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Mushroom poisoning nephrotoxicity

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					From the Department of Nephrology, Sahlgrenska
    University Hospital, Göteborg, Sweden




     Mushroom poisoning:
  Cortinarius speciosissimus

           nephrotoxicity




             Johan Holmdahl




      Institute of Internal Medicine
        Göteborg University 2001
                       Mushroom poisoning:
                         Cortinarius speciosissimus
                               nephrotoxicity

                                  Johan Holmdahl

       Department of Nephrology, Institute of Internal Medicine,
        Göteborg University, Sahlgrenska University Hospital,
                   SE-413 45 Göteborg, Sweden.
              E-mail: johan.holmdahl@medicine.gu.se

                                       Abstract
Cortinarius mushroom nephrotoxicity causes acute and/or end-stage renal failure. Al-
though on average only 1-2 cases yearly require qualified medical treatment for Corti-
narius intoxication in Sweden, the poor long-term outcome after the acute renal failure
in 30-50% of the patients makes this mushroom poisoning one of the most feared.
The overall purpose of the study was to investigate Cortinarius intoxications in mice
and man with the aim of preventing and/or treating acute renal failure and/or end-stage
renal disease.
To achieve this, the LD50 in mice of freeze-dried Cortinarius speciosissimus and Corti-
narius orellanus was determined and shown to be 2.0 g/kg and 3.2 g/kg respectively.
A nephrotoxic substance in Cortinarius speciosissimus was isolated and the 3,3´,4,4´-
tetrahydroxy-2,2´-bipyridine-N,N´-dioxide structure was confirmed by the mass spect-
rum and the NMR spectra.
The LD50 in mice of the nephrotoxic molecule isolated from Cortinarius speciosissimus
was calculated to be approximately 20 mg/kg.
The short and long-term results in five patients transplanted after Cortinarius specio-
sissimus intoxication showed that renal transplantation 6-36 months after the intoxi-
cation does not carry any additional short or long-term risks in comparison with other
renal transplant patients.
The Cortinarius NephroToxicity (CNT) prognostic index was constructed to evaluate
the treatment and outcome in Cortinarius poisoning and made it possible to classify the
patients in three risk-groups: CNT index < 1.1, = 1.1 – 2.1 or > 2.1, indicating a good,
an intermediate or a poor prognosis. The CNT index was based on the serum creatinine
value (µmol/l) before treatment (y) and the number of days after the mushroom meal
(x) according to the formula: CNT index = (y+316) / (x x 102).
The short and long-term results in 30 patients treated for Cortinarius intoxications in
Sweden between 1979 and 1999 showed that treatment with haemoperfusion and/or
haemodialysis 3-8 days after the ingestion of mushrooms does not influence the clinical
course of Cortinarius intoxications.The geographical distribution of 31 cases of Corti-
narius speciosissimus intoxication in Sweden between 1979 and 1999 did not exclude
the possiblity of a causal connection related to the acidification. The chronological dist-
ribution of the 31 cases indicates that the incidence is not increasing. The reason is sug-
gested to be that mushroom-pickers nowadays are more aware of the existence of the
dangerous fungus.
Keywords: Cortinarius speciosissimus, acute renal failure, nephrotoxicity, end-stage
renal failure, renal transplantation, mushroom intoxication, orellanine, haemodialysis,
haemoperfusion, prognostic index.
             Amazing Grace




To Anne, Johanna and Daniel
                                                    Contents

List of publications referred to in this thesis...................................................6
Abbreviations used ............................................................................................7
Introduction .......................................................................................................9
       Historical notes .........................................................................................9
       Epidemiological aspects .........................................................................11
       Toxin structure........................................................................................12
       Toxin action ............................................................................................12
       Extracorporeal treatment ........................................................................12
       Prognosis ................................................................................................13
       Transplantation .......................................................................................13
Introduction to the present studies ................................................................14
Aims of the investigation .................................................................................16
Subjects ............................................................................................................17
Materials and methods....................................................................................19
Results ..............................................................................................................22
       Orellanine: structure and nephrotoxicity (Papers I and II) .....................22
       Renal transplantation (Paper III) ............................................................27
       The Cortinarius NephroToxicity (CNT) index (Paper IV) ....................28
       Patient outcome (Paper V)......................................................................33
       Treatment and clinical course (Paper V) ................................................34
       Epidemiological aspects (Paper VI) .......................................................35
Discussion .........................................................................................................39
Conclusions ......................................................................................................45
Acknowledgements ..........................................................................................46
References .......................................................................................................47
Appendix ..........................................................................................................52
Original paper I ...............................................................................................59
Original paper II .............................................................................................71
Original paper III............................................................................................79
Original paper IV ............................................................................................85
Original paper V............................................................................................119
Original paper VI ..........................................................................................143
List of Papers
This thesis is based on the following papers, which will be referred to in the
text by their Roman numerals:

I.     Holmdahl J, Ahlmen J, Svalander C, Eriksson J and Bucht H (1980). Renal
       damage after intoxication with Cortinarius mushrooms. In: Toxicological
       Aspects, p. 155-163 (Kovatsis, A., Ed.) Thessaloniki, Greece: Technika Stu-
       dio.


II.    Holmdahl J, Ahlmén J, Bergek S, Lundberg S and Persson S-Å (1987). Iso-
       lation and nephrotoxic studies of orellanine from the mushroom Corti-
       narius speciosissimus. Toxicon 25 (2):195-199.


III.   Holmdahl J and Blohmé I (1995). Renal transplantation after Cortinarius
       speciosissimus poisoning. Nephrol Dial Transplant 10:1920-1922.


IV.    Holmdahl J. The Cortinarius NephroToxicity (CNT) index: a method for
       the evaluation of treatment and outcome in Cortinarius mushroom poi-
       soning. Manuscript.


V.     Holmdahl J. Cortinarius mushroom nephrotoxicity in Sweden 1979-
       1999: treatment, short and long-term outcome. Manuscript.


VI.    Holmdahl J and Bohlin A (2001). Cortinarius speciosissimus intoxi-
       cations in Sweden 1979-1999: epidemiological aspects. Windahlia, journal
       of mycology. In press.




6
                Abbreviations used

ARF       acute renal failure
AUFS      absorbance units full scale
BW        body weight
CAPD      continuous ambulatory peritoneal dialysis
CNT       Cortinarius nephrotoxicity
Cr-EDTA   chromium ethylene-diamine-tetra-acetic acid
CFR       chronic renal failure
DMSO      dimethyl sulfoxide
DNA       deoxyribonucleic acid
ESRF      end-stage renal failure
GFR       glomerular filtration rate
HD        haemodialysis
HP        haemoperfusion
HPLC      high-performance liquid chromatography
LD        lethal dosage
NMR       nuclear magnetic resonance
PD        peritoneal dialysis
PE        plasma exchange
RNA       ribonucleic acid
UV        ultraviolet




                                                        7
8
                                                                      Introduction

                               Introduction
Historical notes
The first known written account of intoxications with Cortinarius mushrooms is
probably the poisoning catastrophe in Poznan in 1952 documented by the Polish
physician Stanislaw Grzymala in his work: “Erfahrungen mit Dermocybe orellana
(Fries) in Polen. Massenvergiftung durch den Orangefuchsigen Hautkopf.” (31).
The comprehensive report illustrates the varying clinical pattern, medical history
and pathological-anatomical findings of this puzzling poisoning by Cortinarius
orellanus:
             …“Im ganzen unterlagen der Massenvergiftung mit Pilzen in dieser
             Zeitspanne 102 Personen, von denen 11 starben. Der klinische Verlauf
             der Vergiftung war so ungewöhnlich, dass er trotz der Feststellung des
             Genusses von Pilzen in jedem Falle ernste Zweifel betreffs der Pilz-
             ätiologie aufkommen liess. Besondere Bedenken erweckte die Inkuba-
             tionszeit der Erkrankungen, d. H. die Zeitspanne von dem Genuss der
             Pilze bis zum Erscheinen der ersten Krankheitssymptome, die 3-14
             Tage dauerte.
             …Auf diese Art überschritt sie die Latenzzeit von Vergiftungen durch
             Amanita phalloides und Gyromitra esculenta, die allgemein als längs-
             te Inkubationszeit bei Pilzvergiftungen gilt. Die Vergiftungen wurden
             von folgenden Erscheinungen begleitet: trockenheitsgefühl sowie
             Brennen in der Mundhöhle mit sehr heftigem Durstgefühl, wobei die
             Kranken mehrere Liter Flüssigkeit im Laufe eines Tages zu sich nah-
             men. Weiter beobachtete man Magen- und Darmstörungen sowie
             übelkeit, Erbrechen, Bauchschmerzen, hartnäckige Obstipation und
             bei einigen Kranken Durchfälle. Oft trat Frösteln oder länger andau-
             erndes Kältegefühl ein, jedoch ohne nennenswerte Steigerung der
             Körpertemperatur. Bei fast der Hälfte der Erkrankungsfälle emp-
             fanden die betreffenden Personen heftige Kopfschmerzen und
             Schmerzen in der Lendengegend. In 17 Fällen beobachteten wir in der
             2. oder 3. Krankheitswoche ein juckendes papulöses Exanthem, meis-
             tens bei Kranken mit Obstipation. Bei 24 im Krankenhaus behan-
             delten Fällen war der Verlauf der Krankheit ernst. In fast allen diesen
             Fällen konnten wir eine Nierenschädigung mit Oligurie und Albumi-
             nurie feststellen, manchmal mit Rest-N-Anstieg und Steigerung des
             Harnstoffes im Blut. In der Hälfte dieser Fälle bestanden Ödeme, die
             übrigen verliefen ohne Ödeme. In 4 Fällen kam es zu einer manifesten
             Urämie; fünfmal traten auch meningeale Symptome auf. Bei fast allen
             klinisch behandelten Kranken, sogar bei denen mit leichterer Er-
             krankung, beobachteten wir in der regel eine Beschleunigung der
             Blutsenkungszeit sowie eine mässige bis starke Leukozytose mit
             Linksverschiebung. In allen diesen Krankheitsfällen – auch bei den
             nicht hospitalisierten – haben wir eine Blutdruckerhöhung n i c h t
             beobachtet. Der Verlauf der Krankheit hatte einen langwierigen
             Charakter; auch die Rekonvaleszenz dauerte Wochen bis Monate.
             In den schwersten Fällen trat der Tod frühestens nach 6 und spätestens
             nach 161 Tagen ein, vom Ausbruch der Krankheit gerechnet. In 9 von

                                                                                  9
Introduction

               11 Todesfällen wurde eine Obduktion durchgefuhrt, und zwar durch
               das Institut für gerichtliche Medizin bzw. das Anatomisch-Patholo-
               gische Institut der Medizinischen Akademie in Poznan. In den Vorder-
               grund des anatomisch-pathologischen Bildes treten insbesondere
               Nierenveränderungen im Sinne einer Nierenparenchymschädigung,
               meistens in Gestalt einer schweren Nephrosis toxica oder einer Neph-
               ritis interstitialis – gekennzeichnet durch nekrotische Veränderungen
               der Tubuli, manchmal durch hyaline Entartung der Glomeruli und
               deren Schrumpfung sowie vielfache Infiltrationen mit Lymphozyten
               des Nierenparenchyms.
               English translation:
               “During this period altogether 102 people suffered mushroom poi-
               soning, of whom 11 died. The clinical course of the intoxications was
               so unusual that, although mushroom ingestion could be verified, se-
               rious doubts about the aetiology were raised, particularly because of
               the long incubation time, the time from ingestion of the mushrooms to
               onset of symptoms varying from 3 to 14 days.
               This exceeds the latency times for poisoning by Amanita phalloides
               and Gyromitra esculenta, which up until then had been the longest
               known latency times for mushroom intoxication. Intoxication was ac-
               companied by the following symptoms: dehydration, a burning sensa-
               tion in the mouth and severe thirst, the victim drinking several litres
               of fluids during 24 hours. Gastrointestinal upsets, with nausea, vomi-
               ting, abdominal pain, severe constipation and, in some patients, also
               diarrhoea, were also seen. Shivering or prolonged chills without any
               marked increase of body temperature frequently occurred. Almost
               half of the victims had severe headache and lumbar pain. In 17 cases
               papular exanthema with itching occurred during the second and third
               week of illness. This was particularly common in patients who had
               suffered from constipation. In 24 patients who were treated in hospital
               the course was serious. In all these cases, renal injury with oliguria
               and albuminuria was observed, sometimes with increased residual nit-
               rogen and increased blood urea. Oedema was present in half of these
               patients; the remainder had no oedema. In 4 cases manifest uraemia
               developed and in 5 meningeal symptoms also occurred. In almost all
               the patients treated in hospital, even in milder cases, an increased ESR
               and moderate or severe leukocytosis with left displacement was noted.
               Elevated blood pressure was not observed in any of the patients, even
               those treated in hospital. The course of the illness was prolonged and
               convalescence took weeks or sometimes months.
               In the severest cases, death occurred after at the earliest 6 and at the
               latest 161 days, calculated from the time of onset of symptoms. In 9
               of the 11 deaths, autopsy was performed at a department of forensic
               medicine, viz. at the Department of Anatomy and Pathology at the
               Medical Academy in Poznan. Prominent patho-anatomical features
               were renal changes in the form of severe toxic nephritis or interstitial
               nephritis, manifested as necrotic changes in the tubuli, sometimes
               hyalin deposits in the glomeruli and shrivelling of the latter, and
               extensive infiltration of lymphocytes in the renal parenchyma”.

10
                                                                       Introduction

Through research, Grzymala found that another serious incident of intoxication
with Cortinarius orellanus occurred in the autumn of 1938, when nine persons fell
ill and six of them died.
Although we do not have any written case-studies of intoxication with Cortinarius
until the middle of the 20th century, there are written descriptions of these toxic
mushrooms from as early as the 19th century. The Swede Elias Fries was a pioneer
in the science of mycology. In 1838 he described Cortinarius orellanus in his work
“Epicrisis systematis mycologici. Upsaliæ” (27). The first description of Cortina-
rius speciosissimus (as it is currently named) was possibly given by the British
mycologist Modekai Cubit Cooke in 1883 in “Handbook of British Fungi vol. I”
(16) and it is illustrated in “Illustrations of British Fungi vol. VI” (17) under the
name Cortinarius rubellus. The plate in Illustrations of British Fungi (17) is very
similar but the description in the Handbook (16) does not fit very well. The name
Cortinarius rubellus Cooke may for that reason be disputed. None of these lethal
species are mentioned as poisonous by either Fries or Cooke. It is not until the
second half of the twentieth century that the mycological literature discusses the
connection between these species and the life-threatening acute renal failure which
is significant for intoxications with Cortinarius mushrooms. The first warning in
Sweden in the Journal of the Swedish Medical Association was given in 1975 by
the nephrologist Härje Bucht and the mycologist John Eriksson, when they pointed
out the nephrotoxicity of Cortinarius speciosissimus (9). They referred to six cases
of acute renal failure in Finland.

Epidemiological aspects
In the large genus Cortinarius, which contains more than three hundred species,
there are at least two known poisonous species: Cortinarius orellanus (Elias
Magnus Fries) and Cortinarius speciosissimus (Robert Kühner & Henri Romag-
nesi). They belong to the most poisonous fungi in Europe and typically cause se-
vere renal damage. Cortinarius speciosissimus is a boreal fungus of coniferous fo-
rests and is widely distributed in the northern parts of Europe (i.e. Scandinavia,
northern Scotland) and in the mountainous areas of central Europe. Its prevalence
seems to have increased in the most recent decades in Sweden, especially on acid
soils in the southwestern parts. Cortinarius orellanus is rare in Scandinavia but mo-
re common on the continent of Europe. It grows preferably in deciduous forests.
Cortinarius speciosissimus intoxication has been reported from Finland (34),
Scotland (76), Norway (23); (51), Italy (10), Germany (59) and Austria (26); (36).
Cortinarius orellanus intoxications are reported from Switzerland (24), France
(45); (20); (5), Germany (22); (21), Czechoslovakia (6), Bulgaria (62), Austria (33),
Spain (12), the Czech Republic (3), England (40) and Italy (49).
Cases of poisoning by an unidentified Cortinarius species in Czechoslovakia (77),
in Oregon, USA (50), in Canada (68) and in France (4), by unspecified Cortinarius
species in Italy (83) and in sheep in Norway (84) are also described.
For more information on treatment and outcome of these cases reported outside
Sweden please turn to the appendix.




                                                                                  11
Introduction

Toxin structure
Stanislaw Grzymala isolated and characterised a substance he called orellanine
(30). It was isolated from Cortinarius orellanus and was demonstrated to be neph-
rotoxic in animal experiments. Wieslaw Antkowiak and Wieslaw Gessner purified
the toxin and found the chemical structure to be 3,3´,4,4´-tetrahydroxy-2,2´-bipy-
ridine-N-N´-dioxide (1, 2). The structure of orellanine has been disputed (41).
However, the synthesis of orellanine has been reported (19). A polypeptide neph-
rotoxin (cortinarin A) has been isolated from Cortinarius speciosissimus and partly
characterised (11). By means of high-performance liquid chromatography (HPLC),
cortinarin A and two more substances, cortinarin B and cortinarin C, isolated from
Cortinarius speciosissimus, were separated and quantified (79-81). The chemical
structures of the polypeptide compounds were fully elucidated. Cortinarin A and
cortinarin B were nephrotoxic in laboratory animals.

Toxin action
Different mechanism have been put forward to explain the toxicity of orellanine.
The toxin inhibits the synthesis of proteins, RNA and DNA in Madin-Darby Canine
kidney cells, but direct addition of orellanine to a cell-free system of rabbit reticu-
locyte lysate did not produce any inhibition of protein synthesis. However, when
orellanine is pre-incubated with activating rat liver microsomal systems, this in-
hibition occurs. The in vivo inhibition of protein synthesis is thus most likely due to
a metabolite of orellanine (72). It has also been shown in vitro that oxidation of
orellanine by biological oxidising agents of enzymatic systems at physiological pH
and under anaerobic conditions produces an orthosemiquinone anion radical and
reactive oxygen species. This leads in vitro to a large oxygen consumption and de-
pletion of glutathione, thus rendering cells more susceptible to oxidant damage
(71). Instantaneous oxidation of Fe2+ in the presence of orellanine under air was
shown to be responsible for oxy-radical production concomitant to a stable ferric
complex Fe(III)Or3 formation (Or = orellanine), leading to oxidative DNA breakage
at physiological pH (14); (13).
Fore more information on toxin action please turn to the appendix.

Extracorporeal treatment
There has been a rise and a fall in the utilisation of extracorporeal treatment in
mushroom poisoning in general. In Amanita intoxication, for example, kinetic stu-
dies have shown that haemodialysis, haemoperfusion or plasma exchange cannot
remove a large amount of toxins (38) and therefore a new “antidote”, like N-acetyl-
cysteine, is tested with the expectation of preventing the liver failure (48). So far,
N-acetyl-cysteine has not, been proved to be effective.
In Cortinarius poisoning, haemoperfusion with activated charcoal is recommended
by Fulde et al. in a recent report (28). They used an in vitro model with plasma
mixed with an alkaline aqueous orellanine solution and found that the rate of
adsorption onto activated charcoal was four times higher compared to Amberlite
XAD 4 resin.
Schumacher and Høiland surveyed orellanine and structurally related and well-
known bipyridines such as paraquat and diquat and found that pharmacokinetic
experiments on the nephrotoxic bipyridines suggest that haemoperfusion may be a
rational therapy in intoxicated persons, even several days after mushroom ingestion
(75).
12
                                                                          Introduction

In a review, Michelot and Tebbett present the current knowledge on Cortinarius
poisoning, the toxins incriminated and their mechanism of action (47). They
conclude that treatment of Cortinarius poisoning must be primarily oriented to-
wards the elimination of the toxin from the blood circulation. They write that this is
achieved by haemoperfusion and haemodialysis using the appropriate membranes,
the approach being the same even if the ingestion of the mushrooms was several
days earlier.
In a review article by Köppel, secondary detoxification by plasmapheresis or hae-
moperfusion is proposed but it is stated that it is not clear whether plasmapheresis
has a beneficial effect in orellanus intoxications (42).

Prognosis
The risk of a patient intoxicated with Cortinarius mushrooms developing end-stage
renal failure (ESRF) varies in different reports between 8% (n=26) (5) and 11%
(n=102) (31). However, it is difficult to compare patients in different groups with
varying toxin exposure and distribution according to sex and age. In the specific
case, it is more important to find a method of evaluating the risk of development of
ESRF that is dependent only on the individual sensitivity and the toxin exposure.
Until now, no such prognostic index or criteria have been presented but the need
has been pointed out (82) ; (5).
In a recent review, published in 2001, prognostic criteria such as the amount of
mushroom ingested, early gastrointestinal disorders, early renal insufficiency and a
past history of renal disease are discussed, but no method for the evaluation of the
influence of these factors on the outcome is presented (18).

Transplantation
From a theoretical point of view, an intoxicated patient might serve as an organ
donor as well as an organ recipient. If the intoxication causes brain death but does
not affect other organs, e.g. the kidneys, donation is possible. If the toxin injures the
liver or the kidneys, an organ transplantation can be considered to replace the non-
functioning organ. Liver transplantation after Amanita poisoning has been studied
and recommendations as to when it is safe to transplant have been presented (38).
An evaluation of renal transplantation after Cortinarius intoxication has not yet
been published with the exception of our present paper. Some authors do mention
that a transplantation has been performed after Cortinarius poisoning but either
there is no data on follow-up or the time between the intoxication and the
transplantation is not given (5); (21); (23); (34); (36) ; (76).




                                                                                      13
Introduction to the present studies

              Introduction to the present studies
In the autumn of 1979 two patients, a mother and her son, with acute renal failure
without known cause were admitted to the Department of Nephrology. As an intern,
I became involved in the investigation of these cases. It emerged that the patients
had consumed mushrooms 4-5 days before admission. Since they were living on a
farm with their own waste disposal, the mushroom leftovers could be obtained and
microscopic spore analysis revealed the diagnosis: Cortinarius speciosissimus poi-
soning!
We tried to remove possibly circulating toxin by haemodialysis and haemoperfusion
5-6 days after the ingestion of the mushrooms. To our satisfaction, the creatinine
level normalised and after six months the GFR was normal. These index cases of
Cortinarius intoxication in Sweden are presented in Paper I.
Later the same year, two men were admitted independently and were treated with
haemodialysis and haemoperfusion nine days after the ingestion of mushrooms and
peritoneal dialysis eight days post intoxication respectively (32). These two patients
developed ESRF and were later transplanted.
In order to evaluate the possibility of detecting the toxin in patients and removing it
by haemoperfusion and haemodialysis, we decided to isolate and analyse the toxin,
together with experts at the National Defence Research Institute. These studies are
presented in Paper II.
A French group claimed to have found orellanine in a renal biopsy six months after
the deliberate ingestion of two raw Cortinarius orellanus by a woman with psy-
chiatric problems in a suicide attempt (70). To ensure that no remaining orellanine
could injure a renal graft, the first five Swedish transplanted patients were studied
and the results are presented in Paper III.
The great variety of therapies in Cortinarius poisoning are, as in many other intoxi-
cations, often not evaluated in prospective controlled studies. Extracorporeal
therapies, cyclophosphamide, corticosteroids, probucol and N-acetylcysteine have
been tried in animal experiments and/or on patients and described in reports from
the European countries concerned. In order to make it possible to evaluate the
usefulness of extracorporeal and pharmacological treatment in Cortinarius intoxi-
cations the short and long-term outcome of 23 Swedish patients was analysed and a
prognostic index constructed in a retrospective study. These studies are presented in
Paper IV.
Our first investigations (32) and other reports and reviews (10); (28); (42); (47);
(70); (75) indicated or suggested that treatment procedures such as haemo-
perfusion, plasma exchange and haemodialysis might be effective in preventing the
development of ESRF after Cortinarius mushroom poisoning.
In order to evaluate the usefulness of haemoperfusion and haemodialysis in
Cortinarius intoxications, the short and long-term outcome of 30 consecutive Swe-
dish patients was analysed in Paper V.
Finally, we asked ourselves: Why do we not know of any cases of Cortinarius intoxi-
cation in Sweden before 1979? Either there have been intoxications not recorded or
not recognized as mushroom poisoning or this is a new phenomenon. This may in


14
                                             Introduction to the present studies

turn be a consequence of an increased occurrence of poisonous Cortinarius species
and/or an increased interest in picking mushrooms. In an epidemiological
retrospective collaboration study with the Department of Systematic Botany,
Göteborg University and members of the Swedish Mycological Association and the
Gothenburg Mycological Club, these questions were addressed and the results
presented in Paper VI.




                                                                              15
Aims of the investigation

                     Aims of the investigation
The overall purpose of these studies was to investigate Cortinarius intoxications in
mice and man with the aim of preventing and/or treating acute renal failure and/or
end-stage renal disease.
To achieve this
•     the lethal dose and the renal effects in mice of four Cortinarius species
      (freeze-dried and given by mouth) were investigated.
•     a nephrotoxic substance in Cortinarius speciosissimus was isolated and the
      chemical structure of the molecule was described.
•     the lethal dose and the renal effects in mice of the nephrotoxic molecule
      from Cortinarius speciosissimus were investigated.
•     the short and long-term results of five patients transplanted after Cortinarius
      speciosissimus intoxication were analysed.
•     a prognostic index was constructed and applied in 51 cases of Cortinarius
      nephrotoxicity outside Sweden presented in the literature between 1974 and
      1999.
•     the short and long-term results of 30 patients treated for Cortinarius intoxi-
      cations in Sweden between 1979 and 1999 were evaluated.
•     the geographic and chronological distribution of 31 cases of Cortinarius
      speciosissimus intoxication in Sweden between 1979 and 1999 were studied.




16
                                                                                   Subjects

                                      Subjects
The cases in the present studies have been made known to us in two different ways.
The patients have either been referred to our clinic for treatment or we have been
consulted for advice regarding choice of therapy. Our data have been checked with
the Swedish Poisons Information Centre register, which collects medical records
from all hospitals in Sweden concerning intoxications.
During the years 1979-1999 there have been 18 incidents of confirmed or suspected
intoxication recorded in Sweden, involving 38 subjects in all.
Two cases of Cortinarius gentilis and five cases of Cortinarius limonius ingestion
did not lead to any symptoms or signs of intoxication and are included in Paper V
only (Figure 1).




Figure 1.   The relationship between the different papers and the 38 subjects involved in
            the present study. The arrows do not indicate a chronological flow of subjects.


The remaining 31 subjects were all intoxicated with Cortinarius speciosissimus,
with the exception of one patient who ingested either C. orellanus or C. speciosis-
simus, and are presented in Paper VI. Six of these never showed up for treatment
but they ate the same mushrooms as the patients treated and they all developed
typical symptoms but in a milder form than the ones coming to hospital. This latter
fact may explain their unwillingness to seek help. These six participants are only
included in Paper VI. Three of the 31 subjects were tourists. Two of them came
from Denmark and picked the mushroom in Sweden but were treated in their native

                                                                                          17
Subjects

country. These two patients are only included in Paper VI. One of the tourists came
from North America and was treated for the acute renal failure in Sweden and then
later transplanted in America. This patient is included in Papers IV, V and VI.
The 30 patients treated for suspected or confirmed Cortinarius intoxication are
presented in Paper V. On clinical grounds, these 30 subjects were referred to one
of three groups.
Group I includes all those twelve patients who developed ESRF. There were 7 men
and 5 women. The mean age was 35.2 years (range 14-60 years).
Group II includes those eleven patients who developed acute renal failure (ARF)
and/or typical symptoms after intoxication but not ESRF. There were 6 men and 5
women and the mean age in this group was 35.9 years (range 16-68 years).
Group III includes those seven patients who did not develop ARF or typical symp-
toms. In this group there were 3 men and 4 women and the mean age was 45.6
years (range 17-59 years).
In group I, one patient on CAPD died two and a half years after the intoxication.
She refused blood transfusion (at the time before erythropoietin) and she eventually
died because of anaemia and peritonitis. Ten patients in group I were transplanted
and the first five renal transplantations after Cortinarius speciosissimus intoxi-
cation are presented in Paper III. The other five transplanted patients have been
followed up between one and six years with satisfactory graft function. One patient
with a functioning graft died 19 years after the intoxication because of a ruptured
aortic aneurysm. The five patients in Paper III are also included in Papers IV, V
and VI.
The first two patients in group II are presented in Paper I. They were the first
recorded Cortinarius intoxications in Sweden and have been followed up for 20
years. The two patients in Paper I are also included in Papers IV, V and VI.
All twelve patients in group I and eleven patients in group II are included in Papers
IV, V and VI.




18
                                                           Materials and methods

                        Materials and methods
Collection of the fungi and isolation of the toxic substance
Four different species of Cortinarius mushrooms (Cortinarius orellanus, Cortina-
rius speciosissimus, Cortinarius gentilis and Cortinarius limonius) were collected
in the vicinity of Gothenburg and Lake Vänern on the Swedish west coast. They
were frozen at -20°C and kept under these conditions until freeze-dried for one
week and then homogenised. In Paper I the freeze-dried mushroom homogenate
was mixed with sodium chloride 0.9% before feeding. In Paper II the dried Corti-
narius speciosissimus (44 g) was extracted three times in a total volume of 1800 ml
diethyl ether (Merck p.a.). The fungal residue, not extracted with diethyl ether, was
then refluxed with 500 ml methanol (Merck p.a.) for 30 min and the solvent re-
moved by filtration. This procedure was repeated four times and the combined
methanol extracts evaporated to dryness at reduced pressure to give a residue of
12.1 g. Glass-distilled water (100 ml) was added to the residue and, after centri-
fugation, the precipitate was washed with 5 ml glass-distilled water and with 5 ml
ethanol (99.5% w/v) and then dried at reduced pressure in a dessicator. The light
brownish powder obtained (a total amount of 600 mg) was dissolved in 20 ml of 0.4
M sodium hydroxide. After centrifugation, the pH of the supernatant was adjusted
to 4.5-5.0 with 0.4 M hydrochloric acid. The precipitate was centrifuged and
dissolved in 20 ml of 0.4 M sodium hydroxide. This procedure was repeated three
times and the yellow substance obtained was dried at reduced pressure in a des-
sicator (yield 360 mg). The substance was then refluxed in methanol (20 mg/ml).
After cooling and centrifugation, the solvent was decanted. By repeating this pro-
cedure twice, a white powder was obtained.

Mass spectrum and chromatography
For the dry compound, the mass spectrum was recorded using a Jeol IMS 300 and
the NMR-spectrum using a Jeol FX 90 Q.
The extracted and purified compound was dissolved in ethanol or 0.5 M Tris-HCl
buffer, pH 8.4 and 10-20 µl was injected into a HPLC system by means of a WISP
710 B automatic sample injection module (Waters Associates, Milford, MA,
U.S.A.). Two solvent delivery systems (6000 A, Waters Ass.) were used to pump
the mobile phase through the system at a rate of 2 ml/min and at a pressure of 2100
psi at ambient temperature. The mobile phase was 0.05 M citrate-phosphate buffer,
pH 4.5, 15.4% methanol and PIC B6 1-hexane sulphonic acid (Waters Ass.), 5x10-3
M. The mobile phase was filtered (Millipore 0.2 µm) and degassed before use.
Separation was performed on a Nucleosil C18 analytical column with 5 µm particles
and dimensions of 200x4.6 mm, without a precolumn. The detector used for elect-
rochemical detection was the LC-4 electronic controller, a glassy carbon electrode
(TL-5A) and an Ag/AgCl reference electrode (Bioanalytical Systems Inc., West La-
                                                            .
fayette, U.S.A.). The optimal working potential was 900 mV In some experiments
we also used a Lambda-Max absorbance detector Model 480. All chemicals were
reagent grade, except the methanol, which was HPLC-grade (Rathburn Chemicals
Ltd., Walkerburn, U.K.).




                                                                                  19
Materials and methods

Animals and anaesthesia
A total of 163 male mice (NMRI, ALAB, Sollentuna, Sweden) weighing 25 grams
were used. The animals were housed 4-5 mice per cage. The room temperature was
22° C and the humidity 55+ 5%. Artificial light was the only source of light and the
animals were set on a 12 h light/dark cycle with lights on at 5 a.m. They had free
access to Ewos commercial pelleted diet (R 3) and tapwater.
The mice were anaesthetised with ketamin (Ketalar, Park-Davis, Warner-Lambert
Co., NJ, U.S.A.) 1 mg/g given intraperitoneally.
All experiments were approved by the Animal Ethics Committee of Göteborg
University.

Toxicity studies
In Paper I freeze-dried Cortinarius speciosissimus, Cortinarius orellanus, Corti-
narius gentilis and Cortinarius limonius in 2.5 ml sodium chlorid (0.9%) was
given orally by means of a baby-feeding stomach tube to the mice in doses of 10,
50, 100, 200, 400, 800, 1600 and 3200 mg/kg BW, the volume being constant. Four
mice were used in each group for each species of mushroom. In Paper II the mice
were injected i.p. with the toxin dissolved in 0.5 M Tris-HCl buffer, pH 8.4. The
following doses were given: 320, 160, 80, 40, 20, 10 and 5 mg/kg. Surviving mice
were killed after two weeks. The kidneys were immersed in 10% formaldehyde in
0.1 M phosphate buffer, pH 7.4, and embedded in paraffin. Paraffin sections were
stained with hematoxylin and eosin, Mc Manus or Ladewig stains before micro-
scopy.

Clinical studies
DIALYSIS AND HAEMOPERFUSION
Haemoperfusion (HP) and haemodialysis (HD) was performed through a double
luminal or bilateral percutaneous catheters placed in the femoral vein by the
Seldinger technique or in the subclavian vein or by means of a Scribner´s shunt in
the forearm.
The haemoperfusion was performed with an Amberlite-resin (XAD-4, Extracorp-
oreal) or charcoal filter (Alu-Cart, Gambro) for 3-8 h with a blood flow between
200 and 300 ml/min.
The haemodialysis was performed with a plate or capillary dialyser for 3-8 h with a
blood flow between 200 and 300 ml/min.
Peritoneal dialysis was performed through a Tenckhoff catheter and gravity infusion
and drainage of the dialysis solution was used.

KIDNEY BIOPSIES
Sections from formalin-fixed and paraffin-embedded specimens were cut and
stained before light microscopic investigation. Immunofluorescence and electron-
microscopy were also performed.
Orellanine in renal biopsies was analysed by the Institute of Microbiology at the
University of Innsbruck. The biopsy material was extracted in 50% methanol/water.
The detection of orellanine was carried out with thin-layer chromatography on ce-
llulose plates according to Ruedl et al. (74). The detection limit was 10 nanograms.

20
                                                          Materials and methods

RENAL FUNCTION AND SIZE
Kidney function was measured as GFR using the 4 hours 51Cr-EDTA clearance
technique. 51Cr-EDTA was given as a single bolus injection and the plasma
disappearance curve was used to calculate GFR, expressed as ml/min/1.73m2 body
surface according to Brøchner-Mortensen (8). Serum creatinine (s-creat) was mea-
sured by a modified Jaffé method.
Anuria was defined as urinary output less than 100 ml urine per 24 h. Oliguria was
defined as urinary output 100 to 400 ml per 24 h. Acute renal failure (ARF) was
defined as an assumed rise in serum creatinine to a level of > 200 µmol/l. Chronic
renal failure (CRF) was defined as a persisting serum creatinine level > 200 µmol/l,
but not irreversibly dialysis-dependent. End-stage renal failure (ESRF) was defined
as irreversibly dialysis-dependent. “Recovered” was defined as a fall in serum crea-
tinine to a level < 200 µmol/l.
Kidney size was measured by plain film diagnosis or ultrasound investigation.

STATISTICAL ANALYSIS
The probability of ESRF depending on time to hospitalisation and creatinine value
was calculated by use of the results of a logistic regression analysis.

Epidemiological studies
The mapping of Cortinarius speciosissimus has been done by members of the Swe-
dish Mycological Association (Sveriges Mykologiska Förening) and professional
mycologists. The co-ordinates for each habitat have been stored in a database by the
Swedish Inventory Centre for Mushrooms (61). The map was designed and printed
by Pro Natura (25).




                                                                                 21
Results

                                     Results
Orellanine: structure and nephrotoxicity (Papers I and II)
The approximate LD50 for freeze-dried Cortinarius speciosissimus was 2.0 g/kg
BW mouse and for Cortinarius orellanus 3.2 g/kg. The LD50 for Cortinarius gen-
tilis and Cortinarius limonius were not possible to calculate with the doses given as
they did not exceed 3.2 g/kg. The approximate LD50 for orellanine isolated from
Cortinarius speciosissimus was 20 mg/kg.
Light microscopic changes of tubular cells were seen. No lesions were observed in
the glomeruli.
The mass spectrum recorded with direct inlet and at 70 eV suggests a molecular
peak at m/z 252 (M+), which is also the base peak. Peaks were also obtained at m/z
53 (M-199, 58%), 55 (M-197, 53%), 235 (M-17, 45%), 54 (M-198, 44%), 163 (M-
89, 43%), 137 (M-115, 42%), 70 (M-182, 39%), 220 (M-32, 38%) and 45 (M-207,
36%) (Figure 2).




Figure 2.   The mass spectrum of the purified dry compound from Cortinarius speciosis-
            simus.




22
                                                                              Results

The 1H-NMR spectrum in DMSO-d6 consists of a broad singlet at σ 11.6 ppm and
two doublets at σ 8.27 ppm and σ 7.16 ppm. J = 6.35 Hz (Figure 3).




Figure 3.   1
             H-NMR spectra of the purified dry compond from Cortinarius speciosissimus
            in DMSO-d6.




                                                                                   23
Results

The completely decoupled 13C-NMR spectrum in DMSO-d6 consists of five signals
at σ 110.10, 130.13, 131.88, 150.48 and 155.03 (Figure 4).




Figure 4.    C-NMR spectra of the purified dry compound from Cortinarius speciosis-
            13


            simus in DMSO-d6.




24
                                                                               Results

The simultaneous recordings of HPLC chromatograms of the toxin with a UV-de-
tector and electrochemical detection are shown in figure 5.




Figure 5.   Simultaneous recordings of HPLC chromatograms of purified toxin from
            Cortinarius speciosissimus. Right chromatogram with absorbance detection at
            300 nm and 0.05 AUFS. Left chromatogram with electrochemical detection, a
            working potential of 900 mV and a sensitivity of 500 nA/V.




                                                                                    25
Results

A u.v.-spectrum of our purified substance dissolved in 0.5 M Tris-HCl buffer, pH
8.4, showed λmax at 280 and 308 nm with ε280 = 6.7 x 103 per mole per cm and ε308
= 6.2 x 103 per mole per cm (Figure 6).




Figure 6.   UV-spectra of the toxin isolated from Cortinarius speciosissimus.




26
                                                                                     Results

Renal transplantation (Paper III)
Five patients with end-stage renal failure due to Cortinarius speciosissimus intoxi-
cation were transplanted 6-36 months after the ingestion of mushrooms. All
developed satisfactory graft function during the first 2 weeks after transplantation.
Two patients had no rejection episodes, while two had early and reversible acute re-
jection. The fifth patient had a late cellular rejection 3.5 months after transplanta-
tion and after 2 years had developed a membranous glomerulonephritis with protei-
nuria and declining GFR.
After the publication of Paper III all five patients were followed up between 5 and
20 years and the GFR values are shown in Figure 7.




Figure 7. Glomerular filtration rates (ml/min/1.73 m2) after transplantation in the five
           patients in Paper III with follow-up of 5-20 years. Closed squares denote
           patient no. 1, closed circles patient no. 2, closed triangels patient no. 3, closed
           rhombi patient no. 4 and open squares patient no. 5.




                                                                                           27
Results

The Cortinarius NephroToxicity (CNT) index (Paper IV)
The serum creatinine value obtained for each patient in group I and II before treat-
ment in relation to the number of days from the ingestion of the mushrooms is
given in Figure 8. A straight line was drawn separating the group of patients who
ultimately recovered their renal function (closed circles) from those who remained
in renal failure (open circles).




Figure 8.   The latest serum creatinine level before treatment in all patients in group I and
            II in Paper IV plotted against the number of days from ingestion of the mush-
            rooms. Open circles denote patients developing end-stage renal failure (ESRF,
            group I). Closed circles denote patients with acute renal failure and/or typical
            symptoms after intoxication but no ESRF (group II). The line is drawn where
            the plots from group I and II overlap.


On the premise that there was a relationship between increase in the serum
creatinine level after the ingestion of mushrooms and the extent of the renal injury
that followed, a prognostic index was constructed based on the equation of a
straight line:
y=kx+m                                                     (1)
where y is the serum creatinine concentration in µmol/l, x is the number of days
between the mushroom meal and the creatinine measurement and m is the intercept
with the ordinate. The slope of the line, given by the index k, defines the CNT
index.



28
                                                                              Results

The equation may be rewritten
k = (y - m) / x                                         (2)
The CNT index can be expressed as
k = (y – (-316)) / x                                    (3)
where (-316) is the intercept with the y axis.
The denominator is multiplied by 102 to make the index more user-friendly
k = (y + 316) / (x x 102)                                (4)
and the index for patients can be calculated.
The individual indices for all patients in group I and II were calculated according to
equation (4).
The CNT index equation was applied to 51 cases of Cortinarius nephrotoxicity
outside Sweden reported in the literature between 1974 and 1999 (Table 1).


Table 1. Cortinarius NephroToxicity (CNT) index applied to 51 cases:
_________________________________________________________________
Author          Patient no. CNT             Outcome   Treatment
(pub. year)     (admission index
                day)
_________________________________________________________________
Hulmi (1974)    1 (8)       2.35 (anuria)   ESRF      PD
                2 (11)      2.43 (anuria)   ESRF      HD
                4 (11)      1.69            CRF       HD
Marichal (1977) 1 (9)       2.27 (anuria)   ESRF      HD
Short (1980)    1 (10)      3.26 (oliguria) ESRF      PD/HD
                2 (10)      2.24 (oliguria) ESRF      HD
                3 (11)      0.37            Recovered None
Busnach (1982)  1 (9)       1.62 (oliguria) CRF       PE/HD
                2 (9)       1.23            Recovered PE/HD
Nolte (1987)    1 (5)       2.51 (anuria)   ESRF      HD
Bouget (1990)   1 (11)      1.36            Recovered Steroid/HD
                2 (10)      2.39 (anuria)   ESRF      Steroid/HD
                3 (11)      2.10 (anuria)   CRF       Steroid/HD
                4 (11)      0.63            Recovered None
                5 (11)      2.09 (anuria)   ESRF      Steroid/HD
                6 (11)      1.05            Recovered None
                7 (11)      1.91            Recovered Steroid/HD
                8 (11)      1.41 (anuria)   Recovered Steroid/HD
                9 (11)      0.44            Recovered None
                10 (11)     0.61            Recovered Steroid
                11 (11)     1.45            Recovered Steroid/HD
                12 (13)     1.97            ESRF      Steroid/HD
Delpech (1990)  1 (10)      1.42 (oliguria) Recovered HD/PE
                                                      cont.
                                                                                   29
Results

_________________________________________________________________
Author             Patient no. CNT                 Outcome        Treatment
(pub. year)        (admission index
                   day)
_________________________________________________________________
Raff (1992)        1 (5)         1.34              Recovered      None
Franz (1996)       1 (21)        1.11 (anuria)     ESRF           Probucol/HD
Hölzl (1997)       1 (14)        1.90 (anuria)     ESRF           HD
Horn (1997)        1 (14)        0.80              Recovered      HD
                   2 (11)        1.89 (anuria)     ESRF           Steroid/HD
                   4 (9)         2.31 (anuria)     ESRF           HD
Eigler (1997)      1 (14)        1.26              ESRF           HD
Calvino (1998)     1 (15)        0.53 (anuria)     CRF            HD
Valli (1998)       1 (5)         2.36 (oliguria)   ESRF           PE/HD
                   2 (6)         3.39              ESRF           PE/HD
                   3 (8)         2.02 (anuria)     ESRF           PE/HP/HD
                   4 (8)         1.72              Recovered      PE/HD
                   5 (9)         1.55              Recovered      PE/HD
                   6 (8)         2.38 (anuria)     ESRF           PE/HD
                   7 (8)         1.22              Recovered      PE/HP/HD
                   8 (6)         2.14 (anuria)     ESRF           PE/HD
                   9 (5)         2.22 (oliguria)   ESRF           PE/HD
                   10 (5)        2.39 (anuria)     ESRF           PE/HP/HD
                   11 (6)        1.67              ESRF           PE/HP/HD
                   12 (4)        1.12              ESRF           PE/HD
                   13 (5)        1.25              Recovered      None
                   14 (5)        1.60              Recovered      None
Kilner (1999)      1 (10)        1.35              Recovered      AcetylcysteineHD
                   2 (10)        0.69              Recovered      None
Montoli (1999)     1 (2)         2.07 (oliguria)   ESRF           PE
Bednarova          1 (10)        1.51              ESRF           HD
(1999)             2 (8)         0.91              Recovered      HD
                   3 (9)         1.09              Recovered      HD
___________________________________________________________________
ESRF = end-stage renal failure. CRF = chronic renal failure (serum creatinine > 200
µmol/l). Recovered = serum creatinine < 200 µmol/l. Creatinine levels were converted
from milligrammes per decilitre to micromoles per litre by multiplying by 88.0. PE =
plasma exchange. HP = haemoperfusion. HD = haemodialysis




30
                                                                                Results

The indices of these 51 patients were plotted in three categories according to the
outcome (ESRF, CRF and Recovered) and are shown in Figure 9.




Figure 9.   The Cortinarius NephroToxicity (CNT) indices of 51 patients with Cortinarius
            nephrotoxicity outside Sweden reported in the literature between 1974 and
            1999, plotted in three categories: ESRF = End-Stage Renal Failure, CRF =
            Chronic Renal Failure; serum creatinine > 200 µmol/l and recovered = serum
            creatinine < 200 µmol/l.

All patients suffering from Cortinarius intoxication could then be classified in
three risk-groups:
if the CNT index is < 1.1 the prognosis was good,
if the CNT index is = 1.1-2.1 the prognosis was intermediate and
if the CNT index is > 2.1 the prognosis was poor.




                                                                                     31
Results

The probability of ESRF is shown in Figure 10. The three curves show the value of
time until hospitalisation and serum creatinine giving a probability of 5, 50 and
90% of end-stage renal failure, respectively. For points below a curve the proba-
bility is lower and above it is higher.




Figure 10. The probability of end-stage renal failure. The three curves show the value of ti-
            me until hospitalisation and serum creatinine giving a probability of 5, 50 and
            90% of end-stage renal failure, respectively. For points below a curve the pro-
            bability is lower and above it is higher. The curves have been calculated by use
            of the results of a logistic regression analysis.




32
                                                                               Results

Patient outcome (Paper V)
Group I
A majority of patients in group I experienced nausea, vomiting and/or abdomi-
nal/lumbar pains 1-4 days after ingestion of varying quantities of C. speciosissimus.
Five out of 12 patients had documented polyuria. Seven patients were subjected to
X-ray or ultrasound examination and six had large and swollen kidneys.
On admission to hospital, 3-18 days after the intoxication, acidosis, proteinuria,
haematuria, glucosuria, decreased serum sodium and increased serum potassium
were typical findings.
Percutaneous renal biopsy was performed in two patients on day 10, in two patients
on day 16-20 and in one patient on day 55. The light-microscopic examination
showed tubulo-interstitial nephritis. The glomeruli were undamaged. Cellular
infiltration with lymphocytes, plasma cells and polymorphs was seen on the 10th
day, as well as splitting of the basal membrane of the tubules, swelling of the
tubular cells and fibrosis between the damaged tubules. Electron-microscopic
examination showed irregular profiles of tubules of the thick ascending limbs with
changes in the structure of the epithelial cells and an increase of interstitial
collagen. On day 55 there were few infiltrating cells but marked fibrosis. In two
patients biopsy specimen were analysed for the content of orellanine and it was
postitive in one.

Group II
A majority of the patients in group II experienced thirst, polyuria, nausea and/or
abdominal/lumbar pains 1-14 days after ingestion of varying quantities of C. spe-
ciosissimus. Eight out of 11 patients had documented polyuria. Seven patients were
subjected to X-ray or ultrasound examination and one had swollen kidneys.
On admission to hospital, 5-21 days after the intoxication, proteinuria, haematuria
and slightly decreased serum sodium were typical findings.
Percutaneous renal biopsy was performed in four patients on day 20-24. The
pathological-anatomical diagnosis showed cellular infiltrates, splitting of the basal
membrane of the tubules and flattened tubular cells in one patient, only mild
changes in one patient, interstitial fibrosis and uneven epithelial cells in one patient
and marked interstitial oedema, mild inflammation and glomerular sclerosis in one
patient who was a diabetic. The glomeruli seemed unaffected in the other patients.

Group III
The patients in group III came to hospital before any physical symptoms had
developed.
On admission to hospital, 0-2 days after the mushroom meal, normal serum
sodium, potassium and carbonate were typical findings. One subject had pro-
teinuria and haematuria but this was already present before the intoxication. No
patient had a renal biopsy in this group.




                                                                                     33
Results

Treatment and clinical course (Paper V)
In group I six out of twelve patients were given pharmacological treatment of the
intoxication Three patients were treated with steroids. One patient was treated with
cyclophosphamide. Two patients were treated with vitamin C and E.
In group I (12 patients) haemoperfusion (HP) and haemodialysis (HD) were started
in eight patients 3-8 days after ingestion of the mushrooms. One patient received
treatment twice and seven patients were treated once. Four patients received either
HD or peritoneal dialysis (PD).
All twelve patients in group I developed ESRF and were started on chronic dialysis
treatment. Ten patients were subsequently transplanted.
In group II (11 patients) combined HP and HD were started in four patients 5-8
days after the intoxication. Two patients received treatment three times and two
were treated once. Two patients had HP only once. Two patients had HD only. One
patient was treated with PD. Two patients received no treatment. The first five were
followed up between 7 and 20 years and the GFR values are shown in Figure 11.
Three patients were followed up with normal serum creatinine 1-2 months after the
intoxication. Two patients were followed up with normal GFR 2 months after the
intoxication. One patient with diabetes was followed up with a serum creatinine
level of 241 umol/l 10 months after the intoxication and has now six months later,
the same level of renal function.




Figure 11. Glomerular filtration rates (ml/min/1.73 m2) after intoxication in five patients in
           group II in Paper V with follow-up of 7-20 years. Open circles denote patient
           no. 3, closed circles patient no. 4, closed triangles patient no. 7, closed rhombi
           patient no. 8 and open squares patient no. 9.

34
                                                                            Results

In group III (7 patients) two patients had gastric lavage without visible mushroom
fragments in the return seven hours after the meal and two patients had gastric
lavage with visible mushroom fragments in the return three hours after the meal.
All seven patients were treated with HP and in 3 cases also with HD before any
symptoms had developed. Two patients had HP on three consecutive days. Two
patients had HP on two consecutive days. Three patients had one session of HP and
HD. None of the patients in group III developed any symptoms or signs of in-
toxication.
Thus, HP was done in 21 of the 30 patients. Of these 21, eight developed ESRF, as
compared to four patients in the nine who did not receive HP. Thus, 38% of the
patients treated with HP developed ESRF versus 44% of the patients not treated
with HP.

Epidemiological aspects (Paper VI)
Cortinarius speciosissimus grows preferentially in moist and mossy forests of Nor-
way spruce (Picea abies). The distribution map shows that the species occurs in
almost all the country but it is most frequent in the south-western parts (Fig. 12).
However, the strong agglomeration of localities in north-western Götaland is a
result of a special mushroom inventory investigation. In south-western Sweden the
species is abundant in suitable habitats in certain years.




                                                                                 35
Results




Figure 12. The distribution of Cortinarius speciosissimus in Sweden. Each dot on the
           map represents one habitat. The absence of dots does not exclude the precence
           of Cortinarius speciosissimus.



36
                                                                               Results

Eighteen patients gathered the mushroom in the Västra Götaland region. Six sub-
jects picked the mushroom north-west or north-east of lake Vänern, three in west
Småland and four in north-west Halland (Fig. 13).




Figure 13. The distrubution of Cortinarius speciosissimus intoxications in Sweden from
           1979 to 1999. Each dot on the map represents one person intoxicated. The dots
           are separated when more then one person is affected at the same location.


                                                                                     37
Results

The first six cases came in the autumn of 1979, four in Västra Götaland and two in
Halland. Then there were no intoxications until 1984-85, when seven more subjects
fell ill, six in Västra Götaland and one in the western part of Småland. In 1987 four
patients were affected and all these intoxications occurred in Västra Götaland.
Eight subjects fell ill during the period 1990-91, three in Västra Götaland and five
in Närke. Finally, six cases were reported in 1998-99, one in Värmland, two in
Småland, two in Halland and one in Västra Götaland (Fig. 14). Three of these six
were tourists.




Figure 14. The distribution of Cortinarius speciosissimus intoxications in time in Sweden
           1979 to 1999.




38
                                                                        Discussion

                                 Discussion
Mushroom poisoning in Sweden is continuously monitored by the Swedish Poisons
Information Centre in Stockholm (64), (63), (65). They receive 1,500-2,000 in-
quiries yearly concerning suspected mushroom poisoning. In one year Cortinarius
species were suspected in 89 cases out of 2,024 inquiries (4%) (39). Although on
average only 1-2 cases yearly require qualified medical treatment for Cortinarius
intoxication in Sweden, the poor long-term outcome after the acute renal failure in
30-50% of the patients makes this mushroom poisoning one of the most feared.

Orellanine: structure and nephrotoxicity
The mass spectrum and the NMR spectra indicate that the substance isolated from
Cortinarius speciosissimus is very pure and homogeneous. The spectra also con-
firm the structure, 3,3´,4,4´- tetrahydroxy-2,2´-bipyridine-N,N´-dioxide, found by
Antkowiak and Gessner in Cortinarius orellanus.
The approximate LD50 for the isolated toxin in mice was 20 mg/kg, which corre-
sponds well to the LD50 found for the freeze-dried Cortinarius speciosissimus,
which was 2.0 g/kg, as the yield for 44 g dried mushroom was 360 mg (0.8%).
There is no other toxin with a yield in the same range as is found in Cortinarius
speciosissimus.
Our results have been confirmed by Prast et al., who also identified the 3,3´,4,4´-
tetrahydroxy-2,2´-bipyridine-N,N´-dioxide in Cortinarius speciosissimus (in the sa-
me concentration: yield 0.9%) by mass spectrometric and nuclear magnetic reso-
nance analyses (67). They found the LD50 for the toxin to be 15 mg/kg given i.p. in
mice, but they were not able to find any peptidic toxin.
Tebbett and Caddy, however, isolated 2 major toxic components of Cortinarius spe-
ciosissimus and determined their structures as cyclic polypeptides (81). These
compounds (Cortinarin A and B) were shown in laboratory animals to cause
nephrotoxicity characteristic of Cortinarius mushroom poisoning. They also found
Cortinarius gentilis to contain 0.36 % Cortinarin A expressed as dry weight of
mushroom and Cortinarius orellanus 0.43% and Cortinarius speciosissimus 0.47%
(79). These findings have not been confirmed and must be questioned because the
possible nephrotoxic effect of Cortinarius gentilis is not on the same level as that
of Cortinarius speciosissimus and Cortinarius orellanus. In Paper I we were not
able to identify the LD50 for Cortinarius gentilis or Cortinarius limonius. Contrary
to the opinion expressed in the discussion in Paper I, I would not now consider the
subjective evaluation of the extent of renal damage to be sufficient evidence to de-
scribe Cortinarius gentilis and Cortinarius limonius as nephrotoxic. Furthermore,
Tebbett and Caddy write that renal failure caused by Cortinarius gentilis is
reported in Scotland and Finland (79) ; (81). They refer to Short (76) and Hulmi
(34) but these references do not mention any intoxications with Cortinarius
gentilis. To my knowledge, there have been no Cortinarius gentilis intoxications
reported in the literature.
These objections are supported by the findings reported by Rapior et al. They de-
tected orellanine in Cortinarius orellanus and Cortinarius speciosissimus but not in
Cortinarius gentilis and Cortinarius limonius (69).


                                                                                 39
Discussion

The existence of toxic peptides in Cortinarius was also disputed by Matthies and
Laatsch (46). Matthies et al. were unable to find any substance related to corti-
narins in any extract of mushrooms from various locations collected over a period
of nine years. Matthies et al. concluded that the structural assignment of Tebbett
and Caddy must be incorrect.

Renal transplantation
The short and long-term outcome of the first five Swedish patients transplanted af-
ter Cortinarius speciosissimus intoxication shows no signs of acute or chronic ef-
fects related to the toxin.
The background to the study was the findings by Rapior, Delpech and Andary, who
found orellanine not only in the plasma 10 days after mushroom ingestion but also
in two renal biopsies. I quote word for word: “Orellanine levels in renal biopsies
were 7 µg per 25 mm3 of the first biopsy (13 days after ingestion) and 24 µg per 8
mm3 of the second biopsy (6 months later)” (70). However, in the discussion in the
paper by Rapior, it is said that the renal biopsy taken six months after the in-
toxication contained mainly orelline, and that the amount was equivalent to 24µg of
orellanine per 8 mm3. Orelline is the atoxic degradation product of the toxin
orellanine. This finding gives further support to our results showing no sign of ef-
fects caused by orellanine.
The patients transplanted after the publication of Paper III with a shorter follow-up
have not shown any signs of toxic influence either. Two of these patients had
kidneys donated by their mothers 8-9 months after the poisoning with current
serum creatinine levels of 147 and 222 µmol/l and three had cadaveric organs 9-13
months after the intoxication with current serum creatinine levels between 122 and
236 µmol/l 1-6 years after the transplantation. One patient is still on the waiting-
list.

Cortinarius NephroToxicity (CNT) prognostic index
The CNT index is based on the two pieces of information generally available, the
serum creatinine level before treatment and the time that has elapsed since the
mushroom was ingested. It is shown here that the index could provide a reasonable
idea of the prognosis in this type of mushroom poisoning.
This index may be of value from several points of view. First, as no efficient treat-
ment is available, a prognostic index indicating a good prognosis may help the
physician to abstain from extensive or experimental treatment which may in fact be
dangerous. Second, an index indicating a poor or intermediate prognosis may
prompt him or her to evaluate new therapeutic options. Third, an index indicating
an intermediate prognosis and that recovery is possible may be comforting to the
patient.
The three curves in Figure 15 showing 5, 50 and 90% probability of ESRF run app-
roximately parallel to the straight lines equivalent to a CNT index of 1.3, 1.7 and
2.0 between day 5 and 11 after the mushroom meal. This is also the period when
most patients were hospitalised. Combining the results of the CNT index and the
logistic regression analysis would then result in a narrower interval for the three
risk-groups: if the CNT index is < 1.3 the probability of ESRF is 5% or less, if the
CNT index is = 1.7 the probability is 50% and if the CNT index is > 2.0 the proba-
bility is 90% or more.

40
                                                                                Discussion




Figure 15. The three curves showing 5, 50 and 90% probability of ESRF after Cortina-
           rius intoxication and the straight lines equivalent to a CNT index of 1.3, 1.7 and
           2.0.
Creatinine generation is dependent on age, weight and sex and has been used in
many formulas, e. g. Cockcroft and Gaults (15). In order to adjust the serum crea-
tinine (y) to sex, age (years) and weight (kg), the Cockcroft and Gaults formula
may be used.
The data on weight were not available in the 51 cases reported in the literature and
an “adjusted” index has therefore not been calculated.
The CNT index may also have to be adjusted depending on non-oliguric versus
oliguric ARF, as anuria/oliguria occurred in 76-100% of the patients developing
ESRF versus 19-36% of the patients who did not turn out to be irreversibly dialysis
dependent.
From time to time, optimistic treatment reports are presented showing a dramatic
effect on Cortinarius poisoning. Kilner et al. recently reported on a 66-year-old
lady who 10 days after the consumption of a Cortinarius orellanus soup was found
to have ARF with a serum creatinine level of 1032 µmol/l. She was treated with the
anti-oxidant N-acetylcysteine and prednisolone with subsequent improvement of
renal function (40). However, calculation of the CNT index in this case gave a value
of 1.35, indicating that the patient probably have recovered in any case, particularly
since this is likely a case of non-oliguric ARF as anuria/oliguria is not mentioned in
the report. Thus, this index could be helpful in the evaluation of new modes of
treatment.


                                                                                          41
Discussion

Patient outcome
Cortinarius intoxication is one of the most feared mushroom poisonings because of
the long symptom-free interval and the serious nephrotoxic effect, which is often
irreversible. With this in mind, various active treatment strategies have been tried, in
asymptomatic as well as seriously affected patients with oliguric ARF.
In this retrospective study, 21 patients out of 30 with confirmed or suspected Corti-
narius intoxication were considered to have circulating toxin, i.e. orellanine, in the
blood on arrival at the hospital and were therefore subsequently treated with ha-
emoperfusion. However, our data raise the question if any significant levels of
toxin were present when the extracorporeal treatment was started. Some patients
may possibly not have been exposed to the toxin at all.
Two patients in group III had ingested Cortinarius gentilis and two Cortinarius
limonius. Three patients had ingested either C. limonius or C. speciosissimus. The
absence of symptoms and signs consistent with intoxication may speak in favour of
C. limonius as the species ingested. In a study by Rapior et al. (69), orellanine was
not found in either C. limonius or C. gentilis.
Tebbett et al. claim that a toxic cyclic polypeptide (cortinarin A) is present in C.
gentilis (79) and also claim it to be present in C. limonius (78). Matthies and
Laatsch, however, question the presence of toxic peptides in Cortinarius mush-
rooms (46).
If C. gentilis and C. limonius contain orellanine and/or cortinarin, treatment with
gastric lavage and/or charcoal given 3-7 h after the intoxication in 4 out of 7 pa-
tients in group III may have removed some toxin.
The patients in group I and II all had symptoms and signs consistent with toxin ex-
posure. Orellanine was found in the kidney tissue from one of two patients who
were biopsied 16 (pos.) and 20 (neg.) days respectively after the intoxication. Both
these patients developed ESRF. Rohrmoser et al. found orellanine in two of three
renal biopsies performed 10 (pos.), 60 (pos.) and 180 (neg.) days after the mush-
room meal (73). Rapior et al. detected orellanine in a renal biopsy 13 days after
intoxication (70). Futhermore, Rapior discovered orellanine in plasma 10 days post
intoxication. Recent findings by Rohrmoser et al. contradict this result. They found
no orellanine in 87 clinical samples (blood and urine) from 10 individuals taken
between 2 and 25 days after mushroom ingestion (73).
Six patients had swollen kidneys in group I, but only one in group II. All but one
patient in group I were anuric. In group II no-one was anuric but four were oliguric.
These facts indicate more severe injury of the kidneys and may explain the poor
outcome in group I. The higher incidence of thirst and polyuria in group II might
indicate more isolated tubular damage in the kidneys and therefore a better prog-
nosis.
The patients in group I and II do not differ appreciably in frequency of treatment
with haemoperfusion, 66% and 55% respectively. Haemodialysis was used more in
group I (75% HD) than in group II (55% HD) due to more advanced kidney failure
in these patients. The mean patient delay time from the appearance of symptoms
until the patient came to the hospital was the same in both groups (5.1 versus 5.0
days), but the latency period from ingestion to symptoms was shorter in group I.
This is probably explained by a greater toxin dose and/or different individual


42
                                                                           Discussion

sensitivity. Michelot and Nieminen (47); (56) claim that there is a sex difference in
the sensitivity to the toxin. Group I and II had the same age and sex distribution and
these factors ought therefore not to influence the outcome.
In group II, among the first five patients, the GFR has increased in four subjects
during between 6 and 20 years after the intoxication. In two of these patients (age
24 and 26) the GFR has after 6-10 years started to decrease (1 ml/min/year) during
8 and 10 years. Other studies have shown that there is a gradual decline in GFR in
normal humans, by approximately 4 ml/min per decade below 50 years and 10
ml/min per decade above 50 years of age (29). The fifth patient had a stable GFR
during 7 years and was then lost to follow-up. Thus, the prognosis for patients who
do not turn out to be irreversibly dialysis dependent may be good but after 6-10
years of increasing GFR a shift to a redoubled decreasing GFR rate, as compared to
the normal decline, may be seen.

Epidemiological aspects
The geographical distribution of Cortinarius speciosissimus intoxications in Swe-
den appears to be compatible with the hypothesis that the acidification has increa-
sed the occurrence of Cortinarius speciosissimus in south-western Sweden.
As a consequence of the increasing atmospheric pollution, the soil has become mo-
re acid. In Sweden, especially the south-western part is affected. When measuring
the pH in Halland in south-western Sweden in 1951 and 1987, a reduction in the pH
values was found. The greatest reduction was found in the forest compared with
open ground (44). The reduction of the pH has influenced the fungal flora and
several species seem to have decreased or disappeared. Certain species seem to be
favoured by the low pH and one of them is Cortinarius speciosissimus (7). In many
European countries there have been reports of a negative trend among mycorrhiza
mushrooms as an effect of pollution by nitrogen. Species that have been especially
damaged are chanterelle, cortinariae and hedgehog mushrooms (35).
The pH-values in the humus layer of the forest soil are in general low. This is
mainly due to natural biological acidification processes combined with soils and
rocks giving poor protection against acidification in the main part of Sweden. The
very low pH-values in south-western Sweden may however partly be due to the acid
fall-out from the atmosphere (60).
An investigation of the distribution of mushrooms in south-western Sweden be-
tween the years 1935 and 1955, did not mention Cortinarius rubellus or Cortinari-
us speciosissimus (53). The reason is probably that the name rubellus had long
been forgotten and is not mentioned in any modern literature on fungi. For that rea-
son, it was described again in 1950 as Cortinarius speciosissimus. Some years ear-
lier it was described as Cortinarius speciosus from Switzerland but this name was
found to be illegitimate (already occupied) and had to be changed. The taxonomic
problems in the past make it difficult to estimate the frequency of the species befo-
re 1950.
The distribution of the intoxications in time is shown in figure 13. If the study pe-
riod is divided into an early (1979-1989) and a late period (1990-1999), it can be
noted that the incidence does not increase, with 17 subjects in the first and 14 in the
second period. This may be the result of the attention payed to Cortinarius spe-
ciosissimus since the first reports of the nephrotoxic effect after the Swedish index
cases in 1979. Nowadays Cortinarius speciosissimus is described and illustrated in

                                                                                    43
Discussion

every book on mushrooms and almost all mushroom-pickers know about it. On the
other hand, a random variation may also be an explaination as the total number of
intoxications during the entire period was small.
Three tourists were intoxicated in 1998-99. Obviously, the increasing availability of
information in Swedish on the nephrotoxicity of Cortinarius speciosissimus is not
sufficient to prevent intoxications among foreign visitors.
The occurrence of Cortinarius speciosissimus intoxications in Sweden during the
period from 1979 to 1999 may be due to a combination of an increased prevalence
of Cortinarius speciosissimus and lack of knowledge on the existence of toxic Cor-
tinarius species. Furthermore, there has been an increased demand for mushrooms
in general. The increased prevalence of Cortinarius speciosissimus may be an
effect of the acidification. The increased demand may be due to an increased in-
terest in nature in general and in mushroom-picking in particular.




44
                                                                        Conclusions

                                Conclusions
The approximate LD50 in mice for freeze-dried Cortinarius speciosissimus was 2.0
g/kg and for Cortinarius orellanus 3.2 g/kg.
The mass spectrum and the NMR spectra confirm the structure, 3,3´,4,4´-
tetrahydroxy-2,2´-bipyridine-N,N´-dioxide, for a nephrotoxic substance isolated
from Cortinarius speciosissimus.
The approximate LD50 in mice for the nephrotoxic substance isolated from Cortina-
rius speciosissimus was 20 mg/kg.
The short and long-term outcome of the first five patients transplanted after Corti-
narius speciosissimus intoxication shows that renal transplantation 6-36 months
after the intoxication does not carry any additional short or long-term risks in com-
parison with other renal transplant patients.
A Cortinarius NephroToxicity (CNT) prognostic index based on 23 Swedish pa-
tients and applied to 51 cases of Cortinarius nephrotoxicity outside Sweden
presented in the literature made it possible to classify the patients in three risk-
groups: CNT index < 1.1, = 1.1 – 2.1 or > 2.1, indicating a good, an intermediate or
a poor prognosis.
The short and long-term outcome of 30 Swedish patients shows that treatment with
haemoperfusion and/or haemodialysis 3-8 days after the ingestion of mushrooms
does not influence the clinical course of Cortinarius intoxications.
The geographical distribution of 31 cases of Cortinarius speciosissimus intoxica-
tions in Sweden between 1979 and 1999 does not exclude the possiblity of a causal
connection related to the acidification. The chronological distribution of the 31 ca-
ses indicates that the incidence is not increasing. The reason is suggested to be that
mushroom-pickers nowadays are more aware of the existence of the dangerous
fungus.




                                                                                   45
Acknowledgements

                               Acknowledgements
Many people have in different ways been involved in this work. I want to express my sincere
gratitude to all of you, and in particular I would like to mention:
– Jarl Ahlmén, Mattias Aurell and Gunnar Westberg, my tutors, for their excellent guidance in
scientific thinking and for encouragement and patience.
– Sture Bergek, Ingemar Blohmé, Anders Bohlin, Härje Bucht, the late John Eriksson, Sture
Lundberg, Sven-Åke Persson and Christian Svalander, my co-authors, for valuable contribu-
tions.
– Per-Ola Attman, head of the Department, for the privilege of working at the Department of
Nephrology.
– Jan Carlström, Sture Cedgård, the late Claes Eriksson, Henrik Mulec, Kerstin Nättorp, Erna
Pettersson, Staffan Schön, Per Sjöström and Gunnar Stelin, my colleagues at the Department of
Nephrology in Halmstad, Skövde, Stockholm, Varberg, Vänersborg and Örebro, for fruitful co-
operation.
– Anders Alfredsson, Marita Annerstedt, Krister Delin, Elisabeth Fabricius-Lagging, Marie
Felldin, Katarina Gisslén, Börje Haraldsson, Hans Herlitz, Gert Jensen, Ann-Cathrine Johans-
son, Susanne Ljungman, Öjvind Mattillas, Gunnela Nordén, Gudrun Nyberg, Pia Ottosson, Eva
Palmgren, Ola Samuelsson, Annika Siewert-Delle, Bergur Stefansson and Ola Wulf, my collea-
gues, for friendship and support.
– Betty Blomroos, Christina Gustafsson, Anna-Lisa Leijon, Ingrid Lindholm-Karlsson, Marian-
ne Olovsson, Ann-Mari Persson, Maria Rydén and their nursing colleagues for tender loving
care.
– Inger Alin, Elisabeth Ericson, Inga-Britt Persson and Lisbeth Selvén at the Laboratory of
Nephrology, for excellent laboratory assistance.
– Gunilla Karlsson, Gunilla Nilsson, Gunilla Nyberg and Ann Stenman for expert secretarial
assistace.
– Stig Jacobsson and Nils Hallenberg, at the Department of Systematic Botany, Göteborg Uni-
versity, for sharing their insights in mycology.
– Hans Persson and Christine Karlson-Stiber, at the Swedish Poisons Information Centre in
Stockholm, for valuable and inspiring co-operation.
– Staffan Björck and Bengt Wärleby for expert computer assistance.
– Anders Odén for excellent statistical aid.
– John Gulliver for expert revision of the English text.
– Sune Jäderberg for excellent layout and support.
– My wife Anne for love, support and encouragement and our children Johanna and Daniel for
being the joy of my life.
– Finally, I want to express my gratitude to my parents, Marit and Sten Holmdahl, for loving
care and for providing an environment that has stimulated academic interests.
Finacial support for this research was provided by grants for myself from Njursjukas Förening i
Väst-Sverige, The Göteborg Medical Society, John and Brit Wennerström´s Research Foun-
dation, Magnus Bergvall´s Foundation, Tore Nathorst-Windahls minnesfond and the Medical
Faculty, Göteborg University, and for Mattias Aurell and Gunnar Westberg from the Swedish
State under the LUA-agreement.


46
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                                                                                           51
Appendix

                                  Appendix
Cortinarius speciosissimus intoxications outside Sweden re-
ported in the literature between 1974 and 1999
In Finland in 1974, Hulmi et al. described four cases of intoxication with Cortina-
rius speciosissimus (34). The first patient (s-creat. 1.566 µmol/l day 8) was treated
with peritoneal dialysis and was later transplanted. The second patient (s-creat.
2.357 µmol/l 11 days after intoxication) was treated with haemodialysis and later
transplanted. The third patient had peritoneal dialysis and recovered, with normal
creatinine after 9 months. The fourth patient (s-creat. 1.539 µmol/l day 11) had hae-
modialysis and recovered with creatinine 720 µmol/l after 6 months.
In Scotland in 1980, Short et al. described three previously healthy young adults
who developed gastrointestinal upset after 36-38h, followed by nausea, anorexia,
headache, rigors, severe burning thirst, muscle aching and oliguria (76). One
patient developed polyuria (8 days after the intoxication) which persisted for 3 days
and then recovered completely. The other two patients did not present at hospital
until 10 days after ingestion and severe renal failure had already developed. Both
had a severe interstitial nephritis and neither recovered renal function. They were
maintained on intermittent haemodialysis until they received renal transplants 9
months later. All three patients were on holiday in Northern Scotland in August
1979 where they gathered Cortinarius speciosissimus eaten in a stew by all three.
Some of the mushroom were eaten raw the next day by 2 of the patients.
In Norway, no intoxications were recorded before 1981 according to Jacobsen et al.
(37). In 1982 the first cases were reported (23). A 28-year-old man developed acute
renal failure after the ingestion of small red and yellow mushrooms and he was
transplanted after six months with a kidney from his brother. A 61-year-old man ate
a mixture of mushrooms and after a period of gastroenteritis he developed ARF but
after two days the haemodialysis treatment could be terminated.
In 1983 a 47-year old woman suffered “sudden death” after some days of headache
and diarrhoea. It later turned out that she had ingested Cortinarius speciosissimus
12 days before she died (51).
In Italy in 1983, Busnach et al. described mushroom poisoning by Cortinarius spe-
ciosissimus leading to irreversible renal failure (10). They reported that an expected
poor prognosis had probably been overcome as a result of the very early use of
plasma exchange. In September 1981, a healthy couple, both aged 38, had gastro-
intestinal symptoms 2 days after ingestion of Cortinarius speciosissimus. On day 8
acute renal failure developed and the same day, before dialysis, plasma exchange
(PE) was undertaken. Renal biopsies disclosed a common pattern of tubular nec-
rosis, scattered infiltrates and interstitial oedema. Diuresis and partial recovery
started in the man on day 10, while renal failure persisted over six months in the
woman.
In Germany in 1987, Nolte et al. reported on a 14-year-old boy who was admitted
for vomiting, anorexia, flank pain and leukocyturia/haematuria (59). Shortly after
admission, he developed anuria and acute renal failure so that haemodialysis had to
be started. Pre- and post-renal causes were excluded. There were no signs of acute
glomerulonephritis; liver enzymes were normal. The 123Iodine-Hippuran scan sho-
wed a shock kidney pattern lacking tubular clearance. Renal biopsy revealed an in-


52
                                                                            Appendix

terstitial nephritis with oedema and mixed cellular infiltration. The history was
negative for nephrotoxic agents except for mushroom ingestion. Five days before
admission the boy ate Cortinarius speciosissimus mushrooms. Renal function did
not improve much and renal transplantation was performed after 14 months on
haemodialysis.
In Austria in 1996, Franz et al. reported on a 28-year-old man who thought he had
eaten magic mushrooms but it was later shown that he had eaten Cortinarius orel-
lanoides (=Cortinarius speciosissimus) (26). He was admitted three weeks after the
ingestion of the mushrooms and had a serum creatinine value of 23 mg/dl. He was
treated with haemodialysis, rehydration and probucol (anti-oxidant) but 12 months
after the intoxication the patient still required haemodialysis and was evaluated for
renal transplantation.
Also in Austria in 1997, Hölzl et al. reported on a 23-year-old man who was ad-
mitted to hospital because of acute anuria in August 1995 (36). Fourteen days prior
to admission he had consumed five fruit bodies of raw mushrooms of Cortinarius
speciosissimus. The tentative diagnosis of acute renal failure due to orellanine
intoxication was confirmed by the histological finding of acute interstitial nephritis
in a first renal biopsy one week after onset of anuria. The patient required hae-
modialysis for the following weeks and months and was was then put on peritoneal
dialysis pending renal transplantation. Six months after the onset of symptoms, a
second renal biopsy was performed, which revealed increasing interstitial fibrosis.

Cortinarius orellanus intoxications outside Sweden reported in
the literature between 1976 and 1999
In Switzerland in 1976, Favre et al. presented three cases of intoxication due to in-
gestion of Cortinarius orellanus (24). In all three cases, acute gastroenteritic disor-
ders and hepatocellular damage were observed initially and disappeared spontane-
ously. Histological examination revealed acute tubulointerstitial nephritis.
In France in 1977, Marichal et al. reported two case histories to illustrate that such
cases of poisoning occur in France, where the most dangerous of the Cortina, Cor-
tina orellanus, although not widespread, is also not rare (45).
In Germany in 1977, Fäber and Feldmeier reported on four children treated with
haemodialysis and hydration after Cortinarius orellanus intoxication. One child de-
veloped ESRF (22).
In Czechoslovakia in 1979, Bouska et al. presented a case of fungus poisoning in-
volving nephrotoxic effects (6). Diagnosis was based on the typical course as well
as the postmortem and mycological investigations of intestinal contents. This was
the first death due to Cortinarius orellanus poisoning ever described in Czecho-
slovakia.
In France in 1990, Delpech et al. reported on a 31-year-old woman who was ad-
mitted to hospital 10 days after she had deliberately ingested 2 raw carpophores of
the mushroom Cortinarius orellanus (20). Acute renal failure (creatininaemia
1,100 µmol/l) developed, requiring 6 sessions of haemodialysis, one of plasma-
pheresis and the administration of diltiazem and aminoacids. Eighteen months after
the attempted suicide, the plasma creatinine level was 181 µmol/l.



                                                                                    53
Appendix

Also in France in 1990, Bouget et al. reported on twenty-six young men with no
previous medical history who all ingested mushroom soup, exclusively made with
Cortinarius orellanus (5). They were hospitalised 10-12 days after the incident. On
admission, 12 patients presented with acute tubulointerstitial nephritis with acute
renal failure; 8 required haemodialysis. In addition to symptomatic treatment, 9
patients were given corticosteroids. In this group of 12 patients, 8 recovered rapidly,
and the other 4 suffered from chronic renal failure for several months. In the other
group of 14 patients, initial leukocyturia was observed in 12 cases, although renal
function remained normal during a one-year follow-up.
In Bulgaria in 1995, Osichenko et al. reported on a 40-year-old man intoxicated
with Cortinarius orellanus treated with haemoperfusion and haemodialysis and
recovering with normal renal function (62).
In Germany in 1997, Eigler et al. reported on a 27-year-old man intoxicated by
Cortinarius orellanus (21). At admission 14 days after the mushroom meal, he had
a plasma creatinine value of 1.449 µmol/l and haemodialysis was started
immediately but the acute renal failure could not be reversed. The patient decided
to use CAPD and was later transplanted.
In Austria in 1997, Horn et al. described four patients of differnt age and health sta-
tus who ingested a stew consisting exclusively of Cortinarius orellanus, which had
been mistaken as Cantharellus cibarius (33). The first patient had on admission two
weeks after the intoxication a serum creatinine level of 9.2 mg/dl and was treated
with haemodialysis and recovered, with a serum creatinine level of 1.6 mg/dl ten
months after the event. The second patient (s-creat. 20 mg/dl 11 days after intoxi-
cation) was treated with haemodialysis and steroids without improvement and ten
months after the event remained on chronic haemodialysis. The third patient was
not admitted to hospital and recovered completely without therapy. The last patient
(s-creat. 20 mg/dl 9 days after intoxication) was started on haemodialysis and has
remained on chronic treatment since.
In Spain in 1998, Calvino et al. reported on a young male ex-drug addict who de-
veloped acute tubulointerstitial nephritis after voluntary ingestion of Cortinarius
orellanus which he thought was a magic mushroom (12). He was treated with
haemodialysis and rehydration. Five months after the intoxication the serum
creatinine level was 5 mg/dl and he did not require dialysis.
In the Czech Republic in 1999, Bednarova et al. presented the case-records of three
patients who became intoxicated with Cortinarius orellanus (3). After treatement
with haemodialysis, two recovered and one developed ESRF. Cortinarius orellanus
is very rare in the Czech Republic and is not well known.
In England in 1999, Kilner et al. reported on a 66-year-old Austrian lady and her
dauther who had eaten a soup containing Cortinarius orellanus which she had
picked in the Irish Republic 10 day before admission (40). The lady (s-creat 1.032
µmol/l day 10) was treated with haemodialysis, prednisolone and N-acetylcysteine
and recovered, with s-creat. 168 µmol/l after 2 months. The dauther had s-creat. 376
µmol/l on admission and was obviously recovering without treatment.
In Italy in 1999, Montoli et al. treated a 53-year-old man with plasma exchange 44
h after the ingestion of a mushroom sauce containing Cortinarius orellanus and
Entoloma lividum (49). The s-creat. was 97.5 µmol/l 30 h after the meal and the pa-
tient received three sessions of plasmapheresis but became oliguric on the nineth

54
                                                                          Appendix

day and haemodialysis was started. Seventeen months after the intoxication he was
transplanted.

Cortinarius intoxications – species unspecified
In Czechoslovakia in 1978, Stredova et al. described a 42-year-old man and his
two-year-old daughter developing acute renal failure after ingestion of an unspeci-
fied species of Cortinarius mushrooms (77). The man responded to haemodialysis
but the little girl died.
In Oregon, U.S.A. in 1991, Moore et al. reported two cases of Cortinarius poiso-
ning treated with haemodialysis (50).
In Canada in 1992, Raff et al. described a 20-year-old woman in search of magic
mushrooms who developed acute renal failure after intoxication with an unspeci-
fied Cortinarius mushroom (68). Five days after the ingestion she had a serum
creatinine level of 356 µmol/l and she improved without dialysis or haemoper-
fusion. She did not return for follow-up.
In France in 1993, Bedry et al. reported on a 32-year-old man and his pregnant wife
who presented with weakness, dizziness and vomiting four days after consumption
of mushrooms resembling Tricholoma auratum (4). The initial laboratory data sho-
wed severe rhabdomyolysis and mildly raised hepatic enzymes but no renal in-
sufficiency. The death of the man occurred seven days after the onset of the
symptoms and was due to malignant hyperthermia related to the rhabdomyolysis.
Orellin, a metabolite of orellanine, was afterwards found in the urine of the man.
In Italy in 1998, Valli et al. reported on 27 cases of Cortinarius poisoning between
1987 and 1997 where 14 patients developed ARF (83). Twelve patients were treated
with haemodialysis and plasma-exchange and four patients also received haemo-
perfusion. Nine patients turned out to be irreversibly dialysis dependent (ESRF).

Cortinarius speciosissimus intoxication in sheep
In Norway in 1979, Överås and Ulvund described poisoning in sheep induced by
Cortinarius speciosissimus (84). The flock comprised 60 ewes and lambs of the
Dala breed, grazing a pasture where sheep had been kept for many years. On 13th
August, 4 animals were observed to be ill. The sheep, a lamb, a ram and 2 ewes,
looked depressed and gradually became apathetic. All sheep died or had to be
killed on 15th August.

Cortinarius speciosissimus intoxication in laboratory animals
Lahtiperä et al. studied the effects of Cortinarius speciosissimus on rat kidney by
transmission electron microscopy (43). Suspension of dried mushroom (500 mg/kg
body weight) was administered as a single dose direct into the oesophagus. The
kidneys were fixed by perfusion, and both cortex and medulla were sampled. C.
speciosissimus toxin acted primarily on the epithelial cells of the proximal tubules.
No changes were seen in the glomeruli. The first ultrastructural changes were
observed at 2 days in the renal cortex. The most prominent damage occurred at 5
days, when most of the proximal tubular cells appeared necrotic. Dark bodies
(diameter 0.15-0.5 microns) spaced by 25-45 nm were frequently found in the
damaged tubular cell nuclei. Regeneration of the tubular cells was seen at 10 days.
After 2 months, an increased amount of collagen fibres was seen between tubules.

                                                                                  55
Appendix

The nuclear changes in the damaged tubular cells and the slowly manifesting
toxicity suggest that C. speciosissimus toxin acts on nuclei or nucleoli and/or meta-
bolic pathways associated with them.
Möttönen et al. studied the toxicity of two mushroom species found in Finland,
Cortinarius speciosissimus and Cortinarius gentilis, in the rat (52). Dried, homo-
genised mushroom was given orally by stomach tube. A dose of 500 mg dried
mushroom per kg body weight was used. It was demonstrated that both species
caused renal damage. No damage could be detected in other organs. The renal his-
topathological changes corresponded to those of tubulo-interstitial nephritis. The
sensitivity of different individuals to the fungal toxins varied greatly.
Nieminen et al. studied the nephrotoxicity of the mushroom species Cortinarius
speciosissimus in the rat (55). Dried, homogenised mushroom was given orally by
stomach tube. The development of the kidney damage was followed by both histo-
logical and enzyme histochemical methods. The first signs of kidney damage were
interstitial infiltrates occurring mainly in the outer medullary zone, observed two
days after the administration of the mushroom.
Nieminen et al. also studied the effect of phenobarbitone and phenylbutazone treat-
ment on the renal damage induced by the toxic mushroom Cortinarius specio-
sissimus in female rats (54). Phenobarbitone sodium was given in drinking water
(0.05% solution) for 11 days before the administration of the mushroom. Phe-
nylbutazone was given s.c. in doses of 50 and 100 mg/kg 1 h before the mushroom
administration. Homogenised mushroom was given orally by stomach tube at a do-
se of 250 mg dried mushroom per kg body weight. It was found that the pheno-
barbitone treatment strongly increased the damage induced by C. speciosissimus in
the tubules of the renal cortex but had no effect on the inflammation in the renal
outer medullary zone induced by this toxic mushroom. Phenylbutazone treatment
had no effect on the renal damage induced by C. speciosissimus.
Nieminen also studied the effect of furosemide on the renal damage induced by the
toxic mushroom Cortinarius speciosissimus in female rats (57). Furosemide (50
mg/kg s.c.) was given 15 min before mushroom administration (250 mg dried
mushroom per kg body weight by mouth). It was observed that furosemide clearly
increased the tubular damage induced by C. speciosissimus. In contrast, furosemide
had no effect on the inflammation induced by this toxic mushroom.
Nieminen et al. also showed that a single intraperitoneal dose of cyclophosphamide
(150 mg/kg) given at the same time as an oral dose of Cortinarius speciosissimus
prevented the renal inflammation induced by this toxic mushroom in the male rat
(58). Furthermore, scar formation around dilated collecting ducts was clearly
reduced by cyclophosphamide treatment.

Cortinarius orellanus intoxication in laboratory animals
Prast et al. showed that oral administration of Cortinarius orellanus (Fr.) to male
Sprague Dawley rats caused serious impairment of renal function (66). The signs
observed were similar to those produced in humans who ingest this fungus.
Administration of 2.0 g dried Cortinarius orellanus per kg body weight led to acute
renal dysfunction within 48 h. The pattern of impairment included a reduced glo-
merular filtration rate, decreased renal absorption of water, sodium and potassium,
and proteinuria and glucosuria. The nephrotoxic effect was further characterised by


56
                                                                           Appendix

decreased activities of the brush border enzymes alkaline phosphatase and gamma-
glutamyltranspeptidase in urine, despite a remarkable increase in protein excretion
of predominantly tubular origin. These findings were substantiated by morpholo-
gical changes, which could be detected as early as 12 h after dosing. Detectable
quantities of orellanine were excreted only within the first 24 h after dosing. No im-
pairment of liver function was detected.




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