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Haemorrhagic fever with renal syndrome: evaluation of ELISA for detection of Puumala-virus-specific IgG and IgM
(~ INSTITUT PASTEUR/ELSEVIER Paris 1990
Res.
w • :g:E. ~ On g.
199~, 141, 637-648
HAEMORRHAGIC FEVER WITH RENAL SYNDROME: EVALUATION OF ELISA FOR DETECTION OF F UUMALA-VIRUS-SPECIFIC IgG AND
IgM B. Niklasson (1) (2), E. Tkachenko (3), A.P. Ivanov (3), G. van der Groen (4), D. Wiger (5), H.K. Andersen (6), J. LeDuc (7), T. Kjelsson (1) and K. Nystr6m (s) (1) National Bacteriological Laboratory, S-105 Stockholm;
(2) National Defence Research Establishment, FOA-5, S-172 90,
Sundbyberg (Sweden); (3) Institute of Poliomyelitis and Viral Encephalitides, Academy of Medical Sciences, 142782 Moscow; (4) Prince Leopold Institute of Tropical Medicine, Antwerp, (Belgium); (5) National Institute of Public Health, N-0462 Oslo 4; (6) Aaarhus University, 8000, Aarhus (Denmark); (7) US Army Medical Research, Institute of Infectious Diseases, Fort Detrick, Frederick 21702 MD (USA), and (a) Ume~ Hospital S-901 87 Umea (Sweden)
SUMMARY
IgM and IgG ELISA to Puumala virus were evaluated using sera from patients with haemorrhagic fever with renal syndrome (HFRS) from different geographical regions" Sweden, Denmark, Norway, Belgium and the European USSR. IgM ELISA proved useful in the diagnosis of HFRS in patients from all the regions mentioned above. Specific IgM could be detected as early as day I post onset of disease, and patients remained IgM-positive for several months. Specific IgG ELISA antibodies were also frequently detected in acute sera, and acute-convalescent serum pairs often failed to show a significant titre rise or increase in optical density (OD) values. This limits the use of IgG ELISA in patient diagnosis. Sera collected 2 years after infection revealed higher IgG ELISA OD readings than convalescent sera, and very high values were still detectable l0 to 20 years postinfection. IgG ELISA is therefore useful for the testing of immunity and in seroepidemiological studies.
Submitted September 13, 1990, accepted Octobe~ 30, 1990. Corresponding author: Bo Niklasson, Department of VRoiogy, N~fional BacteriologicalLaboratory, S-105 21 Stockhol,a (Sweden).
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B. N I K L A S S O N E T AL.
Acute and convalescent sera from HFRS patients in Korea and the Asian USSR showed no or only very weak reactivity in the Puumala virus IgG and IgM ELISA. These results are consistent with the "one-way" crossing described earlier. KEY-WORDS:Bunyaviridae, Puumala virus, IgG, IgM, Haemorrhagic fever, Renal syndrome; ELISA, Diagnosis, Scandinavia, USSR, Belgium, Korea.
INTRODUCTION Haemorrhagic fever with renal syndrome (HFRS) refers to a clinical disease characterized by fever and renal dysfunction with or without ha~:morrhagic manifestation (van der Groen, 1985). At least 3 different strains of viruses in the Hantavirus genus of the Bunyaviridae family have been associated with human disease- Hantaan (HTN) virus, urban rat virus and Puumala (PUU) virus (Schmaljohn et al., 1985; Liihdevirta, 1971). Nephropathia epidemica (NE) caused by PUU virus is a mild form of HFRS which occurs in Scandinavia and Finland (Liihdevirta, 1971 ; N'iklasson and LeDuc, 1987). Clinically and serologically related or identical diseases occur in the western USSR and central Europe. Strains from these different regions have been isolated and compared using various serological methods including indirect immunofluorescence test and immunoprecipitation assay. When tested with these methods, all viruses which are associated with the clinical disease NE are indistinguishable (Sheshberadaran et al., 1988). The present study concerns the evaluation of an ELISA for detection of PUU-specific IgM and IgG antibodies using HFRS-patient sera from Sweden, Norway, Denmark, USSR, Belgium and Korea.
MATERIALS AND METHODS Serum samples.
Acute, early-convalescent and late-convalescent phase sera (N = 137) were available from 57 patients. Five patients from Norway (15 sera), 21 patients from Sweden (49 sera), 2 patients from Denmark (5 sera), 6 patients from Belgium (20 sera) and 23 from the European USSR (48 sera) were tested. Serum specimens were drawn during the first month postonset of symptoms and at least once again at a later date.
GMT HFRS HTN
= g e o m e t r i c m e a n titre. = h a e m o r r h a g i c fever with renal syndrome. = H a n t a a n (virus).
PUU NE IFT
= P u u m a l a (virus). - nephropathia epidemica. = i m m u n o f l u o r e s c e n c e test.
P U U M A L A - S P E C I F I C I g M A N D IgG I N H F R S
639
Sera were also collected in Sweden from 7 patients aged l0 to 20 years old following clinical NE infection. During an epidemiological investigation of apparently healthy populations in Sweden, sera from both HFRS endemic (N = 187) and nonendemic (N = 142) areas were collected and tested. Another group included 7 HFRS patients (9 sera) from the Asian USSR and 5 HFRS patients (5 sera) from Korea (kindly provided by H.W. Lee, Institute for Viral Diseases, Korea University, Seoul, Korea). All patients included in this study (excluding apparently healthy population) had a clinical history of HFRS and were antibody-positive in IFT to PUU virus (Niklasson and LeDuc, 1984) and HTN virus (Schmaljohn et al., !985), for European and Asian patients, respectively.
Immune reagents used in ELISA. Antigen was prepared from PUU-virus-infected (strain Vindeln 83-223L) Vero E-6 cells (CRL 1586; ATCC, Rockville, MD) (Niklasson and LeDuc, 1984). Preparation of PUU virus antigen, negative control antigen and rabbit immune serum is described elsewhere (Niklasson and Kjelsson, 1988).
Detection of PUU-virus-specific IgG antibodies by ELISA. A sandwich ELISA was employed as follows. Rabbit anti-PUU-virus Ig diluted 1/400 in coating buffer (0.05 M sodium carbonate pH 9.5 to 9.7) was adsorbed onto 96-well polystyrene microtitre plates (Cooke M29 AR, Dynatech laboratories) at 37°C for lh, followed by virus antigen (undiluted at 37°C for I h), test serum diluted 1/400 in ELISA buffer (phosphate-buffered saline with 0.05 070 Tween-20 and 0.5 °70 bovine serum albumin) at 37°C for 1 h, and swine anti-human-IgG conjugated with alkaline phosphatase diluted 1/200 in ELISA buffer at 37°C for 1 h (Orion Diagnostica, Finland, cat. no 67806). P-Nitrophenol phosphate (Sigma) diluted in diethanolamine buffer (1 M diethanolamine pH 9.8, 0.5 mM MgCl2) was used as substrate. Washing between each step was done 6 times in washing buffer (saline with 0.05 070 Tween-20). The reaction was read after 30 rain at room temperature in a spectrophotometer at 405 run and expressed as the optical density (OD). Optimal dilutions of all reagents used in the ELISA were determined by box titrations. All specimens were tested in duplicate with antigen and negative control antigen. The OD was calculated as the average OD with antigen minus the average OD with negative control antigen. The borderline between positive and negative results was calculated as the mean of the test result of 142 known negative sera (from non-endemic areas and negative by IgG IFT) plus 3 standard deviations. An OD of >0.080 was considered lo be positive. To adjust for plate-to-plate and test-to-test variations in the assay, positive control serum was included in all plates. This control had an OD of 0.700 (in the linear interval of this IgG ELISA). If the positive control serum had an OD between 0.500 and 0.900 the plate was accepted: however, all OD values on the plate were multiplied by a factor so as to set the positive control value at 0.700. The test was run using either a single dilution (1/400) or 2-fold dilutions starting at 1/400. A si.gnificant antibody rise was defined as an OD increase of 100 070 or a 4-fold titre rise using the single dilution and titration methods, respectively.
Detection of PUU-virus-specific IgM antibodies by ELISA. The test format for the IgM ELISA are described in detail in a previous publication (Niklasson et al., 1988). Briefly, a tt-capture ELISA was used to measure specif-
640
B. N I K L A S S O N E T A L .
ic PUU virus IgM. Goat anti-human-IgM (tt-chain specific; Cappel Laboratories, catalogue no. 0201-0201) was adsorbed onto the wells. Test sera diluted 1/400 were added, followed by PUU virus antigen, rabbit anti-PUU-virus Ig, alkalinephosphatase-labelled goat anti-rabbit-IgG (Kirkegaard and Perry, catalog no. 051506), and p-nitrophenyl phosphatase substrate. The optical density was read after 30 rain at room temperature. The OD was read and the borderline between positive and negative was calculated as described above for IgG ELISA. Detection of PUU virus IgG antibodies by WT. Spot-slides with PUU-or HTN-virus-infected ,CNiklassonand LeDuc, 1984) Vera E-6 cells were prepared and stained as described elsewhere (Niklasson and LeDuc, 1987). Test sera were titrated using 2-fold dilutions starting at 1/8. Fluoresceinisothiocyanate-conjugated sheep anti-human-IgG (Wellcome Diagnostics, Temple Hill, Dartford, England, catalogue no. MF03) was used to detect specific IgG.
RESULTS Evaluation of IgG ELISA using sera from Swedish patients. Early-convalescent phase sera (collected day 16 to 41 post-disease-onset) were available from all 21 patients investigated. Acute-phase sera, drawn on days 1-9, were available from 18 of the 21 patients, and late-convalescent phase serum specimens, collected 2 years after the acute infection, were available from 14 of the 21 patients. All these sera were tested by IgG ELISA and titrated to endpoint by IgG IFT. IgG ELISA OD results from all 21 patients over time are seen in figure 1. Figure 2 shows the same sera tested by IgG IFT. Comparison of figures 1 and 2 shows that the IgG ELISA OD mean continues to increase reaching a maximum value at 2 years while the IgG IFT geometric mean titre (GMT) has approximately the same value in convalescent and 2-year sera. To evaluate whether the difference between IgG ELISA and IgG IFT depended on single dilution (ELISA) versus endpoint titration (IFT), 18 sera from 6 patients (acute, early-convalescent and 2-year) were retested in parallel using endpoint titration for both ELISA and IFT. The IgG IFT GMT were 256, 1,024 and 645; IgG ELISA mean OD (1/400 dilution) 216, 420 and 665; and IgG ELISA endpoint GMT 1,008, 1,600 and 3,200 for acute, earlyconvalescent and 2-year sera, respectively. Eleven out of 11 and 7/11 had detectable antibodies in their first serum specimen (collected on days 1-5) when tested by IgG IFT and IgG ELISA, respectively. In these 11 patients with both acute (collected on days 1-5) and early-convalescent scra (collected on days 20-41), only 8 showed a 4-fold or greater titre rise by IFT. Only 6 out of the 11 paired sera tested by IgG ELISA showed an increase in OD of _>100 %.
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Days (D) / Years after onset of disease FIG. 1. - - Acute (day 1-9), early-convalescent (day 16-42) and late-convalescent sera (2-year)
(N=49) from 21 Swedish patients tested by lgG ELISA. O D values for each serum is plotted as well as the m e a n O D o f acute, early-convalescent and late-convalescent sera. O D values are multiplied by 1,000.
All 7 sera collected more than 10 to 20 years postinfection were positive in both assays, with IgG E L I S A OD between 0.650-0.720 and IFT titres of 32-512. Out of 187 sera tested by IgG IFT from an endemic area, 34 were found positive. All 142 sera from the general population living in an nonendemic area were negative by IgG IFT. Identical results were found with IgG ELISA. All 34 positive sera from the general population had an OD of _>0.200 and a IFT titre of _>32.
HFRS patients frf~m the European USSR tested by IgG and IgM ELISA. All sera from HFRS patients from the European USSR were analysed at the Institute of Poliomyelitis and Viral Encephalomyelitides in Moscow using identical reagents. The data are presented separ~te!y since the patient~
642
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10D 20D 30D 40D 50DH2Years Days (D) /Years after onset of disease FIG. 2. - - Acute (day 1-9), early-convalescent (day 16-42) and late-convalescent sera (2 years) ( N = 4 9 ) f r o m 21 Swedish patients tested by lgG IFT. IFT titre (log) for each serum is plotted as well as the G M T of acute, early-convalescent and late-convalescent sera.
sera were tested at 1/128 dilution instead of the 1/400 dilution which was used for all the other sera. In 14 of the 23 patients, the first serum sample was drawn 1-6 days after the onset of symptoms, and a second sample was taken after at least 6 days. A significant titre rise in IgG IFT was demonstrated in 8/14 patients while 11/14 showed a >100 % increase in IgG ELISA. In 7 of the 23 patients, the first specimens were collected on days 7-11 and the second specimens >6 days after the first sample. In these patients, only 2/7 showed a significant titre rise in IgG IFT and only 1/7 demonstrated > 100 °7o increase in IgG ELISA OD. The IgM ELISA OD values for sera from acutely ill patients in European USSR are presented in figure 3. Forty-six out of 48 (96 070)of the sera collected on days 1-27 were positive by IgM ELISA. The 2 negative samples were
P U U M A L A . S P E C I F I C Ig21~A N D IgG I N HFRS
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Acute and convalescent sera from HFRS patients from the European USSR tested for PUU-virus-specific IgM by ELISA.
Sera were tested at a single dilution (1/128). OD values are multiplied by 1,000.
collected on day 1. Only five out of ten sera collected on days 1-3 were positive by IgG ELISA.
HFRS patients from Norway, Sweden, Denmark and Belgium tested by lgG and IgM ELISA. The IgG ELISA results for serum specimens collected in Norway, Sweden (only 21 sera from 9 patients were included), Denmark and Belgium are presented in figure 4. In 11 serum pairs the convalescent sera were drawn between days 12-45 and the acute specimen collected _>10 days earlier. Only 6 of these 11 patients showed an OD increase of _> 100 %. Despite the lack of significant OD increase during this early period, the OD values generally showed a steady increase over the follow-up period. Only 8 of the total 45 patients showed a decrease in IgG ELISA OD at all. The remaining 37 patients showed a continuous increase over time. In
644
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FIG. 4. - - Acute and convalescent serafrom HFRS patients from Norway, Sweden, Denmark
and Belgium tested for PUU-virus-specific lgG ELISA. Sera were tested at a single dilution (1/400). O D values are multiplied by 1,000.
15 patients both serum pairs were collected after day 30. Most of these pairs (14/15) showed an OD increase over time and 2/15 serum pairs showed an increase of > 100 %. IgM ELISA data are presented in figure 5. All of these sera were positive in the IgM ELISA during the first 2 months post onset of HFRS-symptoms. However, by 3-6 months they were in the lower range of significance or negative, and by 12 months postinfection all sera were IgM-ELISA-negative. HFRS patients from the Asian USSR and Korea.
Sera from the Asian USSR and Korea tested by IgG and IgM ELISA were negative or weakly positive and had non-detectable or very low titres to PUU virus by IgG IFT but high titres to HTN virus (76-118) by IFT (data not shown).
PUUMALA-SPECIFIC IgM AND IgG IN HFRS
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and Belgium tested for PUU-virus-specific lgM ELISA.
Sera were tested at a single dilution (1/400). OD values are multiplied by 1,000.
DISCUSSION A diagnostic test with high sensitivity and specificity is essential to define the range of HFRS symptoms as well as the geographical distribution of the different Hantaviruses. IFT for detection of specific Ig or IgG is presently used to confirm the HFRS diagnosis in the majority of routine laboratories, and presence of specific antibodies in acute as well as convalescent sera correlates well with the clinical diagnosis. In the present, as well as in previous studies, a significant proportion of patients with paired sera presented high antibody titres by IgG IFT in their first serum samples (Niklasson and Kjelsson, 1988). Seroconversion or a 4-fold titre rise by IgG IFT is therefore not always found. There is no accepted "standard" way of calculating a significant OD increase by ELISA when comparing results for acute and convalescent sera. Different laboratories use different criteria.
646
B. N I K L A S S O N
ET AL.
In the present study we decided to use an OD increase of _> 100 °70 as the criterion of a titre rise between acute and ~ mvalescent sera when testing at one dilution only. When developing the ask ~y, both endpoint titration and single dilution methods were tried but neither solved the problem of lack of a significant titre rise between acute and convalescent serum specimens (data not shown). IgG ELISA therefore does not appear to offer any advantages over IgG IFT in the diagnosis of NE patients. HFRS caused by PUU or a PUU-related virus is highly endemic in certain parts of Scandinavia, Finland and the European USSR and occurs sporadicaUy in the rest of Europe. Since antibody prevalence rates in highly endemic areas of Sweden may reach more than 30 070 in the older age-groups (Niklasson et al., 1987), *.hepresence of specific IgG may reflect a previous infection and may have nothing to do with the patient's present condition. All acute- and early-convalescent sera (collected on day 2 or later, pos~ onset of symptoms) were positive by IgM ELISA and patients maintained detectable IgM antibody levels for several months. PUU-specific IgG antibodylevels often continued to increase over time. This observation has been documented earlier by Brummer-Korvenkontio et al. (1980). In the present antibody study, we found high titres and high OD values for as long as 20 years postinfection, indicating that this assay is useful for seroepidemiological surveys. The immunological background for this continuous production of specific IgG antibodies is not clear. The presence of high titre specific IgG could be due to continuous expression of virus-specific antigen. However, if infectious virus persists, there are no clinical observations indicating any symptoms after the acute disease. No serologically confirmed case of recurrent infection has been described. It should be noted that the IgM as well as the IgG ELISA showed only very weak reactions or did not detect IgM or IgG antibodies when used on HFRS patient sera from Korea or the Asian USSR. ELISA for detection of human IgG and IgM to prototype HTN (76-118) strain has been developed and evaluated by other investigators (Ivanov et al., 1988; Groen et al., 1989). ELISA systems for detection of specific IgM as well as IgG to HTN virus has also been developed by J. Meegan, J. LeDuc and coworkers at the US Army Medical Research Institute of Infectious Diseases. Ten early convalescent sera from Swedish NE patients (positive by PUU IgM and IgG ELISA) were packed in dry ice and shipped to the USA and tested in the HTN IgM and IgG ELISA. All ten 10 sera were found to be positive by both procedures (data not shown). A serological "one-way" crossing between PUU virus and HTN virus by IFT and immunoprecipitation assay has previously been well documented (Schmaljohn et al., 1985; Sheshberadaran et al., 1988). This pattern also appears to hold true for IgM and IgG ELISA. The present study extends the geographical area were the PUU IgM and IgG ELISA can be used. The first report of serologically confirmed HFRS cases in Denmark is also presented. However, it is evident that IgM and IgG
P U U M A L A - S P E C I F I C I g M A N D igG I N H F R S
647
E L I S A using P U U virus as antigen cannot be used alone in areas were both P U U and H T N viruses cause HFRS. In the geographic areas where both viruses circulate, it would be possible to distinguish between the two serological types by running both H T N and P U U IgM E L I S A in parallel.
RI~SUMI~ FII~.VRE HI~MORRAGIQUE AVEC SYNDROME RI~NAL: I~VALUATION D'UN TEST ELISA POUR LA DI~.TECTION D'IgG ET D'IgM SPI~CIFIQUES DU VIRUS PUUMALA
A l'aide d'on test ELISA, nous avons 6valu6 les IgM et IgG anti-virus Puumala dans le s6rum de sujets originaires de divers pays (Suede, Dannemark, Norv6ge, Belo gique et URSS europ6enne, sujets atteints de fi/~vre h~morragique avec syndrome r6nal (HFRS). Le test ELISA-IgM est utile pour le diagnostic de I'HFRS chez les malades de tousles pays mentionn~s ci-dessus. Les IgM sp~cifiques peuvent ~tre d~tect~es d~s le 24e heure apr~s la d~c!aration de la maladie, et les sujets sont rest~s IgM + durant plusieurs mois. Les IgG sont fr~quemment d~tect~s par ELISA dans les s~rums de malades en phase aigu~; pour les paires de s~rums correspondant aux phases aigui~ et convalescente, il n'y a g~n~ralement pas d'augmentation significative de titre ou d'~16vation des valeurs de la densit~ optique (DO). Cela limite l'utilisation de I'ELISAIgG pour le diagnostic. Les se~'t!msrecueillis 2 arts apr~s l'infection ont des DO plus ~lev~s pour le test ELISA-IgG que les s~rums de convalescents, et des valeurs tr~s ~lev~es sont encore d~tectables 10 ~ 20 ans apr~s l'infection. L'ELISA-IgG est cependant utile pour tester l'immunit~ et pour les ~tudes s~ro~pid~miologiques. Les s~rums de malades HFRS en phases aigu~ et convalescente, en CorSe et URSS asiatique, ont une minime ou nulle r~activit~ au test ELISA-IgM ou -IgG pour le virus Puumala. Ces r~sultats sont en accord avec ceux de l'analyse "one-way cross" pr~c~demment d~crite. MOTS-CLES: Bunyaviridae, Fi~vre h6morragique, Syndrome r6nal, Virus Puumala, IgG, IgM; ELISA, Diagnostic, Scandinavie, Belgique, URSS, Cor6e.
AC KNOWLEDGEMENTS
This project was supported by the US Army Medical Research and Development Command (DAMD 17-86-C-6073).
REF ~!RENCES BRUMMER-KORVENKONTIC~:M., VAHERI,A., HOVl, T., VON BONSDORFF,C.-H., VUORIMIES, J., MANNI, T., PENrTINEN, K., OKER-BLOM, N. & L.~HDEVIRTA, J. (1980), Nephropathia epidemic,a: detection of antigen in bank voles and serologic diagnosis of human infection. J. infect. Dis., 141, 131-134. VANDERGROEN, G. 0985), Haemorrhagic fever with renal syndrome: recent developments. Ann. Soc. beiges M#d. trop., 65, 121-135. GROEN, J. VANDER GROEN, G., HOOFD, G. & OSTERHAUS,A. 0989), Comparison of immunofluorescence and enzyme-linked immunosorbent assays tor the serology of Hantaan virus infections. J. Virol. Methods, 23, 195-203.
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B. N I K L A S S O N E T A L .
IVANOV,A.P., TKACHENKO,E.A., PETROV,V.A., PASHKOV,A.J., DZAGUROVA,T.K., VLAmMmOVA,T.P., VORONKOVA,G.M. & VANDER GROEN, G. (1988), Enzyme immuno assay for the detection of virus-specific IgG and IgM antibody in patients with haemorrhagic fever with renal syndrome. Arch. ViroL, lfl0, 1-7. LAHDEVmTA,J. (1971), Nephropathia epidemica in Finland. A clinical, histological and epidemiological study. Amer. clin. Res., 3, Suppl. 8 (Acad. Diss.). NmLASSON, B. & LEDuc, J. (1984), Isolation of the nephropathia epidemica agent in Sweden. Lancet, I, 1012-1013. NmLASSON, B. & LEDuc, J. (1987), Epidemiology of nephropathia epidemica in Sweden. d. infect. Dis., 155, 269-276. NIKLASSON,B., LEDuc, J., NYSTROM,K. & NYMAN,L. (1987), Nephropathia epidemica; incidence of clinical cases and antibody prevalence in an endemic area of Sweden. Epidemiol. Infect., 99, 559-562. NmLASSON, B. & KmLSSON,T. (1988), Detection of nephropathia epidemica (Puumala virus) specific immunoglobulins M by enzyme-linked immunosorbent assay, d. clin. Microbioi., 26, 1519-1523. SCHMALJOHN, C.S., HASTY, S.E., DALRYMPLE,J . i . , LEDuc, J., LEE, H.W., VON BONSDORFF, C.H., BRUMMERKORVENKONTIO,M., VAHEm, A., TSAI, T.F., REGNERY,H.L., GOLDGAaER,D. & LEE, P.W. (1985), Antigenic and genetic properties of viruses linked to hemorrhagic fever with renal syndrome. Science, 227, 1041-1044. SHESHBERADARAN,H., NIKLASSON,B. & TKACI-IENKOE.A. (1988), Antigenic relationship between hantaviruses analysed by immunoprecipitation, dr. gen. Virol., 69, 2645-2651.
Res.
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199~, 141, 637-648
HAEMORRHAGIC FEVER WITH RENAL SYNDROME: EVALUATION OF ELISA FOR DETECTION OF F UUMALA-VIRUS-SPECIFIC IgG AND
IgM B. Niklasson (1) (2), E. Tkachenko (3), A.P. Ivanov (3), G. van der Groen (4), D. Wiger (5), H.K. Andersen (6), J. LeDuc (7), T. Kjelsson (1) and K. Nystr6m (s) (1) National Bacteriological Laboratory, S-105 Stockholm;
(2) National Defence Research Establishment, FOA-5, S-172 90,
Sundbyberg (Sweden); (3) Institute of Poliomyelitis and Viral Encephalitides, Academy of Medical Sciences, 142782 Moscow; (4) Prince Leopold Institute of Tropical Medicine, Antwerp, (Belgium); (5) National Institute of Public Health, N-0462 Oslo 4; (6) Aaarhus University, 8000, Aarhus (Denmark); (7) US Army Medical Research, Institute of Infectious Diseases, Fort Detrick, Frederick 21702 MD (USA), and (a) Ume~ Hospital S-901 87 Umea (Sweden)
SUMMARY
IgM and IgG ELISA to Puumala virus were evaluated using sera from patients with haemorrhagic fever with renal syndrome (HFRS) from different geographical regions" Sweden, Denmark, Norway, Belgium and the European USSR. IgM ELISA proved useful in the diagnosis of HFRS in patients from all the regions mentioned above. Specific IgM could be detected as early as day I post onset of disease, and patients remained IgM-positive for several months. Specific IgG ELISA antibodies were also frequently detected in acute sera, and acute-convalescent serum pairs often failed to show a significant titre rise or increase in optical density (OD) values. This limits the use of IgG ELISA in patient diagnosis. Sera collected 2 years after infection revealed higher IgG ELISA OD readings than convalescent sera, and very high values were still detectable l0 to 20 years postinfection. IgG ELISA is therefore useful for the testing of immunity and in seroepidemiological studies.
Submitted September 13, 1990, accepted Octobe~ 30, 1990. Corresponding author: Bo Niklasson, Department of VRoiogy, N~fional BacteriologicalLaboratory, S-105 21 Stockhol,a (Sweden).
638
B. N I K L A S S O N E T AL.
Acute and convalescent sera from HFRS patients in Korea and the Asian USSR showed no or only very weak reactivity in the Puumala virus IgG and IgM ELISA. These results are consistent with the "one-way" crossing described earlier. KEY-WORDS:Bunyaviridae, Puumala virus, IgG, IgM, Haemorrhagic fever, Renal syndrome; ELISA, Diagnosis, Scandinavia, USSR, Belgium, Korea.
INTRODUCTION Haemorrhagic fever with renal syndrome (HFRS) refers to a clinical disease characterized by fever and renal dysfunction with or without ha~:morrhagic manifestation (van der Groen, 1985). At least 3 different strains of viruses in the Hantavirus genus of the Bunyaviridae family have been associated with human disease- Hantaan (HTN) virus, urban rat virus and Puumala (PUU) virus (Schmaljohn et al., 1985; Liihdevirta, 1971). Nephropathia epidemica (NE) caused by PUU virus is a mild form of HFRS which occurs in Scandinavia and Finland (Liihdevirta, 1971 ; N'iklasson and LeDuc, 1987). Clinically and serologically related or identical diseases occur in the western USSR and central Europe. Strains from these different regions have been isolated and compared using various serological methods including indirect immunofluorescence test and immunoprecipitation assay. When tested with these methods, all viruses which are associated with the clinical disease NE are indistinguishable (Sheshberadaran et al., 1988). The present study concerns the evaluation of an ELISA for detection of PUU-specific IgM and IgG antibodies using HFRS-patient sera from Sweden, Norway, Denmark, USSR, Belgium and Korea.
MATERIALS AND METHODS Serum samples.
Acute, early-convalescent and late-convalescent phase sera (N = 137) were available from 57 patients. Five patients from Norway (15 sera), 21 patients from Sweden (49 sera), 2 patients from Denmark (5 sera), 6 patients from Belgium (20 sera) and 23 from the European USSR (48 sera) were tested. Serum specimens were drawn during the first month postonset of symptoms and at least once again at a later date.
GMT HFRS HTN
= g e o m e t r i c m e a n titre. = h a e m o r r h a g i c fever with renal syndrome. = H a n t a a n (virus).
PUU NE IFT
= P u u m a l a (virus). - nephropathia epidemica. = i m m u n o f l u o r e s c e n c e test.
P U U M A L A - S P E C I F I C I g M A N D IgG I N H F R S
639
Sera were also collected in Sweden from 7 patients aged l0 to 20 years old following clinical NE infection. During an epidemiological investigation of apparently healthy populations in Sweden, sera from both HFRS endemic (N = 187) and nonendemic (N = 142) areas were collected and tested. Another group included 7 HFRS patients (9 sera) from the Asian USSR and 5 HFRS patients (5 sera) from Korea (kindly provided by H.W. Lee, Institute for Viral Diseases, Korea University, Seoul, Korea). All patients included in this study (excluding apparently healthy population) had a clinical history of HFRS and were antibody-positive in IFT to PUU virus (Niklasson and LeDuc, 1984) and HTN virus (Schmaljohn et al., !985), for European and Asian patients, respectively.
Immune reagents used in ELISA. Antigen was prepared from PUU-virus-infected (strain Vindeln 83-223L) Vero E-6 cells (CRL 1586; ATCC, Rockville, MD) (Niklasson and LeDuc, 1984). Preparation of PUU virus antigen, negative control antigen and rabbit immune serum is described elsewhere (Niklasson and Kjelsson, 1988).
Detection of PUU-virus-specific IgG antibodies by ELISA. A sandwich ELISA was employed as follows. Rabbit anti-PUU-virus Ig diluted 1/400 in coating buffer (0.05 M sodium carbonate pH 9.5 to 9.7) was adsorbed onto 96-well polystyrene microtitre plates (Cooke M29 AR, Dynatech laboratories) at 37°C for lh, followed by virus antigen (undiluted at 37°C for I h), test serum diluted 1/400 in ELISA buffer (phosphate-buffered saline with 0.05 070 Tween-20 and 0.5 °70 bovine serum albumin) at 37°C for 1 h, and swine anti-human-IgG conjugated with alkaline phosphatase diluted 1/200 in ELISA buffer at 37°C for 1 h (Orion Diagnostica, Finland, cat. no 67806). P-Nitrophenol phosphate (Sigma) diluted in diethanolamine buffer (1 M diethanolamine pH 9.8, 0.5 mM MgCl2) was used as substrate. Washing between each step was done 6 times in washing buffer (saline with 0.05 070 Tween-20). The reaction was read after 30 rain at room temperature in a spectrophotometer at 405 run and expressed as the optical density (OD). Optimal dilutions of all reagents used in the ELISA were determined by box titrations. All specimens were tested in duplicate with antigen and negative control antigen. The OD was calculated as the average OD with antigen minus the average OD with negative control antigen. The borderline between positive and negative results was calculated as the mean of the test result of 142 known negative sera (from non-endemic areas and negative by IgG IFT) plus 3 standard deviations. An OD of >0.080 was considered lo be positive. To adjust for plate-to-plate and test-to-test variations in the assay, positive control serum was included in all plates. This control had an OD of 0.700 (in the linear interval of this IgG ELISA). If the positive control serum had an OD between 0.500 and 0.900 the plate was accepted: however, all OD values on the plate were multiplied by a factor so as to set the positive control value at 0.700. The test was run using either a single dilution (1/400) or 2-fold dilutions starting at 1/400. A si.gnificant antibody rise was defined as an OD increase of 100 070 or a 4-fold titre rise using the single dilution and titration methods, respectively.
Detection of PUU-virus-specific IgM antibodies by ELISA. The test format for the IgM ELISA are described in detail in a previous publication (Niklasson et al., 1988). Briefly, a tt-capture ELISA was used to measure specif-
640
B. N I K L A S S O N E T A L .
ic PUU virus IgM. Goat anti-human-IgM (tt-chain specific; Cappel Laboratories, catalogue no. 0201-0201) was adsorbed onto the wells. Test sera diluted 1/400 were added, followed by PUU virus antigen, rabbit anti-PUU-virus Ig, alkalinephosphatase-labelled goat anti-rabbit-IgG (Kirkegaard and Perry, catalog no. 051506), and p-nitrophenyl phosphatase substrate. The optical density was read after 30 rain at room temperature. The OD was read and the borderline between positive and negative was calculated as described above for IgG ELISA. Detection of PUU virus IgG antibodies by WT. Spot-slides with PUU-or HTN-virus-infected ,CNiklassonand LeDuc, 1984) Vera E-6 cells were prepared and stained as described elsewhere (Niklasson and LeDuc, 1987). Test sera were titrated using 2-fold dilutions starting at 1/8. Fluoresceinisothiocyanate-conjugated sheep anti-human-IgG (Wellcome Diagnostics, Temple Hill, Dartford, England, catalogue no. MF03) was used to detect specific IgG.
RESULTS Evaluation of IgG ELISA using sera from Swedish patients. Early-convalescent phase sera (collected day 16 to 41 post-disease-onset) were available from all 21 patients investigated. Acute-phase sera, drawn on days 1-9, were available from 18 of the 21 patients, and late-convalescent phase serum specimens, collected 2 years after the acute infection, were available from 14 of the 21 patients. All these sera were tested by IgG ELISA and titrated to endpoint by IgG IFT. IgG ELISA OD results from all 21 patients over time are seen in figure 1. Figure 2 shows the same sera tested by IgG IFT. Comparison of figures 1 and 2 shows that the IgG ELISA OD mean continues to increase reaching a maximum value at 2 years while the IgG IFT geometric mean titre (GMT) has approximately the same value in convalescent and 2-year sera. To evaluate whether the difference between IgG ELISA and IgG IFT depended on single dilution (ELISA) versus endpoint titration (IFT), 18 sera from 6 patients (acute, early-convalescent and 2-year) were retested in parallel using endpoint titration for both ELISA and IFT. The IgG IFT GMT were 256, 1,024 and 645; IgG ELISA mean OD (1/400 dilution) 216, 420 and 665; and IgG ELISA endpoint GMT 1,008, 1,600 and 3,200 for acute, earlyconvalescent and 2-year sera, respectively. Eleven out of 11 and 7/11 had detectable antibodies in their first serum specimen (collected on days 1-5) when tested by IgG IFT and IgG ELISA, respectively. In these 11 patients with both acute (collected on days 1-5) and early-convalescent scra (collected on days 20-41), only 8 showed a 4-fold or greater titre rise by IFT. Only 6 out of the 11 paired sera tested by IgG ELISA showed an increase in OD of _>100 %.
PUUMALA-SPECIF!C
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Days (D) / Years after onset of disease FIG. 1. - - Acute (day 1-9), early-convalescent (day 16-42) and late-convalescent sera (2-year)
(N=49) from 21 Swedish patients tested by lgG ELISA. O D values for each serum is plotted as well as the m e a n O D o f acute, early-convalescent and late-convalescent sera. O D values are multiplied by 1,000.
All 7 sera collected more than 10 to 20 years postinfection were positive in both assays, with IgG E L I S A OD between 0.650-0.720 and IFT titres of 32-512. Out of 187 sera tested by IgG IFT from an endemic area, 34 were found positive. All 142 sera from the general population living in an nonendemic area were negative by IgG IFT. Identical results were found with IgG ELISA. All 34 positive sera from the general population had an OD of _>0.200 and a IFT titre of _>32.
HFRS patients frf~m the European USSR tested by IgG and IgM ELISA. All sera from HFRS patients from the European USSR were analysed at the Institute of Poliomyelitis and Viral Encephalomyelitides in Moscow using identical reagents. The data are presented separ~te!y since the patient~
642
B. N I K L A S S O N E T A L .
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10D 20D 30D 40D 50DH2Years Days (D) /Years after onset of disease FIG. 2. - - Acute (day 1-9), early-convalescent (day 16-42) and late-convalescent sera (2 years) ( N = 4 9 ) f r o m 21 Swedish patients tested by lgG IFT. IFT titre (log) for each serum is plotted as well as the G M T of acute, early-convalescent and late-convalescent sera.
sera were tested at 1/128 dilution instead of the 1/400 dilution which was used for all the other sera. In 14 of the 23 patients, the first serum sample was drawn 1-6 days after the onset of symptoms, and a second sample was taken after at least 6 days. A significant titre rise in IgG IFT was demonstrated in 8/14 patients while 11/14 showed a >100 % increase in IgG ELISA. In 7 of the 23 patients, the first specimens were collected on days 7-11 and the second specimens >6 days after the first sample. In these patients, only 2/7 showed a significant titre rise in IgG IFT and only 1/7 demonstrated > 100 °7o increase in IgG ELISA OD. The IgM ELISA OD values for sera from acutely ill patients in European USSR are presented in figure 3. Forty-six out of 48 (96 070)of the sera collected on days 1-27 were positive by IgM ELISA. The 2 negative samples were
P U U M A L A . S P E C I F I C Ig21~A N D IgG I N HFRS
643
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Acute and convalescent sera from HFRS patients from the European USSR tested for PUU-virus-specific IgM by ELISA.
Sera were tested at a single dilution (1/128). OD values are multiplied by 1,000.
collected on day 1. Only five out of ten sera collected on days 1-3 were positive by IgG ELISA.
HFRS patients from Norway, Sweden, Denmark and Belgium tested by lgG and IgM ELISA. The IgG ELISA results for serum specimens collected in Norway, Sweden (only 21 sera from 9 patients were included), Denmark and Belgium are presented in figure 4. In 11 serum pairs the convalescent sera were drawn between days 12-45 and the acute specimen collected _>10 days earlier. Only 6 of these 11 patients showed an OD increase of _> 100 %. Despite the lack of significant OD increase during this early period, the OD values generally showed a steady increase over the follow-up period. Only 8 of the total 45 patients showed a decrease in IgG ELISA OD at all. The remaining 37 patients showed a continuous increase over time. In
644
B. N I K L A S S O N E T A L .
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FIG. 4. - - Acute and convalescent serafrom HFRS patients from Norway, Sweden, Denmark
and Belgium tested for PUU-virus-specific lgG ELISA. Sera were tested at a single dilution (1/400). O D values are multiplied by 1,000.
15 patients both serum pairs were collected after day 30. Most of these pairs (14/15) showed an OD increase over time and 2/15 serum pairs showed an increase of > 100 %. IgM ELISA data are presented in figure 5. All of these sera were positive in the IgM ELISA during the first 2 months post onset of HFRS-symptoms. However, by 3-6 months they were in the lower range of significance or negative, and by 12 months postinfection all sera were IgM-ELISA-negative. HFRS patients from the Asian USSR and Korea.
Sera from the Asian USSR and Korea tested by IgG and IgM ELISA were negative or weakly positive and had non-detectable or very low titres to PUU virus by IgG IFT but high titres to HTN virus (76-118) by IFT (data not shown).
PUUMALA-SPECIFIC IgM AND IgG IN HFRS
645
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Weeks (W) / Months (M) after onset of disease FIG. 5. - - Acute and convalescent sera from HFRS patients from Norway, Sweden, Denmark
and Belgium tested for PUU-virus-specific lgM ELISA.
Sera were tested at a single dilution (1/400). OD values are multiplied by 1,000.
DISCUSSION A diagnostic test with high sensitivity and specificity is essential to define the range of HFRS symptoms as well as the geographical distribution of the different Hantaviruses. IFT for detection of specific Ig or IgG is presently used to confirm the HFRS diagnosis in the majority of routine laboratories, and presence of specific antibodies in acute as well as convalescent sera correlates well with the clinical diagnosis. In the present, as well as in previous studies, a significant proportion of patients with paired sera presented high antibody titres by IgG IFT in their first serum samples (Niklasson and Kjelsson, 1988). Seroconversion or a 4-fold titre rise by IgG IFT is therefore not always found. There is no accepted "standard" way of calculating a significant OD increase by ELISA when comparing results for acute and convalescent sera. Different laboratories use different criteria.
646
B. N I K L A S S O N
ET AL.
In the present study we decided to use an OD increase of _> 100 °70 as the criterion of a titre rise between acute and ~ mvalescent sera when testing at one dilution only. When developing the ask ~y, both endpoint titration and single dilution methods were tried but neither solved the problem of lack of a significant titre rise between acute and convalescent serum specimens (data not shown). IgG ELISA therefore does not appear to offer any advantages over IgG IFT in the diagnosis of NE patients. HFRS caused by PUU or a PUU-related virus is highly endemic in certain parts of Scandinavia, Finland and the European USSR and occurs sporadicaUy in the rest of Europe. Since antibody prevalence rates in highly endemic areas of Sweden may reach more than 30 070 in the older age-groups (Niklasson et al., 1987), *.hepresence of specific IgG may reflect a previous infection and may have nothing to do with the patient's present condition. All acute- and early-convalescent sera (collected on day 2 or later, pos~ onset of symptoms) were positive by IgM ELISA and patients maintained detectable IgM antibody levels for several months. PUU-specific IgG antibodylevels often continued to increase over time. This observation has been documented earlier by Brummer-Korvenkontio et al. (1980). In the present antibody study, we found high titres and high OD values for as long as 20 years postinfection, indicating that this assay is useful for seroepidemiological surveys. The immunological background for this continuous production of specific IgG antibodies is not clear. The presence of high titre specific IgG could be due to continuous expression of virus-specific antigen. However, if infectious virus persists, there are no clinical observations indicating any symptoms after the acute disease. No serologically confirmed case of recurrent infection has been described. It should be noted that the IgM as well as the IgG ELISA showed only very weak reactions or did not detect IgM or IgG antibodies when used on HFRS patient sera from Korea or the Asian USSR. ELISA for detection of human IgG and IgM to prototype HTN (76-118) strain has been developed and evaluated by other investigators (Ivanov et al., 1988; Groen et al., 1989). ELISA systems for detection of specific IgM as well as IgG to HTN virus has also been developed by J. Meegan, J. LeDuc and coworkers at the US Army Medical Research Institute of Infectious Diseases. Ten early convalescent sera from Swedish NE patients (positive by PUU IgM and IgG ELISA) were packed in dry ice and shipped to the USA and tested in the HTN IgM and IgG ELISA. All ten 10 sera were found to be positive by both procedures (data not shown). A serological "one-way" crossing between PUU virus and HTN virus by IFT and immunoprecipitation assay has previously been well documented (Schmaljohn et al., 1985; Sheshberadaran et al., 1988). This pattern also appears to hold true for IgM and IgG ELISA. The present study extends the geographical area were the PUU IgM and IgG ELISA can be used. The first report of serologically confirmed HFRS cases in Denmark is also presented. However, it is evident that IgM and IgG
P U U M A L A - S P E C I F I C I g M A N D igG I N H F R S
647
E L I S A using P U U virus as antigen cannot be used alone in areas were both P U U and H T N viruses cause HFRS. In the geographic areas where both viruses circulate, it would be possible to distinguish between the two serological types by running both H T N and P U U IgM E L I S A in parallel.
RI~SUMI~ FII~.VRE HI~MORRAGIQUE AVEC SYNDROME RI~NAL: I~VALUATION D'UN TEST ELISA POUR LA DI~.TECTION D'IgG ET D'IgM SPI~CIFIQUES DU VIRUS PUUMALA
A l'aide d'on test ELISA, nous avons 6valu6 les IgM et IgG anti-virus Puumala dans le s6rum de sujets originaires de divers pays (Suede, Dannemark, Norv6ge, Belo gique et URSS europ6enne, sujets atteints de fi/~vre h~morragique avec syndrome r6nal (HFRS). Le test ELISA-IgM est utile pour le diagnostic de I'HFRS chez les malades de tousles pays mentionn~s ci-dessus. Les IgM sp~cifiques peuvent ~tre d~tect~es d~s le 24e heure apr~s la d~c!aration de la maladie, et les sujets sont rest~s IgM + durant plusieurs mois. Les IgG sont fr~quemment d~tect~s par ELISA dans les s~rums de malades en phase aigu~; pour les paires de s~rums correspondant aux phases aigui~ et convalescente, il n'y a g~n~ralement pas d'augmentation significative de titre ou d'~16vation des valeurs de la densit~ optique (DO). Cela limite l'utilisation de I'ELISAIgG pour le diagnostic. Les se~'t!msrecueillis 2 arts apr~s l'infection ont des DO plus ~lev~s pour le test ELISA-IgG que les s~rums de convalescents, et des valeurs tr~s ~lev~es sont encore d~tectables 10 ~ 20 ans apr~s l'infection. L'ELISA-IgG est cependant utile pour tester l'immunit~ et pour les ~tudes s~ro~pid~miologiques. Les s~rums de malades HFRS en phases aigu~ et convalescente, en CorSe et URSS asiatique, ont une minime ou nulle r~activit~ au test ELISA-IgM ou -IgG pour le virus Puumala. Ces r~sultats sont en accord avec ceux de l'analyse "one-way cross" pr~c~demment d~crite. MOTS-CLES: Bunyaviridae, Fi~vre h6morragique, Syndrome r6nal, Virus Puumala, IgG, IgM; ELISA, Diagnostic, Scandinavie, Belgique, URSS, Cor6e.
AC KNOWLEDGEMENTS
This project was supported by the US Army Medical Research and Development Command (DAMD 17-86-C-6073).
REF ~!RENCES BRUMMER-KORVENKONTIC~:M., VAHERI,A., HOVl, T., VON BONSDORFF,C.-H., VUORIMIES, J., MANNI, T., PENrTINEN, K., OKER-BLOM, N. & L.~HDEVIRTA, J. (1980), Nephropathia epidemic,a: detection of antigen in bank voles and serologic diagnosis of human infection. J. infect. Dis., 141, 131-134. VANDERGROEN, G. 0985), Haemorrhagic fever with renal syndrome: recent developments. Ann. Soc. beiges M#d. trop., 65, 121-135. GROEN, J. VANDER GROEN, G., HOOFD, G. & OSTERHAUS,A. 0989), Comparison of immunofluorescence and enzyme-linked immunosorbent assays tor the serology of Hantaan virus infections. J. Virol. Methods, 23, 195-203.
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B. N I K L A S S O N E T A L .
IVANOV,A.P., TKACHENKO,E.A., PETROV,V.A., PASHKOV,A.J., DZAGUROVA,T.K., VLAmMmOVA,T.P., VORONKOVA,G.M. & VANDER GROEN, G. (1988), Enzyme immuno assay for the detection of virus-specific IgG and IgM antibody in patients with haemorrhagic fever with renal syndrome. Arch. ViroL, lfl0, 1-7. LAHDEVmTA,J. (1971), Nephropathia epidemica in Finland. A clinical, histological and epidemiological study. Amer. clin. Res., 3, Suppl. 8 (Acad. Diss.). NmLASSON, B. & LEDuc, J. (1984), Isolation of the nephropathia epidemica agent in Sweden. Lancet, I, 1012-1013. NmLASSON, B. & LEDuc, J. (1987), Epidemiology of nephropathia epidemica in Sweden. d. infect. Dis., 155, 269-276. NIKLASSON,B., LEDuc, J., NYSTROM,K. & NYMAN,L. (1987), Nephropathia epidemica; incidence of clinical cases and antibody prevalence in an endemic area of Sweden. Epidemiol. Infect., 99, 559-562. NmLASSON, B. & KmLSSON,T. (1988), Detection of nephropathia epidemica (Puumala virus) specific immunoglobulins M by enzyme-linked immunosorbent assay, d. clin. Microbioi., 26, 1519-1523. SCHMALJOHN, C.S., HASTY, S.E., DALRYMPLE,J . i . , LEDuc, J., LEE, H.W., VON BONSDORFF, C.H., BRUMMERKORVENKONTIO,M., VAHEm, A., TSAI, T.F., REGNERY,H.L., GOLDGAaER,D. & LEE, P.W. (1985), Antigenic and genetic properties of viruses linked to hemorrhagic fever with renal syndrome. Science, 227, 1041-1044. SHESHBERADARAN,H., NIKLASSON,B. & TKACI-IENKOE.A. (1988), Antigenic relationship between hantaviruses analysed by immunoprecipitation, dr. gen. Virol., 69, 2645-2651.