2006-mitochondrial dna diagnosis for taeniasis and cysticercosis

8/13/2019 2006-Mitochondrial DNA Diagnosis for Taeniasis and Cysticercosis

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Mitochondrial DNA diagnosis for taeniasis and cysticercosis

Hiroshi Yamasaki a,*, Minoru Nakao a, Yasuhito Sako a, Kazuhiro Nakaya b,Marcello Otake Sato a,c, Akira Ito a

a Department of Parasitology, Asahikawa Medical College, Asahikawa 078-8510, Japanb Animal Laboratory for Medical Research, Asahikawa Medical College, Asahikawa 078-8510, Japan

c Laboratorio de Parasitologia, Escola de Medicina Veterinaria e Zootechnica, Universidade Federal do Tocantins, Araguana-TO, 77804-970, Brazil

Available online 15 December 2005

Abstract

Molecular diagnosis for taeniasis and cysticercosis in humans on the basis of mitochondrial DNA analysis was reviewed. Development and

application of three different methods, including restriction fragment length polymorphism analysis, base excision sequence scanning thymine-

base analysis and multiplex PCR, were described. Moreover, molecular diagnosis of cysticerci found in specimens submitted for histopathology

and the molecular detection of taeniasis using copro-DNA were discussed.

D 2005 Elsevier Ireland Ltd. All rights reserved.

Keywords: Taeniasis; Cysticercosis; Mitochondrial DNA diagnosis; PCR-RFLP; BESS T-base analysis; Multiplex PCR; Biopsied specimen

1. Introduction

Taenia solium, Taenia saginata, and Taenia asiatica are

cestode parasites causing taeniasis in humans. T. solium also

causes cysticercosis in humans; neurocysticercosis is serious

disease characterized by neurologic symptoms including epi-

leptic seizures. BothT. solium and T. saginata are distributed

worldwide, but distribution ofT. asiatica is restricted to Asian

regions. In Asia where these cestodes are distributed sympatri-

cally, differentiation ofT. saginata and T. asiaticais frequently

confused as a result of morphological similarities. Theoretically,

it is possible to differentiate the proglottids of human Taenia

parasites on the basis of morphology, however, it is difficult in

practice. Until recently the only reliable technique for differen-

tiation of taeniid eggs is by DNA-based methods.

In order to improve methods for identifying taeniid

cestodes, various molecular approaches have been developed,

including the use of DNA probes [16], polymerase chain

reaction (PCR) coupled to restriction fragment length poly-

morphism (RFLP) [712], single-strand conformation poly-

morphism (SSCP) [13], PCR-amplified DNA sequences

[7,8,10,14], and random amplified polymorphic DNA

(RAPD)-PCR[1416]. Each of these techniques has advan-

tages and disadvantages, e.g., the use of DNA probes, PCR-RFLP and SSCP are relatively time-consuming; however, PCR

using species-specific primers provides rapid and sensitive and

reliable diagnostic results [17,18]. Most of these studies have

been focused on the differentiation of T. solium from

T. saginata and intraspecific genetic polymorphism.

Recently, mitochondrial DNA analysis ofT. solium revealed

the presence of two distinct Asian and American/African geno-

types[19,20].Therefore, a comprehensive differential diagnosis

based on mitochondrial DNA forT. saginata, T. asiaticaand two

genotypes ofT. solium parasites has been established[2123].

Differential diagnosis of taeniasis can now be achieved using

copro-DNA [23]. In this review, for the comprehensive

differential diagnosis of human taeniid cestodes, PCR-RFLP,

base excision sequence scanning thymine-base (BESS T-base)

reader analysis and multiplex PCR are discussed. The role of

molecular diagnosis of cysticerci found in histopathological

specimens from patients and the detection method of taeniid

DNA using feces from tapeworm carriers are also discussed.

2. Molecular identification of proglottids, cysticerci and eggs

For molecular identification of taeniid eggs, cysticerci and

proglottids, the parasite materials should be stored in ethanol

1383-5769/$ - see front matterD

2005 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.parint.2005.11.013

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

E-mail address: [email protected] (H. Yamasaki).

Parasitology International 55 (2006) S81 S85

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(>80%) after collection. DNA analysis of formalin-fixed

samples for histopathology is also possible, however it may

be difficult to lyse the parasite tissue and to amplify more than

1-kb DNA markers, because of the fragmentation of the DNA

as mentioned in Section 3. For preparation of DNA from

parasite materials, the use of a commercially available kit (e.g.,

DNeasy Tissue Kit, Qiagen) is convenient.

2.1. Polymerase chain reaction coupled to restriction fragment

length polymorphism (PCR-RFLP)

This is a simple method in which a PCR-amplified target

DNA is digested with particular restriction enzymes and used

to compare the subsequent fragment patterns. The method has

been applied for differentiation of T. saginata and T. solium

[611]. In addition, Yamasaki and others [22] reported

comprehensive PCR-RFLP analysis including T. asiatica and

two genotypes of T. solium. On the basis of complete

nucleotide sequences of cytochrome c oxidase subunit1 (cox1) and cytochrome b genes (cob) from human taeniid

cestodes, restriction enzyme sites unique for T. saginata,

T. asiatica, and two genotypes ofT. solium are found. In cases

of cox1, we use TaqI, BamHI, NcoI and DdeI unique for

T. saginata, T. asiatica and Asian and American/African

genotypes of T. solium, respectively. For example, if PCR-

amplifiedcox1is digested withBamHI, the taeniid cestode can

be identified as T. asiatica [22].As in case ofcob , differential

RFLP profiles are provided when Sty I forT. saginata, Vsp I

for T. asiatica, SnaBI and Ssp I for Asian and American/

African genotypes ofT. solium, respectively, are used [22].

2.2. Base excision sequence scanning thymine-base

(BESS T-base) reader analysis

This method was originally developed for the identification

of genetic variations at sites involving thymine bases (T) [24]

and then was applied to the identification of human taeniid

cestodes[21,22]. The principle is as follows: a forward primer

should be designed to include diagnostic nucleotides involving

T (e.g., positions 153, 189, 195, 723, 867, 1065 and 1608 in

cox1) and labeled with fluorescent dye. The BESS T-Base

Reader Kit is commercially available. During PCR amplifica-

tion of a target gene, limiting amounts of dUTP are randomly

incorporated into a PCR product at the T sites. Subsequently,N-glycosylase treatment of the PCR product results in removal

of uracil, creating an abasic site at the location of dUTP

incorporation. Furthermore, endonuclease IV treatment cleaves

the phosphodiester bonds at the abasic sites, generating a DNA

ladder virtually identical to T-sequencing ladder. The sample is

electrophoresed in a 6% polyacrylamide gel containing 8 M

urea, and data obtained are analyzed using GeneScan.

Subsequently, the appearance or disappearance of T-base peaks

serves as diagnostic markers for differentiation of human

taeniid cestodes.

InFig. 1, BESS T-base analysis data with a cox1 fragment

are shown. Most T bases are well-conserved among the taeniid

species, however, several nucleotides are species- andT. solium

genotype-specific (e.g., positions 153, 174, 189 and 195). Anucleotide at position 153 in T. asiatica is T, but nucleotides at

the same position are guanine inT. saginata andT. solium. In

BESS T-base analysis, thus a T-base peak appears at the

position in T. asiatica, but no T-base peaks appear at the

positions in T. saginata and T. solium. In T. saginata,

nucleotides at positions 174 and 189 are T, so that diagnostic

T-base peaks appear at both positions. In the differentiation of

two genotypes ofT. solium, if T-base peaks appear at positions

189 and 195, the taeniid cestode can be identified as the

American/African genotype of T. solium. If a T-base peak

appears at position 195, the T. solium parasite is the Asian

genotype. Comparison of T-base peak profiles allows differ-

entiation of human taeniid cestodes without the need for DNA

sequencing. In BESS T-base analysis, since 100200-bp PCR

products containing diagnostic positions are more convenient

for analysis, the method will be applicable for the accurate

identification of formalin-fixed parasite materials in which

DNA is fragmented.

2.3. Multiplex PCR

Compared to PCR-RFLP and BESS T-base analysis,

multiplex PCR is a simpler and more rapid method. It amplifies

particular genes using multiple primer pairs in a single tube.

Yamasaki and others [23] established multiplex PCR for acomprehensive identification of human taeniid cestodes. For

this purpose, forward primers were designed to be amplified as

PCR products of different sizes unique for T. saginata,

T. asiatica, and two genotypes ofT. solium. Fig. 2 shows a

typical multiplex PCR data with cox1. The diagnostic products

with molecular sizes of 827, 269, 720, and 984 bp are

successfully amplified in T. saginata, T. asiatica, and the

American/African and Asian genotypes of T. solium, respec-

tively. When a mixture ofT. saginata andT. asiatica eggs was

tested by multiplex PCR, two products with molecular sizes of

827 and 269 bp were amplified. The successful amplification

by multiplex PCR depends on the ratio of the forward and

reverse primers and sequences of primers. The primer used for

T. saginata

T. asiatica

T. soliumAmerican

T. soliumAfrican

T. soliumAsian

153 174 189 195

T G T G A T T T T T T T T T G A T T A C T A A T C A T G G A A T

T G T T A T T T T T TT T T G A T T A C TAAC C A T G G A A T

T G T G A T T T A T T T T T G A T T A C T A A T C A T G G T A T

T G T G A T T T A T T T T T G A T T A C T A A T C AT G G T A T

T G T G A T T T A T T T T T G A T T A C T A A C CA T G G T AT

153 174 189 195

T G T G A T T T T T T T T T G A T T A C T A A T C A T G G A A T

T G T T A T T T T T TT T T G A T T A C TAAC C A T G G A A T

T G T G A T T T A T T T T T G A T T A C T A A T C A T G G T A T

T G T G A T T T A T T T T T G A T T A C T A A T C AT G G T A T

T G T G A T T T A T T T T T G A T T A C T A A C CA T G G T AT

Fig. 1. BESS T-base reader analysis with cox1 for human taeniid parasites. T-

base peak profiles from T. saginata, T. asiatica, and American/African and

Asian genotypes ofT. solium are illustrated. Arrows indicate diagnostic T-base

peaks (modified from [21]).

H. Yamasaki et al. / Parasitology International 55 (2006) S81 S85S82


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T. asiatica has been recently modified to reduce the amplifi-

cation of additional products [25].

3. Molecular diagnosis of cysticerci found in

histopathological specimens

The clinical diagnosis of cysticercosis in humans is

performed by imaging diagnosis and serology. Histopatholog-

ical examination of biopsy specimens is also a useful method

for confirmation of the causative cysticercus. However, it is not

always easy to make a definitive diagnosis of the cysticercus as

a result of the preparation of tissue sections and the degree of

degeneration and/or calcification of the tissue. Prior to 2004

there was only a report on the DNA diagnosis of a cysticercus

found in a black bear [26]. More recently, two cases of

neurocysticercosis in humans were confirmed by mitochondrial

DNA analysis of biopsied lesions[27,28].DNA was extracted

from 45 paraffin sections with a 5-Am thickness using 0.02N

NaOH containing proteinase K solution or a commercially

available kit. In the former case [27], a cysticercus character-

ized by suckers and spiral canals was observed. Although the

specimen was fixed with formalin, 1.8-kb and 984-bp cox1

fragments were successfully amplified by conventional PCR

using a primer set[20,21]and DNA sequencing confirmed as

T. solium Asian genotype. In the latter case [28], histopatho-

logical findings were not confirmatory because of the

degeneration of the tissue. However, smaller sizes of cox1andcob fragments (100400 bp) were successfully amplified,

demonstrating the causative agent was the Asian genotype of

T. solium by DNA sequencing. The authors have also reported

a case of systemic intramuscular cysticercosis confirmed by

mitochondrial DNA analysis of extremely calcified cysts [29].

Although the cystic lesions were calcified, had been fixed in

formalin and decalcified for histopathology, a 984-bp cox1

fragment was successfully amplified by conventional PCR,

confirming that the calcified cyst was derived from T. solium

Asian genotype by DNA sequencing of the product.

The amplification of DNA markers from histopathological

specimens seems to be dependent on the preservation and/or

fixation condition of the parasite materials used, probably as a

result of the fragmentation of DNA. Thus, the amplification by

multiplex PCR seems to be difficult and subsequently, DNAsequencing of the PCR product is indispensable for further

confirmation[27 29].

4. Importance of molecular identification of biopsy

specimens

The majority of Taenia species causing cysticercosis in

humans is known to be T. solium. However, there are many

other potentially zoonotic taeniid species including T. taeniae-

formis, T. pisiformis, T. hydatigena, T. serialis, T. multiceps

and Echinococcus spp. [30] and several of these are highly

zoonotic. Taeniid cestodes might infect humans by accidental

ingestion of eggs and be diagnosed or misdiagnosed as

Cysticercus spp., Cysticercus racemosus, Coenurus spp.

Cysticercosis/coenurosis in humans caused by the infection

of such zoonotic species has been reviewed[31]. Cysticercosis

due to Taenia crassiceps has been reported in patients with

HIV-AIDS[3235]. Most recently, a racemose-type cysticer-

cus has been diagnosed as T. solium based on mitochondrial

cox1 and NADH dehydrogenase subunit 1 gene analysis [36].

There were case reports suggesting cysticercosis due to

T. saginata in humans [3739]. Ito [40] speculated on the

possibility of human cysticercosis due to T. asiatica, since both

T. asiatica and T. solium require pigs as the intermediate host.

However, based on the molecular studies, Ito and others [41]now conclude that cysticercosis ofT. asiatica in humans does

not occur similar to its sister species T. saginata.

More recent work by Nakaya and others[42]has shown that

the number of hooks of T. solium developed in non-obese

diabetic/Shi-severe combined immunodeficiency (NOD/Shi-

scid) mice was highly variable from null to 28. Margono and

others[43]also reported variable formation of hookets in 135-

day-oldT. soliummetacestodes, suggesting that the presence of

hooklets is not always an adequate marker for identification of

the species.

Morphological identification of taeniid species is problem-

atic. Data for molecular phylogeny of taeniid cestodes

[20,4446] are expected to be highly informative and useful

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T. saginata T. asiatica T. soliumAmerican/African genotype

T. soliumAsian genotype

Fig. 2. Differential diagnosis of human taeniid cestodes by multiplex PCR. Cox1-fragments with molecular sizes of 827, 269, 720 and 984 bp are amplified in

T. saginata, T. asiatica, American/African and Asian genotypes of T. solium. An asterisk indicates a mixture sample of T. saginata and T. asiatica eggs

(modified from[23]).

H. Yamasaki et al. / Parasitology International 55 (2006) S81 S85 S83


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for molecular identification of taeniid species[26]. In order to

confirm or dispel the notion that human cysticercosis may

sometimes be caused by T. asiatica or T. saginata, or to

provide evidence-based confirmed diagnosis, it is essential to

obtain specimens for molecular identification [47,48]. It is

important to stress the importance of fixing biopsy specimens

in ethanol (or even in formalin for a short time) for adequatepreparation of paraffin sections of such specimens in order to

obtain crucial DNA confirmation[2729],since approximate-

ly 50% of solitary cysticercosis cases [4952] and inactive

cysticercosis with calcified lesions are expected to be sero-

negative[29].

5. Detection of taeniasis using copro-DNA

Taeniasis has been diagnosed based on the coproscopic

examination and morphologic characteristics of tapeworm

proglottids. To date, only few molecular methods for diagnosis

of taeniasis have been reported [12,23,53]. Nunes and others[12] performed PCR to detect taeniid DNA in stool samples,

which had been artificially spiked with T. saginata eggs and

reported a detection limit of 137 eggs (corresponding to 1096

pg DNA). Nunes and others[53]have reported PCR-RFLP to

differentiateT. solium and T. saginata egg in fecal samples of

taeniasis patients. The different DraI-RFLP profiles permit

differentiation of the two taeniid species. The authors reported

that the lower detection limit of the PCR-RFLP was 34 eggs in

2 g stool sediment. Furthermore, a 521-bp cox1 fragment was

detected in 8 out of 12 tapeworm carriers providing diagnostic

sensitivity of 66.6% (5 for T. saginata and 3 forT. solium),

which was higher than previous method with HDP2-PCR

repetitive DNA amplification [12]. Yamasaki and others [23]

have established multiplex PCR for differential diagnosis of

T. saginata and two genotypes ofT. solium carriers using fecal

samples. A detection limit of taeniid DNA was 5 eggs/g feces,

but more reliable results were provided at more than 50 eggs/g.

Multiplex PCR using copro-DNA prepared from fecal samples

of tapeworm carriers yielded 720- and 984-bp cox1 fragments

in T. solium carriers from Guatemala (12 out of 14 samples)

and Indonesia (4 out of 9 samples), respectively [23]. In

T. saginata carriers, 827-bp cox1 fragments were successfully

amplified at a sensitivity of 100% (5 out of 5 cases) [23].

Multiplex PCR has advantages of being faster and easier to

perform when compared to other methods. The characteristics ofhigher sensitivity, species-specific or T. solium genotype-

specific diagnosis, and detection of a tapeworm carrier prior to

patency of the infection are also definite advantages. The

diagnostic marker was detected from a T. solium carrier who

expelled only immature proglottids and was egg-free in stool,

implying that it is possible to detect tapeworm carrier prior to

patency. In areas where taeniasis is endemic, therefore, multiplex

PCR diagnosis will be useful for control programs that aim to

detect and treat tapeworm carriers [23]. Multiplex PCR can

cause non-amplification of target DNA marker because of

competition between template DNA and inhibitory substances

present in feces (personal observation), sample heterogeneity on

stool collection and preservation status of stool samples [23]. A

recent evaluation of detection of T. saginata taeniasis by

multiplex PCR reported that the taeniid DNA was detected in

all 10 tapeworm carriers examined when stool samples were

stored in ethanol properly after collection[25].

6. Conclusions

DNA-based differential diagnosis for human taeniid ces-

todes has become a very powerful tool not only for routine

identification but also for taeniasis/cysticercosis control pro-

grams that aim to detect tapeworm carriers and treat them. It is

also indispensable for definitive diagnosis of cysticercosis

cases in which histopathological findings are not confirmatory.

Acknowledgments

We thank the following researchers: T. Ikejima, P. Deku-

myoy, S.P. Sinha Babu, A. Oommen, G. Singh, DC. Qiu, M.

Wulamu, P.C. Fan, K.S. Eom, V.C.W. Tsang, A. Kassuku, S.S.Afonso, A. Zoli, S. Miura, A. Plancarte, W. Benitez-Ortiz,

C.M. Nunes, M. Vilhena, S. Geerts, J.C. Allan, J. Garcia-

Noval, M. Velasquez-Tohom, P.S. Craig, S.S. Margono and T.

Wandra for providing taeniid parasites and fecal samples; S.

Matsunaga, K. Nagakura, K. Suzuki, T. Nagase and Y.

Kiyoshige for providing biopsy materials. This study was

supported in part by a Grant-in-Aid for Scientific Research

from the Japan Society for Promotion of Science to A.I.

(14256001, 17256002) and a Research Grant from the Ohyama

Health Foundation to H.Y.

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