Journal of Infection (I984), 9, 230-238
Clinical and serological diagnosis o f Nephropathia epidemica, the mild type o f haemorrhagic fever with renal s y n d r o m e Juhani L~ihdevirta,* Jaakko Savola,t Markus Brummer-Korvenkontio,$ Rauni Berndt,t Riitta Illikainent and Antti Vaheri$ * Aurora Hospital, Nordenski6ldinkatu 20, 00250 Helsinki, tPieks~m~ki Regional Hospital, Sairaalantie 14, 76 IOO Pieksiimi~ki and ~Department of Virology, University of Helsinki, Haartmaninkatu 3, 00290 Helsinki, Finland Accepted for publication 5 July I984
Summary The serological diagnosis of Nephropathia epidemica (NE), the.mild European type of haemorrhagic fever with renal syndrome (HFRS), was studied by means of an indirect immunofluorescent antibody technique (IFAT) in a large outbreak in Finland (morbidity 1.4/Iooo population). Acetone-fixed sections of lung of bank voles (Clethrionomys glareolus) naturally infected with Puumala virus served as antigen. Altogether 65 patients were followed serologically for up to 20 months. Serological results obtained by N E - I F A T were compared with the clinical diagnosis of NE. Of the 4I clinically accepted NE cases 40 were serologically positive. Use of the N E - I F A T serological test allowed the diagnosis to be made in seven patients for whom the clinical data were not sufficient for diagnosing NE. The antibody response in NE often appears to be slower than that against Hantaan virus. Practical recommendations are given for the clinical and serological diagnosis of the milder forms of HFRS.
Introduction W H O has recently I r e c o m m e n d e d the use of the n a m e haemorrhagic fever with renal s y n d r o m e ( H F R S ) for a disease k n o w n since the 193o's in various regions of the F a r East, Siberia, Eastern E u r o p e and F e n n o - S c a n d i n a v i a 2 O f the previous local names used for it, the best k n o w n are epidemic haemorrhagic fever ( E H F ) in Japan during the Second W o r l d W a r and later during the K o r e a n war; haemorrhagic n e o p h r o s o - n e p h r i t i s ( H N N ) in the U S S R and Eastern E u r o p e ; K o r e a n haemorrhagic fever ( K H F ) and N e p h r o p a t h i a epidemica (NE) in Scandinavia and Finland. Recently, the term m u r o i d virus n e p h r o p a t h y was also p r o p o s e d for H F R S ) As these names imply, the disease is a haemorrhagic fever, its main manifestation being an acute haemorrhagic t u b u l a r and interstitial nephritis. According to the clinical severity and also to the aetiology o f the disease, two types of H F R S are nowadays distinguished: the K H F type with a mortality of a b o u t 5-IO % and with clear haemorrhagic manifestations, and the E u r o p e a n or N E type with a mortality below 0"5 % and very scanty haemorrhagic manifestations. Correspondence: Dr Juhani L~ihdevirta, Aurora Hospital, Nordenski61dinkatu 20, SF-oo25o Helsinki, Finland. o163-4453/84/o6o23o+o9 $o2.oo/o
© 1984 The British Society for the Study of Infection
Antibodies in Nephropathia epidemica
T h e aetiology of H F R S , at least in the Far East, is the Hantaan virus isolated in r976 by Lee and collaborators 4, 5 from the Korean field mouse, Apodemus agrarius coreae, while the viral etiology of N E (Puumala virus) has only very recently been verified (J. Dalrymple, C. Smaljohn, S. E. Hasty, A.Vaheri, M. Brummer-Korvenkontio and C.-H. von Bonsdorff, manuscript in preparation). T h e first attempt to make a specific serodiagnosis of N E was by means of the indirect immunofluorescent antibody technique ( I F A T ) developed by Lee. 5 This requires the use of sections of lung from field mice and which contain Hantaan virus as antigen. An early study of samples of serum from patients with N E revealed a serological relationship between Hantaan virus and the N E agent. 6 T h e lower titres in respect of the latter agent compared with those related to the K H F agent, however, raised the question of antigenic variance or of differences between the aetiological agents of N E and K H F , features which would also be in keeping with the differences in clinical findingsY, s A more specific method for the serological diagnosis of N E became available when in Finland 9 the N E antigen was found in the lungs of naturally infected bank voles, Clethrionomys glareolus. This antigen is detectable in the lung tissues by immunofluorescence at an intensity similar to that of Hantaan antigen when K H F sera are tested. Furthermore, it gave clearly higher titres for N E sera than did Hantaan antigen. U s i n g this serological N E - I F A T method the validity of the serological diagnosis of N E compared with the clinical diagnosis and vice versa was studied in samples of serum collected from patients during an unusually large local epidemic in central Finland. Materials and methods
T h e series consisted of 65 patients (22 females and 43 males, mean age 35"4 years, ranging from I2 to 7 ° years) with acute fever and clinically (see later) suspected of having N E on admission to the Pieksfimfiki Regional Hospital during the epidemic that took place between August I979 and January I98o. T h e total area from which the patients originated was 2700 km 2, of which 43o km 2 is water, and with a population of 33 ooo inhabitants. T h e area is located in Central Finland within the high risk ' e n d e m i c ' part of the country.l° Between two and six blood samples were taken from each patient while in hospital, during convalescence and during the follow-up period for as long as 2o months after the acute illness. Altogether 239 samples were collected and studied by the N E - I F A T method. Ninety-nine samples were from the first month after onset of fever, 87 were taken between the 2nd and r2th months and 53 during the second year. This was also the case with the serologically negative patients, in order to ensure that they remained negative. T h e series was divided into four groups of patients with respect to the validity of the clinical N E diagnosis (Table I). This grouping was made on the basis of 2o years' clinical experience by D r Lfihdevirta (co-author). T h e clinical picture of NE, especially the chronological pattern of the findings, which is the basis of diagnosis, has been described in detail elsewhere. 7 Briefly stated, N E begins with acute high fever for 3-5 days with headache from the second day onwards. From the third day there are nausea and vomiting, backache and abdominal pains which last for 3-6 days. F r o m the third to fifth day
J. L A H D E V I R T A E T A L .
T a b l e I Correlation between the clinical and serological diagnosis of Nephropathia epidemica (NE) in a series of 65 patients with initial suspicion of N E Distribution of cases according to serological results
Final clinical diagnosis NE diagnosis accepted With typical NE syndrome With some features missing NE diagnosis not accepted No other diagnosis confirmed Other diagnosis confirmedt Total
Diagnostic rise in NE-IFAT titre
High but unchanging NE-IFAT titre
3 r (i + 20 + IO)* 7 (o + I + 6)
2 (o + I + I) o
NE-IFAT antibodies not detected (titre < 20) Total o I (o + o + i)
7 (o + o + 7) o
45 (I + 2 I +23)
,~ Clinical severity of cases (severe + moderate + mild). t These diagnoses are given in the text.
p r o t e i n u r i a , h a e m a t u r i a w i t h l eucocyt uri a, oliguria and azotaemia appear. T h e general c o n d i t i o n begins to i m p r o v e w h e n the p o l y u r i c phase starts in t he s eco n d week. M o s t o f the l a b o r a t o r y values b e c o m e n o r m a l d u r i n g the t h i r d to fifth weeks. T h e first g r o u p , ' t y p i c a l N E cases ', consisted o f patients w hose disease was easy to recognise as N E . I n t he s e c on d g r o u p , some typical features were missing or n o t s o u g h t b u t the diagnosis o f N E coul d be accepted. I n the t h i r d g r o u p , so m a n y features were missing that the diagnosis o f N E was n o t acceptable, and in t he f o u r t h g r o u p , in addi t i on to this, some o t h e r diagnosis co u ld be finally confirmed. T h e I9 clinical and l a b o r a t o r y findings, w hi ch were selected for T a b l e II, are n o t the onl y features o f this d i s e a s e / b u t t h e y w ere chosen as the m o s t p r o m i n e n t and useful findings in clinical practice. As in earlier work, 11 the patients w e r e di vi ded into t hree groups o f clinical severity m a i n l y a c c o r d i n g to the peak value o f s e r u m creatinine. I n m i l d cases it was b elo w 2oo #mol/1, in m o d e r a t e cases b e t w e e n zoo and 8oo #mol/1, and in severe cases if was over 8oo #mol/1. T h e N E - s p e c i f i c i ndi r e c t i m m u n o f l u o r e s c e n t a n t i b o d y test was p e r f o r m e d as p r e v i o u s l y d e s c r i b e d 9 with acet one-fi xed sections o f lung o f P u u m a l a v i r u s - i n f e c t e d bank voles as antigen. All s e r u m dilutions were tested in d u p l i c a t e ; positive a nd negative r e f e r e n c e sera were i n c l u d e d in each bat ch o f tests.
Antibodies in Nephropathia epidemica
The prevalence of I9 clinical and laboratory findings in relation to the N E - I F A T serological tests and compared to the prevalence of findings among the clinical group ' Typical N E syndrome' in the present study and in a previous clinical study/ The most differentiating findings in the present study are underlined (solid line = prevalence 3 x higher in the serologically accepted group than in the serologically negative group; dotted line = prevalence 2 x higher)
Prevalence of findings (%) The present study according to serological results
Findings General Acute high fever Nephrological _S_e_r_u_m_c_r_e atin i_n_e _~_II 5 ~_mol/l_ Proteinuria Polyuria >3ooo ml/day Tenderness over kidneys Backache Erythrocyturia _L _e u_c_o_c_y_t_u_ria_ Haematological Thrombocytopenia < IOO X I0-9/1 Haemoconcentration > 20 g/1 Neurological Headache Vomiting, nausea Myopia Gastrointestinal Abdominal pain Elevation of serum A S A T (GOT) Elevation of serum A L A T (GPT) Circulatory Bradycardia ( < 6o/min) Hypotension ( < i2o m m Hg) E C G changes Mean n u m b e r of findings (total listed I9) per patient
Typical NE Serologically Serologically syndrome in accepted cases negative cases the present (n = 47) (n = I8) study (n = 33)
Previous study 7 (n = 76)
8o 68 53 53 45 15
6x 6 61 28 22 o
97 9I 67 6I 48 15
I oo 62 84 82 74 67
64 53 6
67 33 0
7o 6r 9
90 75 I2
32 30 28 9"0
o ix o 4"4
36 33 36 Io'3
36 40 42
The mean numbers of positive findings per patient for the other clinical subgroups of Table I were: N E diagnosis accepted with some missing features, serologically accepted, 5.6 (7 patients); NE diagnosis accepted with some missing features, serologically negative, 7"o (i patient); NE diagnosis not accepted, not other diagnosis, serologically accepted, 5"o [7 patients); N E diagnosis not accepted, not other diagnosis, seronegative, 4"7 (3 patients); NE diagnosis not accepted, other diagnosis, seronegative, 4"I (I4 patients).
J. LAHDEVIRTA E T A L .
Results Serological findings A diagnostic rise in N E - I F A T titre was detected in 45 patients. In addition, there were two patients with a typical N E syndrome and a high persisting antibody titre. In the first patient the titre was 32o on days 8, 33 and 7I ; in the second patient it was 64o on days 9, 33 and 46, and at 4 months and I8 months after the onset of fever. These high titres in association with a typical clinical N E syndrome were regarded as significant (see Discussion). Both patients have been included in Fig. I, in which the titres of the 47 patients are shown. T h e shape of the curve of geometric mean titres demonstrates the initial rise as the usual primary antibody response, although somewhat slow, but later the titre is rather persistent. In order to illustrate further the serological response, the curves of the titres for 32 patients are presented in Fig. 2. In this figure the patients in the groups on the left [(a), (c) and (e)] had, during the first IO days of illness, an N E - I F A T titre ~<2o, while those on the right [(b), (d) and (f)] had a titre > 2o. T h e r e was no great difference in the antibody response among these groups. T h e patients were further subdivided into groups with declining [(a) and (b)], persistent [(c) and (d)] or increasing [(e) and (f)] N E - I F A T titres after 6 months. As regards the clinical severity of the disease, it was found that the proportion of mild cases was highest (69 %) in the groups with declining titres [(a), (b)], medium (5o %) in the intermediate groups [(c), (d)] and lowest (33 %) in the groups with increasing titres [(e), (f)]. In view of the limited n u m b e r of patients in these groups, however, the correlation observed should be regarded as tentative only. Relation between serological and clinical diagnosis A comparison between the serological and clinical diagnosis is presented in Table I. All the 33 patients with typical N E were serologically positive; 31 had a serological diagnosis and two an unaltered but relatively high titre (see above). All except one (7/8) of the second group (' with some clinical features missing') also had a serological diagnosis but in the third group ('no other diagnosis confirmed'), seven of Io had a diagnostic rise in titre. All of these cases were considered clinically to be mild. T h e other diagnoses in the fourth group of I4 patients were as follows: pneumonia (4 patients); acute bronchitis (3 patients); acute sinusitis (2 patients, one with septicaemia); bronchopneumonia, acute sinusitis and serologically verified Chlamydial infection (i patient); Toxoplasmagondii infection (r patient); diarrhoea following penicillin therapy in a patient with rheumatoid arthritis and coeliac disease (r patient); undefined transitory abdominal and chest pains (i patient); and fever and undefined headache (r patient). Clinical differential diagnosis In order to elucidate the clinical differential diagnosis of NE, the percentage prevalence of I9 clinical and laboratory findings of the whole series in relation to the serological results are set out in Table II. T h e prevalences are compared with those of the present clinical group with typical N E syndrome (see Table I) and of a previous s t u d y ! In addition, Table II shows the mean prevalences
Antibodies in Nephropathia epidemica
320 u. m
8 o =o;o o
~88 o o
o~o o o ~ 8 ~ 08
p--r . . . . . . . . . . . . . . ~'----~;~.......... 20 50 2
~ ~ ~
q - - - i ~ ~i
Months Time a f t e r onset of fever
Fig. I. T h e chronological pattern of N E - I F A T antibody titres in Nephropathia epidemica. T h e figure shows a total of x8x titres representing 47 serologically positive patients of which 45 showed a significant increase in antibody titre. T w o h a d persistent high titres related to attacks of the typical clinical N E s y n d r o m e (see text). T h e geometric m e a n values (solid line) have been calculated at intervals as s h o w n by the parts of the broken line over the time scale.
of findings per patient in the patient subgroups of Table I, illustrating how they differ. Table II forms the basis for discussion of how cases of N E may be recognised clinically. Discussion
This series of patients, originating from the largest recent localised civilian epidemic of N E in Finland, was particularly suitable for the present study because of its size and because of its confinement to one epidemic within the area served by a single hospital. This gave the advantage of being able to perform more coherent clinical studies and serum sampling than is usually possible in series collected from several epidemics and hospitals. Clinically, illnesses in the present series were milder than usual: the distribution of cases according to the severity of disease (severe, moderate and mild) in the 47 serologically accepted cases was 2 % , 47 % and 51%, and in both clinically and serologically accepted cases 2 %, 55 % and 43 %, whereas in the earlier clinical study 7 it was 9 % , 61% and 3o%, respectively. T h e mildness of illnesses in the present series may be regarded also as an advantage, because one aim of this study was to elucidate the differential diagnosis of NE, something which is clinically most difficult to determine in mild cases. Epidemiologically, the present series is of the rural type of HFRS. In Finland the only verified source of infection is the bank vole, a, 12 in which
J. L A H D E V I R T A
I 1 I
, I I
1280 520 8O 2O <20 i
I0 2 0 3 0
2 4 6 8 I0
I0 2 0 30
2 4 6
i t i
[ i i
i i i i
Time a f t e r onset of fever
Fig. 2. T h e curves of the individual N E - I F A T titres of 32 N E patients presented in 6 small figures. In those on the left [(a), (c), (e)] the patients had a titre ~<2o during the first zo days and on the right [(b), (d), (f)] > 2o. In the two upper groups [(a) and (b), 4I % of cases] the patients had a decline in the titre after 6 months, in the middle [(c) and (d), 3T % of cases] an unaltered titre, and at the bottom [(e) and (f), 28 % of cases] an increase in titre after 6 months.
species o f rodent the N E agent (Puumala virus) has been serially passaged in the laboratory. 12 The total morbidity in the present epidemic, z'4/zooo population, is apparently the highest known in Fenno-Scandinavia to date for a localised civilian outbreak. A remarkable simultaneous finding was the extraordinary high infection rate (up to 75 %) with the N E agent among the local bank voles within the epidemic areas. 1~ The corresponding total human morbidity in I965-6 in another area in Finland 7 was o-3/Iooo population, but in one small village was as high as 4 o / I o o o population. Compared to these values, the military epidemic 13,14 in T942 showed a still higher total morbidity o f about IOO/IOOO population. One peculiarity o f N E has been the much lower proportion o f female than male patients. In two earlier studies 7,15 in Finland only 2 0 % and I 2 " 5 % , respectively, were females. The cause of this phenomenon has been unclear, but differences in the rates of exposure have been suggested3 Another explanation is that females may have more mild, atypical and inapparent
Antibodies in Nephropathia epidemica
illnesses. Although the proportion of females in the present series was higher, the findings do not support the latter hypothesis since the proportions were the same among the clinically as among the serologically accepted cases, 32"5 % and 34 %, respectively. T h e good correlation between, on the one hand, the results of the N E - I F A T test and the clinical diagnosis of N E and, on the other hand, the detection of N E infections by serological means only (18 % of total in Table I) indicates that this serological method is useful in the diagnosis of NE. T h e N E - I F A T titres obtained are of the same order as those obtained with the corresponding method in K H F ? Compared to K H F , there are, however, some differences in the antibody response. T h e mean initial rise and the mean decline in titre appear to be slower than those described for K H F ? This may reflect the dissimilarities in the aetiological agents, the Puumala and Hantaan viruses. In the interpretation of the antibody titres the special features of N E - I F A T (Fig. 2) should be considered. For example, the two patients in the present study with high unaltered titres were regarded as having had recent infections because the first samples were taken so late that with a rapid initial antibody response it would have been possible for antibody to reach the plateau as shown in Fig. 2. Furthermore, clinically, the patients had typical features related to a recent illness due to N E rather than to an earlier one. T h a t these patients had pre-existing high titres from an atypical attack of N E months or years earlier, is most unlikely because reinfections are very improbable 7,10 as also are cases of typical clinical N E syndrome caused by another aetiological agent (Table I). A patient with an unrelated febrile disease and with a slowly rising N E - I F A T titre derived from an N E infection months earlier could, however, pose a problem in interpretation. T h e above considerations and Fig. 2 assist in deciding the timing of serum samples for N E - I F A T tests. In order to avoid difficulties in interpretation, the first sample should be taken as soon as possible during the first week after onset of fever and the second sample 2-3 weeks later. A third sample, 2-6 months after the acute phase, may be recommended, because it often gives important information. Also in ordinary practice, without special scientific purposes, the first serum specimen is often not collected until after the first week of illness. T h e present study shows that the use of serological tests is not essential for diagnosing typical clinical cases of N E if the clinician is familiar with the disease. If this is not so, however, N E - I F A T tests serve as a value tool in diagnosis. T h e y are especially needed in atypical and other cases where a clinical diagnosis is impossible. T h e recently recognised and extensive spread of Hantaan virus among rats in city harbours and the wide distribution of the N E agent 1~ make it necessary to look for H F R S in countries where clinicians are not yet aware of these diseases. In order to detect cases of H F R S , serological investigation with the Hantaan ( K H F ) and Puumala (NE) I F A T tests is of primary importance; for a clinical search of cases of H F R S it is also important to know the salient clinical features of its various forms of presentation. Table II serves to give practical advice in the case of NE, the mild type of H F R S , which is probably more difficult to diagnose than K H F especially in the mildest forms of NE. In order to find cases of N E , the clinician should consider the many features, such as
a c u t e h i g h f e v e r , p r o t e i n u r i a , h e a d a c h e a n d t e n d e r n e s s o v e r t h e k i d n e y s , all o f w h i c h b e l o n g to t h e b a s i c clinical s y n d r o m e o f N E , b u t w h i c h are c o m m o n also in o t h e r diseases. I t is p a r t i c u l a r l y u s e f u l to c o n s i d e r s o m e o f t h e o t h e r f e a t u r e s ( u n d e r l i n e d in T a b l e I I ) , s u c h as a z o t a e m i a , t h r o m b o c y t o p e n i a a n d p o l y u r i a , w h i c h c a r r y m o r e w e i g h t in t h e d i f f e r e n t i a l d i a g n o s i s . O n t h e o t h e r h a n d , s o m e f e a t u r e s , s u c h as m y o p i a , w h i l e a l m o s t p a t h o g n o m o n i c , are r a r e . (We thank Ms T y t t i Manni, Ms Helka Saukkonen, B.A., and Ms Seija U k k o n e n for expert technical assistance and Ms Anne Brisk and Ms Satu Cankar for secretarial help. T h i s work was supported by grants f r o m the Sigrid Jus6lius Foundation, Helsinki, and the Medical Research Council of the A c a d e m y of Finland.) References I. Haemorrhagic fever with renal syndrome : Memorandum from a WHO meeting. Bull W H O 1983; 6I : 269-275. 2. Gajdusek DC. Virus hemorrhagic fevers. Special reference to hemorrhagic fever with renal syndrome (Epidemic hemorrhagic fever). J Pediatr 1962; 60: 841-857. 3. Gajdusek DC. Hemorrhagic fever with renal syndrome (Korean hemorrhagic fever, epidemic hemorrhagic fever, nephropathia epidemica)- a newly recognized zoonotic plaque of the Eurasian landmass with the possibility of related muroid virus nephropathies on the other continents. In: MacKenzie JS, ed. Viral Diseases in South-East Asia and the Western Pacific. Sydney: Academic Press, I98Z: 576-594 . 4. Lee HW, Lee PW. Korean hemorrhagic fever. I. Demonstration of causative antigen and antibodies. Korean J lnt Med (Seoul) I976; x9: 371-383. 5. Lee HW, Lee PW, Johnson KM. Isolation of the etiologic agent of Korean hemorrhagic fever. J Infect Dis 1978; x37: 298-308. 6. Lee HW, Lee PW, Lfihdevirta J, Brummer-Korvenkontio M. Aetiological relation between Korean haemorrhagic fever and nephropathia epidemica. Lancet I979; i: I86-I87. 7. Lfihdevirta J. Nephropathia epidemica in Finland. A clinical, histological and epidemiological study. Ann Clin Res 1971; 3 (Suppl 8): I-I54. 8. L/ihdevirta J. Clinical features of HFRS in Scandinavia as compared with East Asia. Scand J lnfect Dis I982; Suppl 36: 93-95. 9. Brummer-Korvenkontio M, Vaheri A, Hovi T, et al. Nephropathia epidemica: Detection of antigen in bank voles and serologic diagnosis of human infection. J Infect Dis 198o; x4I: I3I-I34. IO. Lfihdevirta J, Korpela H. An emerging zoonosis in Scandinavia - Nephropathia epidemica. Nord Vet Med I977; 29: 4o6-412. i I. Penttinen K, Lfihdevirta J, Kekomfiki R et al. Circulating immune complexes, immunoconglutinins, and rheumatoid factors in Nephropathia epidemica, ff lnfect Dis I981 ; I43: 15--21. 12. Brummer-Korvenkontio M, Henttonen H, Vaheri A. Hemorrhagic fever with renal syndrome in Finland: Ecology and virology of Nephropathia epidemica. Scand ff Infect Dis 1982; Suppl 36: 88-91. 13. Stuhlfauth K. Bericht fiber ein neues schlammfieberfihnliches Krankheitsbild bei Deutschen Truppen in Lappland. Dtsch A/led Wochenschr 1943; 69: 439-443, 474-477. 14. Hortling H. En epidemi av f~iltfeber (?) i fmska Lappland. Nord ivied 1946; 3o : lOO1-1oo4. 15. Lfihdevirta J, Elo O. Nephropathia epidemican esiintyminen Suomessa vuosina 1966-1973 (Epidemiology of Nephropathia epidemica in Finland between 1966-1973). Suomen Lii~k~rilehti (Helsinkz~ 1975; 3o : 677-682. 16. Gajdusek DC. Muroid virus nephropathies and muroid viruses of the Hantaan virus group. Scandff Infect Dis 1982; Suppl 36: 96-1o8.