Molecular and serological diagnosis of taeniasis and cysticercosis … · Molecular and serological...
Transcript of Molecular and serological diagnosis of taeniasis and cysticercosis … · Molecular and serological...
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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
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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
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Distribution of Taeniasis/Cysticercosisof T. solium
50,000,000 people 50,000 die annually (MMWR 1993, 42: 1-25)
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No specific symptom
The majority of NCC cases
asymptomatic: no treatment
Clinical Manifestations of NCC
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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
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Epilepsy due to T. solium
Main cause of late-onset epilepsy
Developing countries
Endemic areas for Taenia solium
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Epileptic Seizures
Most common symptom of NCC
70% - 90%
By courtesy of Dr.O.M. Takayanagui
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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
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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!
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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)
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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
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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
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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.
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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
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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.
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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
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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!
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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)
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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)
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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
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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)
?
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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)
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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)
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Pigs and dogs have free access to human feces
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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
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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
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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).
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B
Three human Taenia species are sympatrically occurring in some areas in Thailand
Anantaphruti MT et al. 2007. Emerging Infectious Diseases 13, 1413-1416
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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)
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Tibetan people in the western part of Sichuan, China
(Li TY et al. 2006. Acta Tropica 100, 223-231)
Three species are sympatricallyoccurring!
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T. asiatica has been confirmed from
Taiwan, China, Korea, Indonesia, Philippines, Vietnam, Thailand
through our collaboration projects.
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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
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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
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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.
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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).