Parasitology International 75 (2020) 102036

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Review

Human proliferative sparganosis update T

⁎
Taisei Kikuchi, Haruhiko Maruyama
Department of Infectious Diseases, Division of Parasitology, Faculty of Medicine, University of Miyazaki, Japan

A R T I C LE I N FO A B S T R A C T

Keywords: Proliferative sparganosis is one of the most bizarre and mysterious parasitic diseases ever described. The cau-
Sparganum proliferum sative parasite is Sparganum proliferum, which is a pseudophyllidean cestode distinct from Spirometra tapeworms.
Proliferative sparganosis Here we overview this rare but fascinating disease with the all original case reports on human patients published
Sparganosis in the last 115 years.
Metacestode infection
Proliferative sparganosis is clearly divided into two disease types, cutaneous and internal proliferative
Cestode
sparganosis. Cutaneous type starts with a skin eruption caused by the dermal invasion of a sparganum. Skin
lesion progresses to larger areas of the body if left untreated. Various internal organs and body wall can be
eventually affected. The clinical symptoms of patients in this group are very similar to each other. Molecular
data suggest that cutaneous proliferative sparganosis is caused by S. proliferum of which genetic variation is
limited, regardless of the time or localities of the emergence of patients.
Internal proliferative sparganosis, on the other hand, is much more heterogeneous. Some cases show ag-
gressive infection in internal organs, while others show only restricted lesions. Some of the cases that had been
cited as proliferative sparganosis in the past literature were removed from the list, because they were judged as
cyclophyllidean tapeworm infections. DNA sequencing is mandatory for the definite diagnosis of proliferative
sparganosis.
The Venezuelan strain of S. proliferum is maintained in experimental mice in Japan, which is fully prepared
for the experimental study with advanced technologies in modern molecular biology.

1. Introduction
In 1904, a 33-year-old woman visited the Hospital of the University
of Tokyo, Japan, for the treatment of inguinal hernia on the left side.
However, what attracted the attention of the physicians was the ele-
phantiasis-like skin disease she had been suffering for years.
Dermatologists at the hospital could not reach a diagnosis at the first
visit. Histopathological examination of the excised skin revealed nu-
merous parasites of some kind, surrounded by cystic fibrous tissues.
Worms isolated from surgical specimens actively moved like amoeba in
physiological saline [1].
Professor Ijima at the Department of Zoology, College of Science,
University of Tokyo, examined the parasite, and designated it as
Plerocercoides prolifer, because he considered it as cestode plerocercoids
of unknown identity [2]. In 1907, another patient with very similar
symptoms was found in Florida, the United States, the second patient in
history [3]. Samples collected from this 48-year-old man were sent to
Professor Stiles, who re-designated the parasite as Sparganum proliferum
[4].
Since these earliest cases, infections with S. proliferum have been
sporadically reported [5,6]. The disease is now known as proliferative
sparganosis. In this rare cestode infection, numerous asexually multi-
plying plerocercoids invade various tissues and organs, making the
prognosis poor [7]. In contrast, in ‘ordinary’ non-proliferative sparga-
nosis, only a single or a few Spirometra plerocercoids migrate in con-
nective tissues, showing simple mass effects. Organs, such as lungs,
liver, eyes and central nervous system could be involved, but they are
less common [7–10].
The life cycle of Spirometra has been well documented [9]. Adult
worms live in the intestines of carnivore animals, e.g. dogs and cats,
and freshwater copepods serve as the first intermediate host. Infected
copepods are then ingested by the second intermediate hosts, including
fish, reptiles and amphibians, in which larvae develop into pler-
ocercoids. Humans acquire infection by either drinking water con-
taminated with infected copepods or consuming flesh of undercooked
second intermediate or paratenic hosts [9]. S. proliferum has been
generally believed to possess a similar life cycle. However, no adult
worms nor procercoids have been discovered, and nothing has been

⁎
Corresponding author at: Department of Infectious Diseases, Division of Parasitology, Faculty of Medicine, University of Miyazaki, Kihara 5200, Kiyotake,
Miyazaki 889-1692, Japan.
E-mail address: hikomaru@med.miyazaki-u.ac.jp (H. Maruyama).

https://doi.org/10.1016/j.parint.2019.102036
Received 20 June 2019; Received in revised form 5 December 2019; Accepted 8 December 2019
Available online 10 December 2019
1383-5769/ © 2019 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
T. Kikuchi and H. Maruyama
known as to how they infect humans and other mammalian animals.
Same as non-proliferative sparganosis, proliferative sparganosis is a
zoonosis [11–15].
Biological study on S. proliferum had been severely hampered by the
rarity of the disease. In1980's, however, a major breakthrough was
made by scientists at Universidad Central de Venezuela when they
succeeded maintaining S. proliferum in experimental animals [16]. S.
proliferum plerocercoids were isolated from a 35-year-old patient, and
the worms were inoculated mice, hamsters and a monkey. With the
laboratory-maintained S. proliferum, Japanese researchers determined
DNA sequences of several mitochondrial and nuclear genes, concluding
that S. proliferum is a close but distinct species from S. erinaceieuropaei
[17,18].
Today, more sophisticated techniques are available to investigate
the biology and pathogenicity of S. proliferum. Because it is essential to
know the features of the disease exactly upon starting experimental
study, we overviewed all accessible human case reports of proliferative
sparganosis published in the last 115 years, to update our knowledge on
this extraordinary disease. Here, we would like to present the real
picture of the disease, which should inspire researchers with en-
thusiasm to investigate this enigmatic organism.
2. Literature survey for original case reports
We searched PubMed, Google Scholar and other literature databases
for original case report articles and reviews on proliferative sparganosis
with keywords such as ‘proliferative’, ‘disseminated’, or ‘unusual’ in
combination with ‘sparganosis’, to draw up a list of case reports. We
then obtained PDF files of these reports through document supply ser-
vices provided by the university library network. All case reports we
obtained were published in English, or had English abstract, except for
those published in Japan before 1960. Main texts described in non-
English languages (Thai, Korean, Chinese) were translated into English
by native infectious diseases specialists.
Collected references were further examined carefully if the causa-
tive larvae were spargana with signs of proliferation. Inclusion criteria
for asexual proliferation were 1) disseminated infection with excessive
number of worms, 2) recurrent mass formation after surgical removal,
3) multiple worms with budding and/or branching from the single in-
fection focus. We also included a case with molecular identification
achieved by DNA sequencing. References were removed from the list
when causative cestode larvae were judged cyclophyllidean tapeworms.
3. Clinical picture
3.1. Disease types
Extensive literature search and referring to the original case reports
enabled us to extract eighteen proliferative sparganosis cases, either
confirmed or suspected, from the body of literature (Tables 1 and 2)
[1–7,19–36]. Having analyzed original description, we came to re-
cognize two distinct groups in proliferative sparganosis.
One group consists of patients who have skin eruption as the initial
symptom caused by the invading parasite in the dermis, followed by the
spread of the infection to larger areas of skin. The other group of pa-
tients complain of nodules or masses in deep connective tissues or in-
ternal organs, without any sign of dermal involvement. In the following
sections, these two disease types are referred to as cutaneous pro-
liferative sparganosis and internal proliferative sparganosis, respec-
tively. Characteristic features of these two distinct disease types are
summarized in Table 3.
3.2. Cutaneous proliferative sparganosis
Eight patients are included in this group (Cases 1, 2, 3, 4, 5, 6, 8,
and 17 in Tables 1 and 2). Male patients are six, while females two. All
2
Parasitology International 75 (2020) 102036
patients are adult, ranging from 24 to 62 years old. Five were found in
Japan, and three in the Americas. First six cases were before 1925, next
one in 1981, and the latest in 2014.
In five of the eight cases, the primary skin lesion was reportedly in
the lower half of the body; thigh or abdomen, from where infection
progressed. Seven cases developed disseminated skin infection
(Fig. 1A). However, for unknown reasons, skin of the face was intact
even in the advanced stage. In three cases, spargana were picked up by
scratching the diseased skin, or worms could be squeezed out from the
skin (Fig. 1B). At least four patients in this group further experienced
invasion of spargana into various parts of the body, such as peritoneal
cavity, retroperitoneum, and lungs (Table 2). The progression of the
disease appeared to have taken years, before reaching the terminal
stage (Table 1).
The latest patient in this group reported in 2014 deserves detailed
description (Case 17). A 61-year-old European man admitted to the
hospital after a holiday travelling through Bolivia, Brazil and Paraguay.
Upon his return to Germany, he noticed an itching reddish patch on his
right chest. From this skin eruption, a single sparganum was removed,
which had no evidence for budding or branching. The sparganum was
identified as S. proliferum by DNA sequences [35].
Proliferative sparganosis has been recognized as a fatal nasty in-
fectious disease, which is presumably due to the fatal cases in the first
half of the 20th century. In cutaneous proliferative sparganosis, seven
out of the eight patients did die. However, early diagnosis enabled by
the modern medical technology, and immediate treatment would pos-
sibly save patients, because the progression of the disease is slow. The
first case in Tokyo or that in Florida might not have been lost, if worms
had been surgically excised in the very early stage of infection.
3.3. Internal proliferative sparganosis
Ten patients are grouped in this category (Tables 1 and 2). Male
patients dominate, and eight patients are from Asian countries, in-
cluding Japan, Taiwan, South Korea, China, and Thailand. Ages of the
patients are similar to those of the cutaneous group, but with one pe-
diatric case in China. Clinical symptoms of this type are caused by
pathological conditions related to nodules, tumors or masses in body
wall or internal organs. No sign of dermal infection exists.
In seven of the ten cases, the primary infection foci were in the
lower part of the body, such as groin, lower abdomen, or pelvic cavity.
Masses especially large ones coincided abscess formation. Tumors in
some cases were invasive, extending regionally or distantly. In two
cases (Case 13 and 14), new lesions appeared in the distant locations,
one in the lung and the other in the brain, in which ‘hematogenous
metastasis’ was suggested. Unfortunately, distant lesions were revealed
by computer tomography (CT) only, and the presence of worms was not
directly demonstrated. One of these cases, Case 14, was successfully
cured by praziquantel [33].
One of the most characteristic features of this group is involvement
of bones. In four cases (Cases 10, 13, 15, 18), bones were destroyed by
the invasion of worms. Three cases had bone lesions only, with no re-
gional or distant tumor progression (Cases 10, 15, 18). As long as the
present eighteen cases are concerned, it appears that cutaneous infec-
tion and bone infection are mutually exclusive (Table 3).
Although fatal cases could be found in this group (Case 7, 11, and
13), the overall prognosis seems better than that of the cutaneous type
(Table 3). This could be due to the shorter periods before the diagnosis.
In the internal group, the median period between the onset and the
diagnosis was 1.5 years (6 months to 5 years), whereas it was 7 years
(3 weeks to 23 years) in the cutaneous group (Table 3). Considering
that all patients in the internal proliferative sparganosis group were
diagnosed after 1970, this earlier diagnosis must have owed much to
the development of non-invasive medical imaging technologies, e.g. CT,
ultrasonography, and magnetic resonance imaging (MRI).
 T. Kikuchi and H. Maruyama

Table 1
List of reported proliferative sparganosis cases.
Case no Author Year of Age/sex Estimated place of Primary lesions Lesions in chronic phase Onset to diagnosis
diagnosis infection

1 Ijima (1905), Kondo & Yamamura 1904 33/F Tokyo, Japan Eruption in left thigh Elephantiasis condition in whole body except for face, head, neck and upper 5 years
(1908) extremities
2 Gates (1908), Stiles (1908) 1907 48/M Florida, USA Eruption in left shoulder Acne-like condition of whole body, masses in trunk muscles, lungs, scrotum, 23 years
peritoneal cavity, brain and spinal cord
3 Usui (1909), Kodama (1917), Yoshida 1907 36/M Tokyo, Japan Eruption in neck Nodules in skin of whole body, oral mucosa, retroperitoneum, lymph nodes, 7 years
(1914) mediastinum, viscera
4 Inoue (1911a, 1911b), 1911 55/M Kyoto, Japan Eruption in right thigh Dermal and subcutaneous nodules in whole body except for face and head 12–13 years
Yoshida (1914)
5 Akanuma (1920) 1919 62/M Kyoto, Japan Eruption in lower abdomen Nodules in skin of abdomen and extremities, abdominal cavity, left lung, 12 years
kidneys
6 Tashiro (1921, 1924) 1921 24/F Kumamoto, Japan Eruption in left thigh Elephantiasis condition in lower extremities, pubic region, masses in 6 years

3
abdominal wall, pleural cavity, lungs, peritoneum, pelvis
7 Beaver & Rolon (1981) 1977 24/M Paraguay Unspecifiable Masses in mediastinum and abdominal wall unspecifiable
8 Moulinier et al. (1982) 1981 35/M Venezuela Eruption in abdomen Nodules in skin of whole body except for face, palms, soles, and genitalia 7 years
9 Cho et al. (1985) 1981 35/M South Korea Left inguinal swelling Subcutaneous tumors in left groin, left thigh, and right scrotum unspecifiable
10 Liao et al. (1984) 1982 26/F Taiwan Mass in lumbar vertebrae – 4 years
11 He & Huang (1984) 1982 11/M Guangdong, China Mass in lower abdomen Nodules and masses in abdominal wall, abdominal cavity, viscera, < 1 year (?)
mesenteric lymph nodes
12 Lo et al. (1987) 1984 43/F Taiwan Mass in lower spinal cord – 6 months
13 Aoshima et al. (1989) 1987 47/M Tokyo, Japan Mass in right pelvic cavity and Nodules in lungs (CT) 10 months
Nakamura et al. (1990) pelvic bone
14 Jirawattanasomkul & Noppakun 2000 32/M Thailand Nodules in extremities, palms, Nodule in brain (CT) 4–5 years
(2000) and armpit
15 Settakorn et al. (2002) 2001 51/M Thailand Mass on right tibia – 2 years
16 Meric et al. (2010) 2010 45/M Reunion Island Nodules in skin, scrotum, and Intra-abdominal masses, mesenteric nodules 4 years
liver
17 Schauer et al. (2014) 2014 61/M Bolivia/Brazil/Paraguay Eruption in chest wall – 3 weeks
18 Laovachirasuwan et al. (2015) 2015 63/M Thailand mass in left distal humerus – 5 months
Parasitology International 75 (2020) 102036
 T. Kikuchi and H. Maruyama

Table 2
Summary of clinical features and molecular diagnosis.
Case Disease Progression Non-morphological examination Treatment Concomitant
conditions
No Year/countrya Primary lesion in Large skin Primary lesion in Single Visceral Bone Fatal outcome Antibody DNA sequencing
skin lesion lower body lesion lesion lesion positive

1 1904/Japan ✔ ✔ ✔ ✔ surgical operation inguinal hernia

(skin restoration)
2 1907/USA ✔ ✔ ✔ ✔ removal of cutaneous mass
3 1907/Japan ✔ ✔ ✔ ✔ ✔ none
4 1911/Japan ✔ ✔ ✔ ✔ skin biopsy inguinal hernia
5 1919/Japan ✔ ✔ ✔ ✔ ✔ none inguinal hernia
fasciolosis
6 1921/Japan ✔ ✔ ✔ ✔ ✔ surgical operation
(skin restoration)

4
7 1977/Paraguay ✔ ✔ removal of abdominal mass
8 1981/Venezuela ✔ ✔ ✔ ✔ ✔ mebendazole, praziquantel gynecomastia
9 1981/South Korea ✔ removal of spargana inguinal hernia
10 1982/Taiwan ✔ ✔ ✔ spinal surgery
11 1982/China ✔ ✔ ✔ supportive care
12 1984/Taiwan ✔ ✔ spinal surgery
13 1987/Japan ✔ ✔ ✔ ✔ ✔ removal of pelvic mass
14 2000/Thailand ✔ removal of nodules, albendazole,
praziquantel
15 2001/Thailand ✔ ✔ ✔ incisional biopsy
16 2010/Reunion ✔ removal of abdominal mass HIV positive
Island
17 2014/BO/BR/PY ✔ ✔ skin biopsy
18 2015/Thailand ✔ ✔ ✔ incisional biopsy, albendazole,
praziquantel

a
Abbreviations for countries, BO: Bolivia, BR: Brazil, PY: Paraguay.
Parasitology International 75 (2020) 102036
T. Kikuchi and H. Maruyama
Table 3
Summary of cutaneous and internal proliferative sparganosis.
Cutaneous proliferative sparganosis
(n = 8)
Age of patients: median (range) 42 (24–62)
Male/female 6/2
Regions Asia (5), Americas (3)
Period between onset and diagnosis: median (range) 7 years (3 weeks-23 years)
Primary lesion skin eruption
Skin infection 8/8
Bone infection 0/8
Fatality 87.5% (7/8)
4. Diagnosis
4.1. Morphological features of S. proliferum
Diagnosis of proliferative sparganosis was entirely relied on the
morphological examination until recent development of molecular di-
agnosis. Spargana in typical cases are relatively small, up to 10 mm
long and about 1 mm wide. They are thread-like, vermiform or oval,
with branching and budding from the sides (Fig. 1C). Multiplication
appears by transverse fission. They are encapsulated singly or in groups
of two or three, but free worms can also be observed [2,4]. In tissue
sections, their structure is similar to that of Spirometra spargana,
showing two well-defined areas, the tegument and the parenchyma
with readily recognizable calcareous corpuscles [5].
Detailed morphological study was conducted by the Venezuelan
researchers [37]. The surface of the tegument was covered with mi-
crotriches, and in all larvae, single or multiple tegument-covered par-
enchymal cavities were observed. The shape and length of the cavities
were extremely variable and were either empty or containing a
homogeneous material (Fig. 2). These parenchymal cavities appeared
the so-called ‘nutritive substance’ observed by Ijima and other early
researchers [2,4,26]. The excretory system was composed of numerous
randomly arranged ducts covered by uniformly distributed peduncu-
lated microvilli. Tissues of spargana were relatively well differentiated,
though nerve cords were not observed [37].
In internal type, spargana are globular or egg-like forms with oc-
casional peripheral budding. They also have large empty vacuoles or
channels, which were either vesicles formed by invagination of the
outer wall, or excretory channels lined with a distinct membrane [5,6].
One striking feature was found in the larvae of Case 16 (Reunion Is-
land). In tissue sections, extremely long hair-like microtriches were
observed on the surface of the tegument [7]. Because this ‘hairy’ te-
gument has not been observed in any proliferating or non-proliferating
spargana [38], parasites of Case 16 should be molecularly examined.
Non-proliferative sparganosis could be difficult to differentiate from
proliferative sparganosis, when infection is heavy. Spirometra spargana
might distribute various parts of the body, giving a similar clinical
picture of proliferative sparganosis [39,40]. Basically, multiple worms
are not found in a single infection focus in non-proliferative sparga-
nosis, even when infection is disseminated. DNA sequencing of excised
spargana is absolutely required for the definite diagnosis of sparganosis.
4.2. Proliferative sparganosis and metacestode infections
In the diagnosis of cestode larva infections, some authors were
cautious enough not to assert their worms S. proliferum. For example, La
Chance et al. stated their cestode parasite was an undifferentiated
sparganum or tetrathyridium [41]. In 1981, Beaver and Rolon re-ex-
amined histological sections of larvae, which had been reported as
proliferative sparganosis from Japan and Taiwan [42,43]. They con-
cluded that larvae from these cases were of cyclophyllidean metaces-
tode and undifferentiated sparganum or tetrathyridium, respectively
5
Parasitology International 75 (2020) 102036
Internal proliferative sparganosis
(n = 10)
39 (11–63)
8/2
Asia (8), Americas (1), others (1)
1.5 years (6 months-5 years)
nodules/masses in connective tissues, or internal organs
0/10
4/10
30.0% (3/10)
[5]. Therefore, we removed these two cases from the list of proliferative
sparganosis in the present article.
It has been well known that, in addition to Echinococcus species,
some cyclophyllidean tapeworms exhibit asexual proliferation in hu-
mans at the metacestode stage. The racemose type cysticercus is a kind
of Taenia solium metacestode, which is characterized by cysts or mul-
tiloculated clusters lacking scolex, preferentially occurring in the sub-
arachnoid space or the cerebral ventricles. They resemble ‘bunches of
grapes’, and are responsible for the most severe clinical form of cysti-
cercosis [44–47].
Coenurosis is another zoonosis caused by infection with coenuri of
Taenia (Multiceps) multiceps, T. serialis, T. brauni, and T. glomerata.
Canids are the definitive hosts and a variety of mammals including
rodents, horses, cattle, and sheep serve as intermediate hosts. In his-
tology, coenuri can be recognized by the presence of many proto-
scolices in each coenurus [48,49].
Recently, another group of metacestode infection was reported from
Canada and the United States. The patients had numerous nodules in
the lungs and liver. Abdominal imaging in one of the patients revealed a
large heterogeneous central hepatic lesion with multiple satellite no-
dules. Liver biopsy revealed a degenerating three-layered membrane
without scolices. Tissue sections of one of the patients contained
hooklets. DNA sequencing of mitochondrial 12S rDNA, or cytochrome C
oxidase subunit 1 (Cox1), indicated that the patients were infected with
Versteria tapeworm [50,51]. The genus Versteria belongs to Family
Taeniiddae, whose definitive hosts are mustelids (carnivores of the fa-
mily Mustelidae) in North America [52]. Interestingly, these two and
another Versteria-infected patient described below, had immunological
disorders [50,51]. Disseminated Versteria infection could possibly be
another example of opportunistic helminthic infection [53].
4.3. Antibody test
Antibody test was performed with cestode antigens in three cases
(Case 8, 13 and 18). In Case 13, binding of patient serum to Spirometra
antigen was demonstrated in immuno-electrophoresis, although no
control antigens, such as cysticercus or cysticercoid antigens, were in-
cluded [6]. In Case 18, specific binding of patient serum was shown in
enzyme-linked immune-sorbent assay (ELISA) and immuno-chromato-
graphy test (ICT). Patient's serum was positive for the binding to Spir-
ometra antigen, but negative for cysticercus antigen [36]. In Case 8
(Venezuela), attempts were unsuccessful to demonstrate specific anti-
bodies against crude S. proliferum antigen in counter immuno-electro-
phoresis and double immunodiffusion test [27].
4.4. Molecular diagnosis
At the end of the 20th century, Professor Kojima at the Institute of
Medical Science, University of Tokyo, Japan, asked the Venezuelan
researchers, Drs. Oscar Noya and Belkisyole Alarcon de Noya, for a
portion of laboratory-maintained S. proliferum plerocercoids. They ac-
cepted his request, making it possible for Professor Kojima's group to
T. Kikuchi and H. Maruyama
Fig. 1. Skin eruption and isolated spargana from the Venezuelan proliferative
sparganosis patient (Case 8).
A: Dermal nodules on chest wall, B: S. proliferum plerocercoid squeezing out of
the skin, C: Isolated S. proliferum spargana.
(For Fig. 1A, similar photographs of the same patient appear in reference [24],
Copyright cleared)
clone and sequence genes of S. proliferum. They determined sequences
of genes for mitochondrial NADH dehydrogenase subunit 3 (NADH3),
mitochondrial tRNA, Cox1, and a portion of nuclear-coded succinate
dehydrogenase iron‑sulfur protein subunit (sdhB). Based on these se-
quences, they concluded that S. proliferum was a distinct species be-
longing to the order Pseudophyllidea [17,18]. Researchers have now
reached an agreement that S. proliferum is clearly an independent spe-
cies closely related to Spirometra tapeworms [54,55].
For the diagnostic purpose, nucleic acid amplification and DNA
sequencing was first employed in 2014. Genomic DNA was extracted
from surgically removed plerocercoid and sequences for Cox1, NADH3,
6
Parasitology International 75 (2020) 102036
Fig. 2. Tissue sections of S. proliferum spargana maintained in the laboratory
(H.E. staining).
Numerous cavities lined with tegument and abundant excretory system can be
observed. The shape and length of parenchymal cavities are variable either
empty or containing a homogeneous material, what Ijima designated as ‘nu-
tritive substance’. Bar; 200 μm (A), 100 μm (B).
and mitochondrial 12SrRNA genes were determined. This patient (Case
17) was diagnosed as having proliferative sparganosis, in spite of the
lack of apparent signs of sparganum proliferation [35], though there
remains uncertainty about the diagnosis of this case, as will be dis-
cussed below.
5. Experimental study
Experimental study with S. proliferum in the early days was con-
ducted by Dr. Usui, University of Tokyo, and Dr. Tashiro, Kyushu
University, with samples from Cases 3 and 6, respectively. They fed
dogs, cats, rabbits, and monkeys, with plerocercoids to see if any adult
worms could be obtained. They also transplanted plerocercoids to test
asexual proliferation in animals. Feeding experiments were un-
successful, but they confirmed asexual multiplication of the spargana in
animals [19,25,26].
More than half a century later, very similar infection experiments
were done in Venezuela, with plerocercoids isolated from Case 8. This
time, experimental mice were used for the inoculation, and survival and
multiplication of spargana were confirmed [16]. Since this experiment,
S. proliferum has been maintained in mice by serial passages, which was
transferred to Professor Kojima in Japan as described above.
T. Kikuchi and H. Maruyama Parasitology International 75 (2020) 102036

6. Discussion the molecular diagnosis of parasites. However, pitfalls are everywhere.

Having gone through all the accessible case reports, it is clear that
proliferative sparganosis is divided into two clinical types. One is cu-
taneous, and the other is internal. The cutaneous type starts with a skin
eruption caused by the dermal infection with spargana. Skin lesion
progresses to larger areas of the body, and various organs and body wall
could be eventually affected. Clinical symptoms of the patients in this
type are very similar to each other. In contrast, clinical picture of the
internal type is much more heterogeneous. Some cases show aggressive
infection in internal organs, while others show only restricted lesions,
e.g. single bone infection.
Morphological diagnosis of proliferative sparganosis may not be
very easy. In 1976, Connor et al. reported ‘mutated spargana’ in a 58-
year-old man in Pennsylvania, the United States [56]. Although this had
been cited repeatedly as a proliferative sparganosis case [7,41], Beaver
and Rolon (1981) re-examined tissue sections and concluded that the
patient was infected with cysticerci or cysticercoids [5]. The true
identity was clarified by molecular diagnosis decades later. In 2003,
Olson et al. successfully amplified and sequenced 18S rDNA V2 region
of DNA extracted from paraffin-embedded tissue sections. The sequence
indicated that the parasite was a Versteria tapeworm [57]. We re-
evaluated the reported sequence with more recent cestode sequence
data available in the public databases. We confirmed that the parasite
from the case of Connor et al. (1976) is certainly Versteria sp. belonging
to the order Cyclophyllidea (Fig. 3).
Because of the limitation of morphological examination, it is the
molecular techniques that should finally give the correct diagnosis.
DNA sequences of S. proliferum have been examined in three cases so
far. Sequences for NADH3, Cox1, mitochondrial tRNA, and a portion of
sdhB were determined in worms of Case 8, the Venezuelan case. Then,
sequences for NADH3, mitochondrial tRNA and the conserved region of
Cox1 of worms of Case 3, the second case in Japan, were shown to be
100% identical to those of Case 8 [17,18]. The most recent example is
Case 17 in 2014, in which DNA for NADH3 and the conserved region of
Cox1 were amplified and sequenced. According to the case report, they
had higher similarity to Case 8 sequences than to S. erinaceieuropaei
sequences [35].
DNA sequences might seem straightforward for the application to
For the diagnosis of proliferative sparganosis, available data are too
fragmentary and we do not know how S. proliferum is genetically di-
verse and how it relates to Spirometra species complex. In spite that
sequences of genes from Case 8 and Case 3 were reportedly identical,
the sequence for 18S rDNA of S. proliferum from a dog in Florida [14]
was 98.4% identical to Case 8 spargana (Kikuchi T. et al., unpublished
observation. Sequence data of the Venezuelan S. proliferum have been
deposited with links to BioProject accession number PRJDB8966/
PRJEB35374 in the DDBJ/EBI/NCBI BioProject database). S. proliferum
genomes should naturally have certain variations.
As for the difference between S. proliferum and Spirometra, Almeida
et al. (2016) showed that S. proliferum of Case 8 sit in the middle of
Spirometra species complex, based on the sequences of conserved region
of Cox1 [58]. At least three clades were identified in Spirometra species
complex, two South American and one Asian, and S. proliferum was
within one of the two South American clades. In the present review, we
included Case 17 in the cutaneous proliferative sparganosis as described
above. However, this diagnosis remains uncertain. Because the case
report does not provide the sequence data of the worm, nor the ac-
cession number of the Spirometra sequences they used for the compar-
ison. If the authors compared sequences of their isolate with Asian se-
quences, the difference they observed could be the difference between
the South American and the Asian Spirometra isolates.
What makes it worse is the difficulty in constructing a phylogenetic
tree. While mitochondrial Cox1sequences indicate S. proliferum in the
middle of the Spirometra clades [58], 18S rDNA suggests S. proliferum
stands outside the Spirometra clades [59]. This inconsistency is possibly
due to the difficulties in constructing a phylogenetic tree with mi-
tochondrial sequences [60]. Unfortunately, most of the submitted se-
quences are of mitochondria and the number of genes sequenced are
small. From the next proliferative sparganosis-suspected case on, larger
sequence data, not only mitochondrial but nuclear as well, are defi-
nitely required. Recent techniques, such as next generation sequencing
combined with whole genome amplification [61,62] should help us
obtain larger amount of data.
One of the major riddles about S. proliferum is its life cycle. Nobody
has ever discovered the definitive host for this enigmatic tapeworm.
With regard to this point, it is puzzling that dogs and cats cannot be the

Taenia saginata (AB731616.1) Fig. 3. Phylogenetic tree of a metacestode from Pennsylvanian case.
1
Taenia asiatica (AB731617.1) DNA sequence of 18S rDNA V2 region were compared among cyclo-
1
phyllidean and pseudophyllidean tapeworms. Phylogenetic tree
Taenia solium (AB731615.1)
0.95
clearly indicates that the patient reported by Connor et al. (1976) was
Echinococcus multilocularis (AB731634.1)
1 infected with Versteria metacestode, but not S. proliferum. 18S rDNA
0.15 Echinococcus granulosus (AB731639.1) V2 sequence for S. proliferum was obtained in our laboratory (data
AY193876.1 (Olson et. al) deposited with links to BioProject accession number PRJDB8966/
0.99
0.99
Versteria mustelae (AB731633.1) PRJEB35374 in the DDBJ/EBI/NCBI BioProject database).
Hydatigera krepkogorski (AB731632.1)
0.84 1
Hydatigera taeniaeformis (AB731628.1)

0.99
Anonchotaenia macrocephala (KF685935.1)
1
Anonchotaenia cf. brasiliensis (KF685938.1)
0.11
Avitellina centripunctata (JQ609343.1)
Parorchites zederi (KF705621.1)
1
0.98
Raillietina echinobothrida (AY382316.1)
Hymenolepis diminuta (AF124475.1)
1
Hymenolepis microstoma (AJ287525.1)
Schistocephalus solidus (AF124460.1)
0.94
Diphyllobothrium latum (DQ181941.1)
1
Spirometra erinaceieuropaei (D64072.1)
0.51
0.98
Sparganum proliferum
Mesocestoides corti (AF286984.1)

0.03

7
T. Kikuchi and H. Maruyama
definitive hosts. Feeding experiments failed [19,25,26], and dogs and
cats with proliferative sparganosis have been reported [12–15]. Con-
sidering that carnivores are definitive hosts of all known Spirometra
tapeworms, one has to assume a critical change in S. proliferum adap-
tation, that has resulted in the major host switching or even the loss of
the definitive host. Whole genome sequencing followed by gene anno-
tation and protein family analysis should possibly reveal what has
happened in this tapeworm's genome during the course of evolution.
From the Institute of Medical Science, University of Tokyo, the
Venezuelan S. proliferum has been transferred to Dr. Kuramochi at the
National Museum of Nature and Science, Japan, and further to
Professors Kanuka and Ishiwata at the Department of Tropical
Medicine, Jikei University School of Medicine, Tokyo. Now, the
Venezuelan strain of S. proliferum is maintained in biosafety level two
animal facilities in these research institutions. More than one hundred
years ago, Professor Ijima wrote in his report;
Now a remarkable fact about the present Plerocercoid is that it is
capable, at a certain advanced stage of its intra capsular life, of pro-
liferating by a process of budding. . . . So far as my knowledge goes, a
budding Plerocercus or Plerocercoid seems not to have been known
before, at least not with certainty.
We are ready to start experiments to elucidate how and why these
strange cestode larvae proliferate to cause a devastating disease, with
advanced technologies in modern molecular biology.
7. Conclusions
1. Proliferative sparganosis is clinically divided into cutaneous and
internal types.
2. DNA sequencing of parasites is definitely required for the diagnosis
of proliferative sparganosis.
3. Genome databases should be much more enriched with correctly
identified cestodes for the precise diagnosis of not only proliferative
sparganosis, but non-proliferative sparganosis and other related
metacestode infections as well.
Acknowledgement
We thank Professor Yukifumi Nawa, Khon Kaen University,
Thailand, and Professor Masahide Yoshikawa, Department of Pathogen,
Infection and Immunity, Nara Medical University, Japan, for literature
acquisition and discussion. We also thank Professor Tamaki
Okabayashi, Dr. Sangchul Hyun, and Dr. Amy Hombu for the transla-
tion of Thai, Korean, Chinese case reports into English, respectively. We
appreciate Professor Somei Kojima much for supplying photographs of
the Venezuelan patient, and Professor Kenji Ishiwata, Department of
Tropical Medicine, Jikei University School of Medicine, Japan, for
providing stained tissue sections of laboratory-maintained S. proliferumt
for photomicrographs.
This study was supported by the Japan Society for the Promotion of
Science (KAKEN 26460510), and Japan Agency for Medical Research
and Development (JP19fk0108095h0001).
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