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NEPHROPATHIA EPIDEMICA

C.-H. VON BONSDORFF, M. BRUMMER-KORVENKONTIO, T. HOVI, J. LAHDEVIRTA, N. OKER-BLOM, K. PENTTINEN, P. SAIKKU, A. VAHERI
Department of Virology, and Third Department of Medicine, University of Helsinki, Haartmaninkatu 3, SF-00290 Helsinki 29, Finland.
INTRODUCTION

Nephropathia epidemica (NE) is a clinical entity with a close relationship to the hemorrhagic fever with renal syndrome. The disease has been described by Myhrman (2) and Zetterholm (2) in Sweden. In Finland the first and also largest outbreak occurred during World War II, when during three months over 1000 German and 60 Finnish soldiers in a small area in northern Finland near the Russian border fell ill (3,4). The total number of notified cases of NE in Finland after the war is about 1000 (5,6), in Sweden 400 (7,8) and 60 in Norway (9,10). The seven cases described by Hansen (11) from Denmark as "atypical acute nephritis" may also be regarded as NE.

CLINICAL FEATURES

The clinical picture of NE (for detailed description see Lähdevirta (5)) is similar much less severe than that of the hemorrhagic fever with renal syndrome. The hemorrhagic manifestations are scanty, the hematuria is usually microscopic and lasts only for 2 to 6 days. The mortality is less than 1/200.

The disease has an acute onset. Without prodromal signs the fever rises within a few hours over 39ºC. Headache, nausea, vomiting, somnolence and other neurological symptoms (in a few cases meningismus) follow thereafter. On the 3rd to 6th day the renal involvement develops manifesting as backache, abdominal pain, tenderness over the kidneys, proteinuria (mostly 1 to 1.5 g/1) and oliguria. In most cases microscopical hematuria (5 to 10 erythrocytes per field), leucocyturia and azotemia is seen, as well. The number of blood platelets is often between 50.000 and 150.000 sometimes only 20.000 per mm³. The oliguria (rarely anuria) phase of duration is followed by one week's polyuria and hypostenuria. The decreased concentration capacity of kidneys persists for several weeks. With the onset of the polyuric phase the patients' condition shows surprisingly rapid improvement. The mean duration of the hospitalization is 17 days. The persistent sequalae slight if any. NE seems to be a multisystemic infection and evidence for carditis, hepatitis and meningoencephalitis has also been obtained sometimes. We have found rheumatoid factor activity to be transiently present in the serum of NE patients during the early phase of the disease (Fig. 1). The timing of the activity suggests that it may, like in certain other acute infections (12), be an indication of temporary presence of immune complexes in circulation.

Fig. 1. Occurence of anti IgG globulins in NE patients. Samples from 14 patients have been plotted according to the time of onset of the disease. The mean titers are indicated by the line. In a control group (30 blood donors) two had a titer of 8, the others were negative.


Fig.2. The age and sex distribution of NE patients in Finland (in 1966-1973) (According to Lähdevirta and Elo, 1975)

HISTOPATHOLOGY
According to Kuhlbäck et al. (13) and Lähdevirta (5) the renal histopathological picture of NE is an acute, tubular and interstitial hemorrhagic nephritis with a mild glomerulitis with the interstitial extravasation being the most prominent finding. These findings have been recently confirmed by electron microscopy (14) and by immunohistology. The latter work also suggests that immune complex deposition in kidneys may have a role in the pathogenesis of NE. A direct vascular damage by the plausible agent has not been demonstrated. The renal affection is transitory and morphological sequalae are very slight (16).
EPIDEMIOLOGY

Population distribution. The epidemiology of NE has special features. The great majority of patients (over 80%) are men, mostly at the age of 15 to 35 years (Fig.2). Females and children contract the disease less often and when ill show in average milder symptoms. Perhaps the infection is many times totally subclinical among them. The incidence of NE among the rural population is four times greater than in cities. During the epidemic among the German troops in northern Finland in 1942 the morbidity in a front line combat unit was 48% while no cases were seen in the army staff.

Geographical distribution. NE has a pronounced regional occurrence. Fig. 3 is the map of the home residents of the patients and Fig. 4 shows the morbidity in different parishes in Finland. The morbidity is highest (maximum 1.3%) in certain parishes located in the Lake Finland.

Seasonal distribution. NE has a pronounced early winter incidence. Well over half of the infections occur during November-January (Fig. 5). Most of the cases appear some weeks after the cooling of the weather on autumn.

Annual distribution. For the first time NE was encountered in Finland 1942, then in 1957. After that a periodicity of 3 to 4 years has been noted. The cases have been most numerous during the winters 1965/66, 1969/70, 1972/73 and 1976/77. The numbers of hospitalized patients in the last mentioned winters were 177 and 104.

ETIOLOGY

Several investigations have suggested that rodents are the natural reservoir of hemorrhagic fever with renal syndrome (see e.g. Smorodintsev et al. (17), Casals et al.(18), Trencséni and Keleti (19)) Lee (20) has presented in the preceding paper that the etiological agent of Korean hemorrhagic fever has been found in the field mouse (Apodemus agrarius coreae). In Finland (and also in Sweden (8)), the highest NE incidences seem to coincide with the years of a high prevalence of voles (Fig.6). Some other rodents have the same periodicity as the voles and so it is possible that other species also may harbour the agent in nature. However, A. agrarius occurs only in the southeastern corner of Finland (not at all in Scandinavia) and A. flavicollis in the southern and central parts of Fennoscandia. Among the voles the bank vole (Cletrionomis glareolus) seems to be the best candidate. It has spread over the whole of Fennoscandia (excluding northern Lapland). It seems, like NE, to avoid regions of open fields, being a dweller of forests and bush vegetation. In the autumn, when the temperature drops, bank voles often move into barns getting into contact with each other and with men. This may well be the expansion phase of disease during which the infection spills over the human beings. Presumably later in the winter the gathered voles have passed through the infection and the disease dies out, - human cases are few in the late winter. It is possible that the mites maintain the agent at that time (21,19). The sporadic cases during the summer are for the most part urban dwellers spending their vacation in summer cottages or camping military troops close to microfoci in nature.

More direct evidence for a role of the bank vole has also been obtained. Two persons at the Department of Virology contracted NE one month after dissecting voles collected from the endemic area. There also occurred an unusual family outbreak in the heart of the city of Helsinki. The son of the family had a terrarium with bank voles collected from the endemic area. Five out of the six members of the family contracted NE one after another within two weeks (22).



Fig.3 The geographical distribution of house residencies (= the sites of contraction ?) of the NE patients in Finland (June 1966 - May 1977)


Fig. 4. The morbidity to NE in different parishes in Finland (in 1966 - 1973). (According to Lahdevirta and Elo 1975)

Fig. 5. The seasonal distribution of NE cases in Finland (in 1966-1973). (According to Lahdevirta and Elo 1975)

Fig. 6. Annual occurrence of NE cases in Finland as compared to the population density of voles (symbols: H=high, D=decreasing, I=increasing, L=low density of vole populations, according to Myllymaki (24) and Myllymaki et al. (25))

ISOLATION ATTEMPTS

The agent of NE is unknown. Some preliminary reports on a successful isolation have been published (e.g. Hoorn et al. (23) in Sweden) but they have been left without confirmation. In Finland the isolation attempts were started in 1964. The efforts were enforced during 1976/77 and concentrated in two different approaches :

1) to search for NE patients at the earliest possible phase of the disease,

2) to collect rodents from the farms where recent cases of the disease have occurred.

Samples from the patients. The samples collected for the isolation attempts are: serum, white cells (both crude buffy coat fraction and lymphocyte cultures), urine (sediment and a concentrate prepared by initial untrafiltration followed by ultracentrifugation), renal biopsy, cerebrospinal fluid, throat swab and faeces. The samples were collected within the first days of the disease (renal biopsies, however, more than a week after onset). Some samples from the cases of the family outbreak (see above) were obtained even before the disease.

Samples from the rodents. The material includes the pets of the above mentioned family (four bank voles), and both voles (Clethrionomys glareolus and Microtus agrestis) and mice (Apodemus flavicolIis) from the endemic area. Blood, urine and different organs (kidney, lung, spleen, liver, heart, salivary gland with regional lymphnodes) were used as source material for isolation attempts.

Isolation methods. All the samples of patients and rodents were inoculated both into experimental animals and cell cultures. The animals were: newborn and weaned laboratory mice, guinea pigs, bank, field and grey-side (Cl. rufocanus) voles.

The inoculation route was ip, for the mice also ic (and intranasally sometimes). The primary cell cultures were made from the organs of the inoculated animals at the time they fell ill or some weeks to six months after the inoculation if they kept well.

Besides the experimental animals several cell lines (listed in table 1) were used for isolation attempts.

TABLE I

CELLS USED IN ISOLATION ATTEMPTS


(Origin of established lines is given in parentesis. The other cell types represent adherent primary or early passage cell cultures from the tissues).


Main types:


Additional types:


Human amnion

Human embryonic skin

Green monkey kidney

Mouse macrophages

Mouse embryo, total

Mouse embryo viscera


Chick embryo


Human glia

Field vole kidney

Bank vole kidney

McCoy, X-ray irradiated (Mouse)

Lymphoid line (Bovine)

Lymphoid line (Porcine)

 

Types used in passages:


HeLa (Human)

Vero (Green monkey)

Kidney line DK (Dog)
Salivary gland SVG line (Dog)

RK 13 (Rabbit)


BHK21/WI-2 (Hamster)


Human embryonic kidney

PK 15 (Porcine) HAK (Hamster)

Pt K2 (Potorous tridactylis)

Singh's Aedes albopictus (Mosquito)


Each sample was immediately inoculated in approximately ten different cell cultures, all of the cell types were not constantly available. Three to four blind passages were done.

Attempts to demonstrate the presence of the NE agent primarily carried out by immuno-fluorescence. Cell smears or sections of frozen organs were fixed with acetone (15 min 4ºC) and stained by convalescent sera from NE patients using either direct or indirect method. Additional methods for detecting a possible agent were : 1) electron microscopy, 2) immune electron microscopy using convalescent sera, 3) normal immune adherence using human convalescent serum, guinea pig complement and red cells, 4) immune diffusion with human convalescent sera,

5) interference in cell cultures using Semliki Forest virus as the challenge, and 6) labelling the cultures with radioactive adenine followed by sucrose gradient centrifugation of the supernatants.

In addition primary cell cultures have been prepared from the organs of the voles from endemic area. These cell cultures have been studied as the above mentioned cultures.

Isolation results. In two instances the possible presence of a NE antigen in the initial samples has been detected by immunofluorescence. A buffy coat sample from the member of the "NE-family" taken one week before the onset of the disease showed a clear granular cytoplasmic fluorescence in 10% of all the white blood cells (Fig. 7). At the day of onset of the disease about 1% of the cells were positive in two observed patients. Samples taken after the onset were negative. The unspecified tagging of immunoglobulins cannot be totally excluded. However, when mononuclear leucocytes from

one of the immunofluorescence-positive buffy coats were cultured for 5 days in vitro and then inoculated into newly prepared cultures of mouse peritoneal macrophages, an atypical CPE was noted after six days. A similar CPE was reproduced in eight sequential passages by transferring supernatant of the infected cultures onto fresh macrophages. These primary findings were confirmed by reisolating twice the cytopathogenic agent in macrophage cultures. Infectivity titres in these passages remained relatively low, only up to 10^4 per ml. Infected macrophage supernatant also induced CPE in bank vole kidney cells and in kangaroo rat kidney cell line (Pt K2). A partial neutralization of infectivity by two logs in the macrophage system could be demonstrated by using unheated convalescent serum from one of the patients while the respective preinfection serum showed no effect on the CPE. Attempts to show antigens of the agent in the cells by direct or indirect immunofluorescence have not yielded convincing results so far.

Another positive finding of the immunofluorescence was noted in primary cell culture made from the organs of a bank vole of the endemic area. There was a weak but clear cytoplasmic fluorescence in a small fraction of the cells by indirect method. Also here the possibility of an unspecific staining cannot be totally excluded. It is worth mentioning, however, that the two laboratory infections described above most likely were contracted when these primary cultures were prepared.

CONCLUDING REMARKS

Nephropathia epidemica (NE) is less severe than the related disease in the Far East, the hemorrhagic fever with renal syndrome. Especially the hemorrhagic manifestations are in the former disease scanty. The number of hospitalized patients is 20 to 200 per year in Finland, the respective numbers in the Scandinavian countries are smaller. Because the etiologic agent of NE is unknown, the number of undiagnosed, atypical or subclinical infections cannot be estimated.

The incubation time has been supposed to be about three weeks. The onset with high fever is acute without prodromal signs. The clinical picture is dominated by the renal affection : proteinuria, oliguria (later polyuria), microscopic hematuria and azotemia. The interstitial extravasation is the most prominent finding in the renal biopsies. The disease is, however, a multi-systemic infection. Evidence of carditis, hepatitis and meningoencephalitis has been noted rather often. The increased activity of the rheumatoid factor has been found during the early phase of NE. Sequelae of death are very rare. Reinfections have not been observed.

The greatest majority of patients are young rural men who fall ill in the early winter. The periodicity of NE coincides with the fluctuations of the population densities of voles in Finland. Our isolation studies have concentrated on the voles from the NE endemic area and on material derived from a family outbreak apparently caused by pet voles

The results, while still preliminary, suggest that non-cellular patient samples (urine, plasma, etc) yielded negative results but that cellular patient samples (buffy coat) and cellular samples from infected bank voles may yield positive findings.


Fig. 7 The cytoplasmatic fluorescence (direct method) seen in the buffy coat cells of a sample taken one week before the onset of NE.

REFERENCES
1. Myhrman,G. (193 4) Nord.med.Tidsskr., 7, 793-794.
2. Zetterholm,S. (1934) Läkartidningen, 31, 425-429.
3. Stuhlfaut,K. (1943) Dtsch. Med. Wochenschr., 69, 439-443, 474-477.
4. Hortling,H. (1946) Nord. Med., 30, 1001-1004.
5. Lähdevirta,J. (1971) Ann. clin. Res., 3, Suppl. 8.
6. Lähdevirta,J. & Elo,O. (1975) Suomen Lääkärilehti, 30, 677-682.
7. Ornstein,K. & Söderhjelm,L. (1965) Med. Welt., 17, 898-901.
8. Nyström,K. (1977) Acta med. Scand., Suppl. 609.
9. Tungland,B. (1954) Nord. Med., 51, 635-638.
10. Mouland,G. (1975) T. norske laegeforen, 95, 425-429.
11. Hansen,E. (1958) Ugeskr. Laeger, 120, 908-913.
12. Penttinen,L., Wager,O., Pyrhönen,S., Leinikki,P. & Teppo,A.M. (1974) Lancet, i, 367.
13. Kuhlbàck,B_ Fortelius,P. & Tallgren,L.G. (1964) Acta Path.Microbiol.Scand., 60, 323.
14. Collan,Y., Lähdevirta,J. & Jokinen,E.J. (1978) Virchows Arch. A. Path. Anat. Hist. 377, 129-144.
15. Jokinen,E.J.,Lähdevirta,J. & Collan,Y. (1978) Clin.Nephrol., 9, 1-5.
16. Lähdevirta,J., Collan,Y., Jokinen,E.J. & Hiltunen,R. (1978) Acta Path.Microbiol. Scand. Sect. A. (in press)
17. Smorodintsev.A.A., Chaudakov,V.G. & Churilev,A.V. (1959) Haemorrhagic nephrosonephritis. Pergamon Press, London.
18. Casals,J., Hoogstraal,H., Johnson,K.M., Shelekov,A., Wiebenga,N.A. & Work,T.H. (1966) Amer.J.Trop.Med.Hyg.,
1. 15, 751-764.
19. Trencséni,T. & Keleti,B. (1971) Clinical aspects and epidemiology of haemorrhagic fever with renal syndrome. Akadémiai Kiado, Budapest.
20. Lee,H.W. (1978) in Ebola Virus Haemorrhagic Fever. Ed. S.R.Pattyn. Elsevier/North Holland, Amsterdam.
21. Chumakov,M.P. (1963) J.Hyg.Epidem.Microbiol.Immunol., 7, 125-135.
22. Oker-Blom,N., von Bonsdorff,C.H., Brummer-Korvenkontio,M., Hovi,T., Penttinen,K., Saikku,P., Vaheri,A. &
2. Lähdevirta,J. (1978) Proc. 2nd Int. Symp. Arctic Arbovir., Mont Gabriel, Canada (in press)
23. Hoorn,B., Bergman,S., Nyström,K. & Söderhjelm,L. (1971) Lancet, 1144.
24. Myllymäki,A. (1977) EPPO Bulletin (in press)
25. Myllymäki,A., Christiansen,E. & Hansson,L. (1977) EPPO Bulletin (in press)


DISCUSSION
J A. Bryan : Is it your feeling that in your family outbreak all became ill because of contact with the rodents or do you think it was person to person transmission ?
C.H. von Bornsdorff : The incubation period is too short for a person to person transmission. Person to person transmission has never been observed. Rodent-man contact is always responsible.
J.G. Breman : Did you look for antigen in the voles responsible for this household outbreak ?
C.H. von Bornsdorff : We did, but I think too late. We did not investigate them until March and that was too late. Furthermore, cultures were made and we did not make frozen sections.
J. Casals : We learned from our trips in the Soviet Union that they have two varieties of the disease : the one in the Far-East which is certainly the same as the one in Korea, associated with Apodemus agrarius whereas in the European part of the Soviet Union. particularly on the middle Volga and further south, the disease is clinically milder with low mortality somewhat similar to what you described in your country and in Sweden. The latter is associated with Clethrionomys glareolus. There is no evidence as far as we know, whether those two agents are the same or related. But the disease is really very different, the mortality in the Far East of the Soviet Union is from 5 to 20% whereas in the European region it is no more than 1 or 2%.

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