Molecular and serological diagnosis

of taeniasis and cysticercosis in Asia and the Pacific

A. Ito and ColleaguesAsahikawa Medical College

JITMM2007 Bangkok 30 Nov 2007

sponsored by JSPS-Asia・Africa Science Platform Fund

Neurocysticercosis of Taenia solium

is one of the most lethal

parasitic diseases worldwide and on the list of neglected infectious diseases WHO 2005

Neurocysticercosis of Taenia solium

is one of the most lethal

parasitic diseases worldwide and on the list of neglected infectious diseases WHO 2005

Distribution of Taeniasis/Cysticercosisof T. solium

50,000,000 people 50,000 die annually (MMWR 1993, 42: 1-25)

No specific symptom

The majority of NCC cases

asymptomatic: no treatment

Clinical Manifestations of NCC

Epileptic Seizures 62 Intracr. hypertension 34 Meningitis 29 Mental disorders 11 Vasculitis 2 Spinal 0.5 Combined 37

Clinical pictures %

Clinical Manifestations of NCC of T. solium

No symptoms specific to NCC of T. solium

Epilepsy due to T. solium

Main cause of late-onset epilepsy

Developing countries

Endemic areas for Taenia solium

Epileptic Seizures

Most common symptom of NCC

70% - 90%

By courtesy of Dr.O.M. Takayanagui

Treatment of NCC patients based on crucial diagnosis

Chemotherapy vs Surgery

Most of the symptoms of NCC become evident when the parasite has just been damaged by the host immune responses or by treatment with metacestocidal drug such as

praziquantel through mass screenings

No symptoms specific to NCC of T. solium

How to suspect NCC?1) Neurologic disorders:

no symptoms specific to NCC

2) Neuroimaging: not always typical

3) History of traveling to and/or living in T. solium endemic areas

Then,

4) Serology!

Differential Serodiagnosis of Cysticercosis by Immunoblot(modified from Ito A et al. 1998. Am J Trop Med Hyg 59: 291-294)

Cysticerocosis

Cystic Echinococcosiswith high titers

Cysticercosisconfirmed at suspected

CDC, USA in Japan*

Alveolar Echinococcosiswith high titers

(kDa)

80

50

3328

18

* T. solium: not indigenous in Japan

Gold standard for cysticercosis is

immunoblot using specific antigens for identification of patients

(CDC, USA and AMC, Japan)

0.00.10.20.30.40.50.60.70.80.91.01.11.21.31.41.5

Abs

orba

nce

(405

nm)

Native GPs Ag1V1/Ag2

Comparison of native GPs and recombinant chimeric antigens (Sako Y et al. 2000. JCM 38)

healthy personscysticercosis patients

Similar sensitivity in ELISA and IB between these two antigens

Cysticerocosis

Native GPs 65/70 92.9%

Ag1V1/Ag2 66/70 94.3

How to diagnose NCC?

Neuroimaging: not always typical

Serology: sensitivity not always 100%

Surgery suspected brain tumor

Histopathology

Molecular identificationIto A et al. Neurocysticercosis: clinical manifestation, neuroimaging, serology and molecular confirmation of histopathology specimens. Southeast Asian J Trop Med Public Health 2006; 37 (Suppl 3): 74-91

Molecular identification of metacestodein the histopathological specimens

No antibody Response : ID?

Solitary NCC? Calcified NCC?

With or without hooklets?

Are the causative agents really cysticerci of T. solium?

Other zoonotic taeniid species?

The majority of such cysts may be expected to be due to The majority of such cysts may be expected to be due to T. soliumT. solium. . However, molecular confirmation is essential nowadays.However, molecular confirmation is essential nowadays.

THA

IND

CHI2

CHI1

IRI

TAN

MOZ

MEXPER

BRA

CAM

ECU

BOL

1000

1000

0.02

E. multilocularis(outgroup)

T. saginata

T. asiatica

T. solium

993

808

Cytb (1068 bp)

Latin America

&Africa

Latin America

&Africa

Asia Asia

E. multilocularis(outgroup)

T. saginata

T. asiatica

THA

IND

CHI2

CHI1

IRI

TAN

MOZ

MEX

PER

CAM

BRA

ECU

BOL

1000

985

1000

1000

0.01

T. solium

Cox1 (1620 bp)

Two genotypes of Two genotypes of T. soliumT. solium worldwideworldwide

Nakao M et al. 2002. Parasitology 124, 657-662

African/ AmericanGT

Asian GT

0.5

1.0

kb

0.7

Case 1

1.5

T. so

lium (A

sian)

T. so

lium

(Amer

ican)

M

1.0

kb

0.5

1.5

Case 1

M

984 bp1.8

PCR-amplification of cox1

PCR : 94C, 30sec; 58C, 30sec; 72C, 90sec; 35 cycles

53-year-old, Japanese woman; a solitary cyst in the left frontal lobe; repeated travel histories to India and Southeast Asian countries from 1993 to 2001.

Case 1

Such cases may retrospectively be analyzed as to where they

were exposed to eggs based on subtyping of mtDNA.

This case is suspected to have been exposed to T. solium eggs

in Bali, Indonesia.

A long-standing puzzle has been

that adult taeniid tapeworms

expelled from people in Asia-Pacific

seemto be T. saginata, the beef tapeworm,

although these people eat

pork rather than beef(from Ito et al. 2003. Lancet 362, 1918-1920).

T. saginata vs T. asiatica

How many human Taenia species are distributed in Asia-Pacific?

Now we have three in Asia-Pacific

Taenia solium, Taenia saginata and Taenia asiatica.

Historically we recognized only two in the world

T. solium and T. saginata!

Multiplex PCR for differentiation of three human taeniid species

1.01.5

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 M31 32

T. saginata T. asiaticaT. solium

AsianT. solium Amer/Africa

827 bp 269 bp 720 bp

Lane 11 : taeniid egg sample from Yunnan, China

(mixture of T. saginata and T. asiatica)

0.5

984 bp1

( Yamasaki H et al. 2004. J C Microbiol 42, 548-553)

Three countries where we have confirmed three species are briefly introduced.

1) Indonesia where three species are distributed basically separately due to the barriers of the religion (Wandra et al. 2006 Trans R Soc Trop Med Hyg 100)

2)Thailand where three species are sympatrically occurring (Anantaphruti et al. 2007 Emrg Infect Dis 13)

3) China where three species are sympatricallyoccurring (Li et al. 2006 Acta Trop 100)

Bali

1000 km

Bali

1000 km

Timor

Papua T. soliumJakarta

Sumatra

Java

Lake Toba

North Sumatra T. asiatica T. solium ?

Kalimantan

Flores

Bali T. saginata T. solium

Taeniasis/Cysticercosis in Indonesia

Immunoblots of NCC suspected patients’sera from Bali 2005

A B

A:GPs prepared by isoelectric focusing

B: recombinant AgN P N P

International collaborative projects

Blood vs CSF no difference (unpublished)

*

*: not NCC but malignant tumor (Feb 2006)

(see Sudewi RAA et al. 2007. Trans R Soc Trop Med Hyg 101: in press)

?

Taenia saginata in Bali

very common in local residents(Wandra T et al. 2006. Trans R Soc

Trop Med Hyg 100: 346-353)

Taenia saginata in Bali

very common in local residents(Wandra T et al. 2006. Trans R Soc

Trop Med Hyg 100: 346-353)

PNGPAPUA

Jayapura5

41

2

3

PNGTaenia soliumtaeniasis/cysticercosis:

highly endemic

Wamena1990’

Enarotali1970’

Nabire2000’

Manokwari2000’

Merauke

OK Teddy Mine 19976

(from Wandra T et al. 2003. Emerg Infect Dis 9, unpublished)

Pigs and dogs have free access to human feces

The dog was suspected to be infected with T. solium cysticerci

by highly specific antibody-ELISA and IB (Ito A et al. 2002. J Helminthol 76)

Traditional tongue examination for porcine cysticercosis

Taeniasis of T. asiaticais still endemic in Samosir

island, north Sumatra!(Wandra et al. SEAJTMPH 2007: 38,

Trop Med Health in press)

Taeniasis of T. asiaticais still endemic in Samosir

island, north Sumatra!(Wandra et al. SEAJTMPH 2007: 38,

Trop Med Health in press)

T. asiatica

In Indonesia,

there is no area where three human taeniid species are

sympatrically occurring (Wandra et al. Parasitol Int 2006: 55 Supplement; SEAJTMPH 2007:

38; Trop Med Health in press).

B

Three human Taenia species are sympatrically occurring in some areas in Thailand

Anantaphruti MT et al. 2007. Emerging Infectious Diseases 13, 1413-1416

2 Taenia solium Taenia saginataor

Taenia asiatica

Three worms were expelled from one patient in Thailand 2004 (Anantaphruti MT et al. 2007 EID 13, 1413-1416)

Tibetan people in the western part of Sichuan, China

(Li TY et al. 2006. Acta Tropica 100, 223-231)

Three species are sympatricallyoccurring!

T. asiatica has been confirmed from

Taiwan, China, Korea, Indonesia, Philippines, Vietnam, Thailand

through our collaboration projects.

Metacestodes of Taenia saginata in the peritoneal cavity of NOD/Shi-scid mouse after 23 weeks of

infection with in vitro hatched oncospheres

How to use viable eggs of Taenia species

Huge number of metacestodes of

Taenia asiatica

after 19 weeks of inoculation with

in vitro hatched oncospheres

in a NOD/Shi-scid mouse

from Nakaya K et al. Parasitol Int 2006; 55Supplement

We have to re-evaluate all T. saginata worms in the world when the patients were from

Asia-Pacific or having visiting or living history in Asia-Pacific.

“A long-standing puzzle that adult taeniid tapeworms expelled from people in Asia-Pacific

seem to be T. saginata, the beef tapeworm, although these people eat pork

rather than beef”

has been resolved

not to be T. saginata but to be T. asiatica.

Summary

1. Three human Taenia species are occurring in Asia-Pacific. Taeniasis may be detected by several tools.

2. Molecular identification of these human Taeniaspecies is feasible.

3. Serology is highly useful for detection of NCC.

4. Confirmation of NCC is based on neuroimaging, serology, histopathology and molecular identification.

5. T. saginata in the world as well as in Asia-Pacific should be re-evaluated by molecular tools (Ito et al. 2003. Lancet 362, 1918-20).