sevier.com/locate/parint

Parasitology International

Case report

A case of intramuscular cysticercosis diagnosed definitively by

mitochondrial DNA analysis of extremely calcified cysts

Hiroshi Yamasaki a,*, Teruaki Nagase b, Yoshiro Kiyoshige c, Mamoru Suzuki d, Kazuhiro Nakaya e,

Yukio Itoh f, Yasuhito Sako a, Minoru Nakao a, Akira Ito a

a Department of Parasitology, Asahikawa Medical College, Asahikawa 078-8510, Japanb Department of Otolaryngology, Yamagata Prefectural Shinjo Hospital, Shinjo, 996-0024, Japanc Department of Orthopaedic Surgery, Saiseikai Yamagata Hospital, Yamagata, 990-8545, Japan

d Gunma University, Maebashi 371-8511, Japane Animal Laboratory for Medical Research, Asahikawa Medical College, Asahikawa, 078-8510, Japanf Department of Forensic Medicine, Juntendo University School of Medicine, Tokyo, 113-8421, Japan

Received 25 October 2005; received in revised form 15 November 2005; accepted 26 November 2005

Available online 10 January 2006

Abstract

A case of obsolete intramuscular cysticercosis diagnosed definitively by mitochondrial DNA analysis of extremely calcified cysts was reported.

X-ray and computed tomography findings highly suggested cysticercosis due to Taenia solium; however, no direct evidence of cysticercosis was

obtained through serological or histopathological examinations. Mitochondrial DNA analysis of a histopathological specimen confirmed the

causative agent to be the Asian genotype of T. solium.

D 2005 Elsevier Ireland Ltd. All rights reserved.

An 83-year-old Japanese man developed pharyngeal pain

beginning 8 October 2003 after falling downstairs on 29

September 2003. He was referred to the Saiseikai Yamagata

Hospital, Yamagata, Japan, on 11 October 2003 with

complaints of dysphagia, dehydration and lumbago, and

diagnosed as having a lumbar compression fracture by the

Department of Orthopaedic Surgery and also acute subman-

dibular sialadenitis by the Department of Otolaryngology. On

admission, systemic X-ray examination revealed the existence

of numerous elliptic and calcified cysts measuring approxi-

mately 12�5 mm scattered in the musculatures throughout

the body, particularly in the lower trunk and extremities (Fig.

1A). Interestingly, no cysts were found in the brain. Some

subcutaneous cysts were tangible; however, the patient had no

pain. Computed tomography (CT) images also showed

numerous calcified cysts with high-density areas scattered in

the skeletal muscles of the chest, buttocks and thighs, and

1383-5769/$ - see front matter D 2005 Elsevier Ireland Ltd. All rights reserved.

doi:10.1016/j.parint.2005.11.057

* Corresponding author. Tel.: +81 166 68 2421; fax: +81 166 68 2429.

E-mail address: hyamasak@asahikawa-med.ac.jp (H. Yamasaki).

cysticercosis was diagnosed based on these imaging findings

(Fig. 1B).

A blood sample and calcified cysts were collected from the

patient. DNA examination of the excised cysts was performed

after obtaining the patient’s approval. No antibodies were

detected by immunoblot using a recombinant Taenia solium

antigen (data not shown). The calcified cysts excised from the

gluteal region were extremely solid (Fig. 1C) and the

characteristic morphology of T. solium cysticercus was not

confirmed due to the degeneration and marked calcification of

the tissue (Fig. 1D).

In order to confirm whether the calcified cysts were really

derived from T. solium or not, DNA analysis was performed.

The calcified cysts were fixed with formalin, decalcified using

the Plank-Rychlo method, dehydrated and embedded in

paraffin. Three cysts were sectioned individually with a 5-Amthickness and DNA was extracted from four sections per

sample using a DNA Isolator PS kit (Wako Pure Chemicals,

Osaka, Japan). T. solium DNA prepared previously [1] and

human DNA prepared from peripheral blood using a QIAamp

DNA Blood Mini Kit (Qiagen, Hilden, Germany) were used as

positive controls. As target genes for T. solium, cytochrome c

55 (2006) 127 – 130

www.el

Fig. 1. Imaging and histopathological findings. (A) A plain X-ray findings

showing numerous calcified cysts scattered in the soft tissue at level of

abdomen and buttocks. The shape of the calcified cysts varies from elongated,

plump oval or elliptic configurations. (B) A CT scan images showing

calcifications with high density-areas at thigh level. (C) The cysts presenting

with a rice grain-shape, measuring approximately 10–12�4–5 mm. (D)

Cystic tissue stained with hematoxylin–eosin. No characteristic labyrinth-like

structure was observed. The tissue was encapsulated with an extremely solid

shell-like structure (an arrow in the inlet). A bar in the inlet=1 mm.

H. Yamasaki et al. / Parasitology International 55 (2006) 127–130128

oxidase subunit 1 gene (cox1) and cytochrome b gene (cob)

were amplified. Primers used for cox1 (AC number AB066485)

were as follows: F4 (5V-ATATTTACTTTAGATCATAAGCG-3V), R5 (5V-TCAAAAAACGCAGAACTAAATTTACGA-3V),F6 [3] and R6 [3]. For cob amplification, F2 [2], R2 [2], Cytb/F

[4] and Cytb/R [4] were used. For the human DNA markers,

microsatellite markers of TH01 locus in the tyrosine hydrox-

ylase gene [5] and D1S80 locus [6] were amplified using

primers TH01/F (5V-GTGGGCTGAAAAGCTCCCGATTAT-3V, AC number D00269) and TH01/R (5V-ATTCAAAGGG-TATCTGGGCTCTGG - 3 V) a n d MCT11 8 / F ( 5 V-GAAACTGGCCTCCAAACACTGCCCGCCG-3V, AC num-

ber D28507) and MCT118/R (5V-GTCTTGTTGGAGATG-CACGTGCCCCTTGC-3V), respectively. DNA sequencing

was performed on an ABI PRISM 310 Genetic Analyzer.

The results of PCR are shown in Fig. 2. In the PCR

using F6/R6 and F4/R5 primers for cox1, 138-bp and 635-

bp fragments were successfully amplified from all cyst

samples, respectively (panels A and B). As in the case of

cob, 126-bp fragments (panel C) were amplified using F2/

R2; however, amplification of 1.3-kb products using Cytb/F

and Cytb/R primers was unstable (data not shown). With

regards to the detection of human DNA markers, 180–195-

bp fragments of the TH01 locus were successfully

amplified (panel D), but not 369–801-bp fragments of

the D1S80 locus, even in the nested PCR (data not shown).

The reason could be due to the fragmentation of

chromosomal DNA by formalin-fixation. It has been

reported that microsatellite markers were detected, but not

minisatellite DNA markers, when formalin-fixed specimens

were tested [7].

Subsequently, it was demonstrated that the calcified cysts

were derived from T. solium cysticercus and the causative T.

solium was the Asian genotype [1,4] by DNA sequencing of

PCR-amplified cox1 and cob fragments (data not shown). The

present case was originally diagnosed as being an obsolete

systemic cysticercosis based on imaging findings and a history

of living in China [8]. The patient stated that he had frequently

eaten a variety of uncooked meat approximately 60 years ago

in north China where cysticercosis/taeniasis is endemic until

now.

In general, a diagnosis of subcutaneous or intramuscular

cysticercosis is performed by surgical excision or biopsy of the

cystic lesions [9–12], imaging diagnosis [13], sonography

[14–16] or fine needle aspiration cytology [17–21]. We report

here the first case of disseminated intramuscular cysticercosis

confirmed definitively by mitochondrial DNA analysis of

extremely calcified cysts. We also reported two cases of

solitary neurocysticercosis confirmed by mitochondrial DNA

analysis using histopathological specimens [2,22]. In the first

case [22], the characteristic morphology of T. solium cysticer-

cus was observed, but not in the second case [2]. As

demonstrated here, mitochondrial DNA diagnosis using histo-

pathological specimens is a powerful tool for a definitive

diagnosis of cysticercosis cases where the cystic lesions have

degenerated and/or calcified and the characteristic morphology

is not confirmatory. The DNA diagnosis using such histopath-

ological specimens would be also applicable for identification

of other parasites as extensive DNA sequence data are

currently available.

Acknowledgments

This study was supported in part by a Grant-in-Aid for

Scientific Research from the Japan Society for Promotion of

0.7

0.50.4

0.3

kb

1.01.5

0.1

0.2

0.5

0.3

kb

1.01.5

138 bp

1st PCR nested PCR

0.1

0.2

0.5

0.3

kb

1.01.5

126 bp

1st PCR nested PCR 1st PCR nested PCR

0.1

0.7

0.2

0.50.4

0.3

kb

1.0

1.5

190 bp180 bp

*

635 bp

1st PCR nested PCR

*

Blank

T. soli

um

Sample

1

Sample

2

Sample

3

Blank

Blank

T. soli

um

Sample

1

Sample

2

Sample

3

Sample

1

Sample

2

Sample

3

Sample

1

Sample

2

Sample

3

M

Blank

Blank

Blank

T. soli

um

Human

DNA

Sample

1

Sample

2

Sample

3

Sample

1

Sample

2

Sample

3

Sample

1

Sample

2

Sample

3

Sample

1

Sample

2

Sample

3

M M

M

A B

C D

Fig. 2. PCR-amplification of T. solium and human DNA markers. Panel A, 138-bp cox1 fragments amplified using F6/R6 primers; panel B, 635-bp cox1 fragments

amplified using F4/R5 primers; panel C, 126-bp cob products amplified using F2/R2 primers; panel D, 180–195-bp TH01 locus. PCR for T. solium genes was

performed according to a previously described protocol [1]. For amplification of the human TH01 locus, the PCR protocol consisted of 30 cycles at 94 -C for 1 min,

at 60 -C for 30 s and at 72 -C for 30 s. Ex Taq DNA polymerase Hot Start version (TaKaRa Bio Inc., Shiga, Japan) was used. For the nested PCR, 2 Al of the firstPCR products were used as template DNA samples. Blank, PCR mixture without any template DNA. Panels A, C and D show the electrophoretic profiles using 4–

20% gradient polyacrylamide gels and panel B shows electrophoresis on a 1% agarose gel. M indicates 100-bp ladder DNA size markers (Promega). Asterisks

indicate sequenced samples.

H. Yamasaki et al. / Parasitology International 55 (2006) 127–130 129

Science (grants 14256001 and 17256002) to A.I. and a

Research Grant from the Ohyama Health Foundation to H.Y.

References

[1] Yamasaki H, Nakao M, Sako Y, Nakaya K, Sato MO, Mamuti W, et al.

DNA differential diagnosis of human taeniid cestodes by base excision

sequence scanning thymine-base reader analysis with mitochondrial

genes. J Clin Microbiol 2002;40:3818–21.

[2] Yamasaki H, Nakao M, Sako Y, Nakaya K, Ito A. Molecular

identification of Taenia solium cysticercus genotype in the histopath-

ological specimens. Southeast Asian J Trop Med Public Health 2005;

36(Suppl 4):131–4.

[3] Yamasaki H, Allan JC, Sato MO, Nakao M, Sako Y, Nakaya K, et al.

DNA differential diagnosis of taeniasis and cysticercosis by multiplex

PCR. J Clin Microbiol 2004;42:548–53.

[4] Nakao M, Okamoto M, Sako Y, Yamasaki H, Nakaya K, Ito A. A

phylogenetic hypothesis for the distribution of two genotypes of the pig

tapeworm Taenia solium worldwide. Parasitology 2002;124:657–62.

[5] Edwards A, Civitello A, Hammond HA, Caskey CT. DNA typing and

genetic mapping with trimeric and tetrameric tandem repeats. Am J Hum

Genet 1991;49:746–56.

[6] Kasai K, Nakamura Y, White R. Amplification of a variable number of

tandem repeats (VNTR) locus (pMCT118) by the polymerase chain

reaction (PCR) and its application to forensic science. J Forensic Sci

1990;35:1196–200.

[7] Kobayashi R, Itoh Y. Analysis of DNA extracted from formalin-fixed

and paraffin-embedded tissues by polymerase chain reaction (PCR)

and its application in a paternity case. Curr Top Forensic Sci 1997;1:

256–9.

[8] Nagase T, Kiyoshige Y, Suzuki M. A case of obsolete systemic

cysticercosis cellulose. Clin Parasitol 2004;15:24–6 (in Japanese).

[9] Wortman P. Subcutaneous cysticercosis. J Am Acad Dermatol 1991;25:

409–14.

[10] Matsushima H, Hatamochi A, Shinkai H, Shimizu M, Hatsushika R. A

case of subcutaneous cysticercosis. J Dermatol 1998;25:438–42.

[11] Miura H, Itoh Y, Kozuka T. A case of subcutaneous cysticercosis

(Cysticercus cellulosae cutis). J Am Acad Dermatol 2000;43:538–40.

[12] Uthida-Tanaka AM, Sampaio MC, Velho PE, Damasceno BP, Cintra ML,

de Moraes AM, et al. Subcutaneous and cerebral cysticercosis. J Am Acad

Dermatol 2004;50:14–7.

[13] Yamashita P, Kelsey J, Henderson SO. Subcutaneous cysticercosis. J

Emerg Med 1998;16:583–6.

[14] Mani NBS, Kalra N, Jain M, Sidhu R. Sonographic diagnosis of a solitary

intramuscular cysticercosis cyst. J Clin Ultrasound 2001;29:472–5.

[15] Asrani A, Morani A. Primary sonographic diagnosis of disseminated

muscular cysticercosis. J Ultrasound Med 2004;23:1245–8.

[16] Vijayaraghavan SB. Sonographic appearances in cysticercosis. J Ultra-

sound Med 2004;23:423–7.

H. Yamasaki et al. / Parasitology International 55 (2006) 127–130130

[17] Verma K, Kapila K. Fine needle aspiration diagnosis of cysticercosis in

soft tissue swellings. Acta Cytol 1989;33:663–6.

[18] Vuong PN. Fine needle aspiration cytology of subcutaneous cysticercosis

of the breast. Case report and pathologic discussion. Acta Cytol 1989;33:

659–62.

[19] Rajwanshi A, Radhika S, Das A, Jayaram N, Banerjee CK. Fine needle

aspiration cytology in the diagnosis of cysticercosis presenting as palpable

nodules. Diagn Cytopathol 1991;9:517–9.

[20] Arora VK, Gupta K, Singh N, Bhatia A. Cytomorphologic panorama of

cysticercosis on fine needle aspiration. A review of 298 cases. Acta Cytol

1994;38:377–80.

[21] Kapila K, Sahai K, Verma K. Semi-quantitative analysis of soft-tissue

reactions in fine needle aspirates from tissue cysticercosis. Cytopathology

2003;14:208–11.

[22] Yamasaki H, Matsunaga S, Yamamura K, Chang CC, Kawamura S, Sako

Y, et al. Solitary neurocysticercosis case caused by Asian genotype of

Taenia solium confirmed by mitochondrial DNA analysis. J Clin

Microbiol 2004;42:3891–3.