as Japanese encephalitis by MACELISA or HI were 26% and 33%, respectively. Detection of IgM anti-JE activity by the antibody- capture immunoassay is superior to the HI method for establishing a diagnosis of acute Japanese encephalitis using dried blood specimens. Effective programmes for public health disease surveillance and control require accurate yet simple and inexpensive laboratory diagnostic methods. Encephalitis, presumably due mainly to Japanese encephalitis (JE) virus, is a disease problem of epi- demic proportions in Thailand. In 1974, the Ministry of Health of the Royal Thai Government instituted a new nationwide surveillance system for this disease. In this system, filter-paper strips are saturated with the patient's blood, allowed to dry, and afixed to a standardized report form which is mailed to the Virus Research Institute of the Ministry of Public Health in * The views of the authors of this article do not purport to reflect the positions of the US Department of the Army or the Department of Defense. Requests for reprints should be addressed to the Academic Affairs Branch, Walter Reed Army Institute of Research, Washington, DC, 20307, USA. ' Chief, Department of Virus Diseases, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand. 2 Director, Virus Research Institute, Department of Medical Sciences, Ministry of Public Health, Bangkok, Thailand. 3Senior Medical Scientist, Virus Research Institute, Bangkok, Thailand. 4Medical Scientist, Division of Provincial Health (Laboratory Services), Department of Medical Sciences, Ministry of Public Health, Bangkok, Thailand. 5Senior Medical Scientist, Department of Virology, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand. 6 Chief, Department of Virology, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand. Bangkok. The blood is eluted from the filter-paper strips, and the eluates are examined for a rise in titre between the acute and convalescent samples by haemagglutination inhibition (HI) assay. In a trial using specimens obtained from dengue-infected children, HI titres obtained using serum or filter- paper eluates were shown to be essentially identical (1). Although this system has worked well for the sur- veillance of Japanese encephalitis in Thailand, one problem was that a fourfold or greater rise in titre was detected in less than 50%7o of the reported clinical cases of encephalitis (2). In those cases failing to show a fourfold rise in HI titre, the etiology of the encephalitis remained undetermined. Two main possibilities exist: either the HI test as performed on the filter-paper specimens is relatively insensitive and most of the cases are in fact due to Japanese encephalitis, or there are other important unrecog- nized etiologies of acute encephalitis in Thailand. Recently we devised simple solid-phase immuno- assays for the independent measurement of JE virus-specific IgM or IgG in the patient's blood or cerebrospinal fluid (3, 4). In the study reported here, we compared the results of testing filter-paper eluates by antibody-capture ELISA with the results obtained by the more conventional HI method. 4615 1037- 1038 D. S. BURKE ET AL. MATERIALS AND METHODS Blood specimen collection and handling Acute (S1) and convalescent (S2) blood samples were obtained from patients clinically diagnosed as having acute viral encephalitis by physicians in provincial hospitals throughout Thailand during the year 1983. Blood was obtained from the antecubital vein or by fingertip lancination. Filter-paper stripsa were saturated with whole blood and allowed to air dry at ambient temperatures, then stapled to a standard report form which was mailed to the Virus Research Institute in Bangkok. The total interval between the first bleeding and receipt of the filter paper strips at the laboratory was typically 2-3 weeks. Once received at the laboratory, the strips were stored at 4 °C until testing, which was usually within 7 days. The strips were cut into four parts, three parts being retained at the Virus Research Institute for the HI tests, and one part was sent to the Armed Forces Research Institute of Medical Sciences in Bangkok where the ELISA tests were done. The HI tests and ELISA tests were completed entirely independently. HI tests The filter-paper strips were eluted into 0.6 ml of pH 9.0 borate saline buffer containing 12.5%o acid- washed kaolin b and incubated overnight before centrifugation. The specimens were then adsorbed with goose red blood cells and tested against 8 HA units of sucrose-and-acetone-extracted mouse brain JE virus antigen, according to the methods of Clarke & Casals as adapted to microtitration plates (5, 6). The lowest dilution of eluate examined was equivalent to a 1: 20 dilution. Paired eluates were tested on the same plate. A patient was diagnosed as having acute Japanese encephalitis if either (a) there was a fourfold or greater rise in titre between the acute and con- valescent samples or (b) the absolute titre in either the acute or convalescent specimen was equal to or greater than 1: 640. ELISA Filter-paper strips were eluted into 0.2 ml of phosphate-buffered saline at pH 7.2 for an equivalent dilution of 1:20 and tested in antibody-capture assays for JE virus IgM and IgG activity according to previously described methods (4). Briefly, the specimens were applied into wells of polystyrene microtitration plates that had previously been a Nobuto, from Toyo Roshi Kaisha Ltd, Nikobashi, Chuo-ku, Tokyo, Japan. bFisher Scientific Co., Pittsburgh, PA, USA. sensitized with either goat anti-human mu chain (for IgM antibodies) or anti-human gamma chain (for IgG antibodies). After washing to remove all blood proteins except for the captured immunoglobulin, the following reagents were added and incubated in succession: (1) 50 HA units of sucrose-acetone extracted suckling mouse brain JE virus antigen, (2) peroxidase-labelled human flavivirus hyperimmune IgG, and (3) substrate. Colour changes were quantitated with an automated plate reader. Two wells containing a weak positive control (WPC) serum (1: 100 dilution) and two wells containing a negative control (NC) serum (1: 100 dilution) were included on each plate. Paired specimens from the patients were tested on the same plate without knowledge of which was the first or the second sample. The results were expressed as dimensionless "MAC ELISA units" or "GAC ELISA units", as previously described (4). These units are defined as the ratio of the absorbance of the test sample (AbsT) minus the absorbance of the negative control (AbsNC), divided by the absorbance of the weak positive control (AbsWPC) minus the absorbance of the negative control: Units = (AbsT-AbsNC)/(AbsWPC-AbsNC) One hundred units was the cut-off for determining a positive test in the MAC ELISA (a colour intensity equal to or greater than the WPC colour). A patient was,diagnosed as having acute Japanese encephalitis if either the acute or the convalescent serum contained 100 or more units of JE virus IgM activity. The level of IgG anti-JE activity was not used in establishing a diagnosis but was measured solely to facilitate interpretation of the HI and ELISA tests. RESULTS Specimen collections Paired acute and convalescent filter-paper blood samples from 243 clinically diagnosed cases of acute encephalitis formed the study sample. All specimens were collected between the 62nd and 280th days of the year 1983. The number of cases from the North, North-east, Central, and South regions of Thailand were 158, 62, 17, and 6, respectively. Eighty-nine percent of the study cases were under the age of 21 years, and 4807 were under the age of 10 years. The patients had been ill for an average of 5.6 ± 4.9 days (mean ± one standard deviation, n = 197) when the first sample of blood was obtained. The average interval between the first and second samples was 7.8 ± 4.1 days (n = 243). 1039SURVEILLANCE OF JAPANESE ENCEPHALITIS BY ELISA HI titres The distribution of HI titres in filter-paper eluates is shown in Fig. 1. The modal titre was 1: 80 in the acute specimens and 1: 60 in the convalescent specimens. Sixty-three patients had an HI titre of 1: 640 or greater in the convalescent eluate. Sixty-five eluate pairs showed a fourfold or greater rise in titre. On the criterion of either a fourfold titre rise or a titre of 1: 640 or greater in the convalescent sample, 89 patienis (37%) could be assigned a diagnosis of acute Japanese encephalitis by HI serology. IgM and IgG anti-JE activity The distribution of cases by IgM and IgG anti-JE activity in acute and convalescent filter-paper eluates is also shown in Fig. 1. Although the modal IgM anti- JE activity in both acute and convalescent specimens was between 0 and 100 units, 74 acute specimens and 127 convalescent specimens had greater than 100 units of JE virus IgM activity. In 10 cases the activity in the acute specimen was greater than 100 although the convalescent specimen's activity was less than 100. Overall, using a cut-off value of 100 units, 137 patients (56%) could be assigned a diagnosis of acute Japanese encephalitis by MAC ELISA. The modal IgG activity in both acute and convalescent specimens was 0 to 100 units, but only 61 acute, compared with 132 convalescent, specimens had activity levels greater than 100 units. 1001 50 r; JE Hi titre O 1n 1 _ a20 t0 *0 *0 160 320 640 200 2560 5120 10240+ IgG anti-JE units 050 z zo0 0-99 o00-i1 300-31 500-514 roo-r9 oo+ 200-2" 400-4" S00-M 000-091, 100-1 i ~~~~~~~~~~IgManti-JE units .o 0-99 100-199 300-3n 500-59 roo-r9s16200-299 400-420 *00-699 900-s 900 Fig. 1. Histogram distributions of test results. Open bars = acute specimens; closed bars = convalescent specimens. Top: JE HI titres. Middle: IgG anti-JE virus units. Bottom: IgM anti-JE virus units. Table 1. Comparison of Hi and IgM anti-JE test results HI result Negative Positive a Both IgM anti-JE test result: Negative 82 24 106 Positiveb 72 65 137 Both 154 89 243 X= 15.84; P< 0.0001 a Criteria for positive result: fourfold or greater Hi titre rise or S2 HI >1:640. b Criteria for positive result: Si or S2 JE MAC ELISA > 100 units. Comparison of diagnoses by HI titre versus ELISA activity As shown in Table 1, there was agreement in the diagnosis in 147 cases (82 patients negative by both tests and 65 patients positive for Japanese encephalitis by both methods, x2 = 15.84, P < 0.001). In 24 cases, the ELISA for JE virus IgM activity was persistently negative while the HI diagnosis was Japanese en- cephalitis, and in another 72 cases the ELISA for JE virus IgM was positive while by HI there was no evidence for acute JE infection. Table 2 shows the results with the two methods for cases in which SI was obtained during the JE epidemic period in 1983 (day 170 through day 229; June 19 through August 17) and for cases in which SI was obtained outside the JE epi- demic period (before day 170 or after day 229). By the HI method, 38% of specimens obtained within the epidemic season and 3301 obtained outside the season were positive. Using the ELISA method the propor- tions were 63% and 26%, respectively. Of the 24 cases positive by HI but lacking JE virus IgM, 18 (75 %o) had relatively high levels of IgG anti-JE activity (> 100 units). Of 72 cases positive by ELISA but negative by HI, only 11 (15%7e) had high JE virus IgG activity. Among the 72 JE virus IgM-positive but HI-negative cases, the proportions with detectable JE virus IgM in the first specimen only, in both specimens, and in the second specimen only were 6 (801o), 36 (50%o), and 30 (4801o), respectively. Among these 72 cases there were 15 that also had a third specimen submitted; all 15 (10001o) were positive for JE virus IgM. Correlations between HI titres and ELISA activity in individual specimens Table 3 shows the Pearson correlation coefficients between the results obtained by the three assays. When the rises in activity were compared, only those D. S. BURKE ET AL. Table 2. Test results on specimens obtained before, during, and after the 1983 encephalitis epidemic Days in 1983 when specimens (Si) were obtained Before Days Days Days Days Days Days Days Days After day 160to 170to 180to 190to 200to 210to 220to 230to day 160 169 179 189 199 209 219 229 239 240 Total Epidemic activitya 0 0 +/0 + + + + + +/0 0 0 No. tested 10 10 21 33 34 61 31 20 1 1 1 2 243 No. of IgM positive 2 5 10 19 25 44 22 6 3 1 137 (20)" (50) (48) (58) (74) (72) (71) (30) (27) (8) (56)b No. of Hl positive 4 5 7 16 14 23 9 6 1 4 89 (40) (50) (33) (48) (41) (38) (29) (30) (9) (33) (37) a Relative increase in incidence of reported cases of encephalitis above the baseline: 0 = at baseline, + / 0 = slight increase, + = definite increase, + + = marked increase. b Figures in parentheses are percentages. Table 3. Correlations between the HI titre and ELISA activity in individual casesa Variable # 1 Variable # 2 r S1: IgM anti-JE S2: IgM anti-JE 0.68 Si: IgG anti-JE S2: IgG anti-JE 0.67 S1: log JE HI S2: log JE HI 0.73 S2: IgM anti-JE S2: IgG anti-JE 0.21 S2: IgM anti-JE S2: log JE HI 0.30 S2: IgG anti-JE S2: log JE HI 0.66 A: IgM anti-JE A: log JE HI 0.41 A: IgG anti-JE A: log JE HI 0.65 a S1 and S2 refer to the first and second specimens, respec- tively; A refers to the change between S1 and S2. cases that had relatively low activity in the SI specimen by all three tests (SI IgM anti-JE < 250 U, S2 IgG anti-JE < 250 U, and SI JE HI < 1: 640; n = 184) were analysed. DISCUSSION In our previous studies (3, 4) we observed that the intensity of the reaction in antibody-capture immuno- assay systems was independent of the dilution of the serum specimen assayed over a wide range of dilutions (from undiluted to 1: 100 for IgM assays and from undiluted to 1:10 000 for IgG assays). In these ranges of dilutions saturation kinetics apply, i.e., all the heavy-chain specific "antibody capture sites" on the solid phase bind the immunoglobulin. Unbound excess immunoglobulin is simply washed out of the system along with other blood proteins. We expected that variations in the amounts of IgM eluted from filter-paper strips would not appreciably influence the intensity of the reaction and predicted that the antibody-capture approach would be well suited for the diagnosis of Japanese encephalitis using this type of specimen. In this study we compared theMAC ELISA and the HI methods for measuring JE-specific antibodies in eluates of dried blood on filter papers. Overall, there was a statistically significant agreement in results obtained by the two methods, yet many discrepancies were found in the assignation of diagnoses in individual cases. Although the HI method has been used for over 30 years as the standard assay for the serodiagnosis of arbovirus infections and has previously been reported to be useful in sero- epidemiologic studies using dried blood, we believe that most of the discrepancies between the two methods compared here were due to false positive and false negative results using the HI method rather than with the ELISA method. Our reasons for this inter- pretation are given below. In 24 cases the HI method established a diagnosis of Japanese encephalitis while the MAC ELISA was per- sistently negative. The proportion of total cases which were HI-positive but ELISA-negative was higher during the non-epidemic periods (7/43, 160o) than during the epidemic period (17/200, 8%1o), while the opposite was true for ELISA-positive but HI-negative cases (4/43, 9%o during the non-epidemic periods versus 68/200, 34%7o during the epidemic period). This suggests that the specificity of the ELISA is superior to that of the HI test. It is likely that most of these 24 cases were biological rather than laboratory false positives, for in most cases (22/24, 91%Wo) relatively high levels of IgG anti-JE activity were present as measured by GAC ELISA, and 18/24 (750o) showed 1040 1041SURVEILLANCE OF JAPANESE ENCEPHALITIS BY ELISA a rise of > 100 GAC units between acute and con- valescent samples. In individual cases the HI titre correlated much better with the IgG than the IgM and anti-JE activity, and rises in the HI titre correlated better with rises in IgG anti-JE activity than with rises in IgM anti-JE activity. IgG antibodies have greater cross-reactivity than do IgM antibodies in reactions with flaviviruses (7), so some of these HI rises may have been caused by infections with flaviviruses other than JE virus. All four dengue virus serotypes are endemic among the human population in Thailand, and other flaviviruses (Tembusu, Weselsbron, Langat) have been isolated in Thailand (8). More than 10%o of the human population of northern Thailand may become infected with one or more flavi- viruses during any given year (9). In 72 cases (300/o of the total number of cases tested and 53%o of all positives by ELISA), JE virus IgM activity was detected by ELISA while the HI test showed no evidence for acute JE virus infection. In the IgM antibody-capture system, false-positive test results are rare except in patients with high levels of serum rheumatoid factor or in patients undergoing a primary dengue infection. In our previous studies we discovered that false positives due to rheumatoid factor or dengue always gave radioimmunoassay positive-over-negative (P/N) ratios lower than 15.0. In this study we established the cut-off for a positive value at 100 units, equal to a P/N ratio of 15.0. Because we were evaluating the MAC ELISA as a screening test for epidemiological information, we did not attempt to retest the specimens with border- line positive MAC ELISA test results (30 to 99 units, equivalent to P/N ratios in the range 5.0 to 14.9) with dengue antigen and control antigens. By so doing, we probably sacrificed some sensitivity for simplicity and specificity. Most (61/72, 850/o) of the IgM-positive, HI-negative cases did not show an appreciable (> 100 GAC ELISA units) rise in JE virus IgG activity between specimens; this suggests that the absence of an HI titre rise was a biological false negative rather than a laboratory error. Even when suitably timed, paired blood specimens were obtained by venepunc- ture, followed by ideal processing and careful storage, 15-200/o of JE patients showed no rise in HI antibodies. Such patients have other unequivocal evidence for acute JE infection (high levels of IgM anti-JE activity in both serum and CSF). IgM is reported to be highly labile when dried blood specimens are stored and tested (10, 11). In the filter- paper specimens used in this study, much of the IgM anti-JE activity may have been lost prior to testing, most probably owing to lability under the conditions of shipment. Additional losses of IgM probably occurred when the specimens were adsorbed with kaolin for the HI test; along with non-specific inhibitors of haemagglutination, kaolin adsorption preferentially removes IgM but not IgG (12). Whereas the HI titre reflects the quantity of antibody in a specimen, the antibody-capture ELISA, under the saturation conditions used here, reflects the specific activity (antigen bound per mole of isotype). A large decrease of the amount of IgM eluted would therefore be expected to lead to a proportionate decrease in the HI titre response without appreciably affecting the MAC ELISA result. In more detailed studies at one provincial hospital (data not shown) we found that cases of "en- cephalitis" that could not be confirmed as due to JE virus were typically relatively mild and were probably a collection of illnesses of various etiologies, such as aseptic meningitis due to enteroviruses, encephalo- pathy due to dengue, and true encephalitis due to measles or following rabies immunization. Although only 53% of all cases tested showed evidence of acute infection with JE virus by MAC ELISA, during the peak of the encephalitis epidemic season 720/o of cases were positive. If a cut-off value of 30 units had been used rather than 100 units, over 80%o of reported cases during the peak epidemic season would have been confirmed as due to JE virus. These findings, we believe, indicate that this virus was the only signifi- cant cause of the 1983 epidemic of encephalitis in Thailand. The results of this study have led us to conclude that the MAC ELISA approach probably has greater specificity and sensitivity than the HI test for screening of filter-paper eluates to establish a diagnosis of acute JE infection. The MAC ELISA also compares favourably with the HI test as regards reproducibility, cost, and ease of performance. We have recently completed preliminary trials using the antibody-capture immunoassays to test for virus- specific IgM antibodies to dengue, hepatitis A, and hepatitis B in filter-paper eluates, and obtained uniformly encouraging results. RESUME AMELIORATION DE LA SURVEILLANCE DE L'ENCEPHALITE JAPONAISE PAR DETECTION DE L'IgM SPECIFIQUE DU VIRUS DANS DES PRELEVEMENTS DE SANG DESSECHES Depuis 1974, le Ministere de la Sante de Thailande assure la surveillance a 1'echelon national de l'encephalite virale aigue. La methode d'inhibition de l'hemagglutination (IH) etait utilisee pour faire le diagnostic de laboratoire de 1042 D. S. BURKE ET AL. I'infection par le virus de 1'encephalite japonaise (EJ) par detection des anticorps dans des prelevements desseches de sang de malades elues a partir de bandes de papier filtre. Dans des rapports precedents, une augmentation de quatre fois ou plus des titres en IH de virus EJ avait et trouv6e dans moins de 50%lo des cas cliniques. Dans l'etude presentee ici, une epreuve immuno-enzyma- tique avec capture d'anticorps (AC ELISA) a e compar6e avec la methode d'inhibition de l'hemagglutination pour le diagnostic de laboratoire de l'encephalite japonaise aigue. Des prelevements apparies, requs par la poste, de 243 malades atteints d'encephalite ont et examines. Les malades etaient atteints depuis 5,6 ± 4,9 jours lors du pre- mier prelevement de sang, et l'intervalle entre le premier et le deuxieme prelevement etait de 7,8±4,1 jours (moyenne± ecart type). Une aliquote de chaque eluat de papier filtre a e absor- bee avec du kaolin et des hematies d'oie, puis examinee par IH en commencant a une dilution de 1/20. Le diagnostic d'encephalite japonaise aigue etait pose s'il y avait soit a) une augmentation de quatre fois ou plus du titre entre les prelevements de phase aigue de convalescence, soit b) si le titre absolu dans l'un des deux prelevements etait egal ou superieur a 1/640. Une autre aliquote, non absorbee, a e examinee A la dilution unique de 1/20 par des epreuves immuno-enzyma- tiques bien normalisees avec capture d'anticorps pour l'IgM du virus EJ (JE MAC ELISA) et pour l'IgG (JE GAC ELISA). Le diagnostic d'encephalite japonaise aigue etait pose si l'intensite de la couleur dans le JE MAC ELISA (en utilisant un des eluats du sang du malade) etait au moins egale A celle obtenue en utilisant un serum temoin connu, faiblement positif pour les IgM du virus EJ. Au maximum de l'epidemie d'encephalite de 1983, dans 727o des 61 cas le diagnostic d'encephalite japonaise a e pose par la methode MAC ELISA, contre 380o seulement avec la methode IH. Pendant les periodes non epidemiques, sur les 43 cas observes, le diagnostic a e pose dans 26% des cas par la methode MAC ELISA et dans 33%o des cas par la methode IH. La labilite des anticorps IgM pendant la dessic- cation et l'envoi postal, associee A la migration selective des IgM par rapport aux IgG pendant le processus d'extraction sur kaolin, a probablement diminue la reactivite de la methode IH mais pas celle de la methode JE MAC ELISA, plus sensible. La detection des IgM anti-virus EJ par epreuve immuno- enzymatique avec capture d'anticorps est de ce fait supe- rieure A la methode IH pour etablir le diagnostic d'encepha- lite japonaise aigue en utilisant des prelevements de sang desseches. REFERENCES 1. Top, F. H. JR., ET AL. 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