Cysticercosis,A Neglected Tropical Disease

Michael Parkhouse Instituto Gulbenkian de Ciência PortugalInstituto Gulbenkian de Ciência, Portugal

Mil C té Eli b th F BIOMED V l• Milagros Cortéz, Elizabeth Ferrer, BIOMED, Venezuela,• Teresa Gárate, Luis Miguel González, Elizabeth Ferrer, ISCIII, 

Madrid EspañaMadrid, España• Hugo. García, UPCH, R. Gilman, PRISMA , E. González, UASMP, 

PeruPeru• Leslie Harrison, Edimburgo, Escocia• Edda Sciutto UNAM Mexico

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• Edda, Sciutto, UNAM, Mexico

N l t d T i l DiNeglected Tropical Diseases

• Non‐reported diseases of the marginalised poor in developing countries, both in rural areas and city slums

C• Causes‐ Unequal distribution of wealth and power‐ Exploitation, national and internationalExploitation, national and international

•Consequences‐Multiple disadvantages, social, educational and health

•Solution‐ Short term: community basedL t liti l‐ Long term: political

Consequences of Neglected Tropical DiseasesConsequences of Neglected Tropical Diseases

• Social and economic impact on family, community d iand region

• Reduce regional development (tourism)

• Vulnerable groups, particularly women, young and old

• Low mortality, high morbidity

The “top ten” human parasites (WHO)The “top ten” human parasites (WHO)InfectionsInfections DeathsDeaths

(thousands per year)(thousands per year)( p y )( p y )

Ascaris 900,000900,000 2020

Hookworm 800,000800,000 5555

Malaria 800,000800,000 15001500

Trichuris 500,000500,000 --

Amoeba 480,000480,000 7575Amoeba ,,

Filarias 280,000280,000 --

Schistosomes 200,000200,000 750750

Giardia 200,000200,000 --

Trypanosomes 25,00025,000 6565

Leishmania 1,2001,200 11

LOW MORTALITY HIGH MORBIDITYLOW MORTALITY, HIGH MORBIDITY

Morbidity in neglected tropical diseasesy g p

DebilitationDebilitationAnaemiaDi hDiarrhoeaMalnutritionInfertilityImpaired physical and mental performanceImpaired physical and mental performanceDisfigurementStigmatisationReduced probability of marriage/familyReduced probability of marriage/family

El control de las enfermedades infecciosasEl control de las enfermedades infecciosas

Ed ióEducación

Control de Control de vectores

Importancia del diagnósticoImportancia del diagnóstico

El control de las infecciónes Tropicales Desatendidasp

Ed ióEducación

Control de Control de vectores

Importancia del diagnósticoImportancia del diagnóstico

Difficulties in the treatment of NTD’scu t es t e t eat e t o s

• Low priority in healthcare and research budgets• Low priority for vaccine development

chronic infections of the poor‐ chronic infections of the poor‐ complex immunity, often without obvious vaccine targets‐ vaccine budget $5 billions low compared with “pharma” budget ofg $ p p g$350 billions NB “Zantac” , $3.5 billions in 1993‐ a good vaccine destroys its market

bl b d b d• Problem exacerbated by AIDS and TB

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CYSTICERCOSIS: A NEGLECTED TROPICAL DISEASE

• Predominantly a disease of rural poor, but now in urban areas due to population movementp p

• Principal risk factors‐ Pigs: increasing unsupervised, “clandestine” consumption of pork‐ Availability and quantity of clean water‐ Disposal of human faeces eg fertilisation of‐ Disposal of human faeces eg. fertilisation of vegetables with “aguas negras” in Mexico

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Two pigs in la Higuera, Bolivia, where they killed Che Guevara

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Taenia saginata and Taenia soliumTaenia saginata and Taenia solium

A t f id bl t l• Account for considerable meat loss.• Taenia saginata – Cosmopolitan in distribution, but has a

higher prevalence in cattle rearing countries of the tropics andg p g psub‐tropics.

• Taenia solium – Geographically more restricted but is veryprevalent in the countries of the former Soviet Union centralprevalent in the countries of the former Soviet Union, centraland south America, Africa and Asia. It is of low prevalence ineurope(>5%).

• Their presence is a strong indicator of poor standards of• Their presence is a strong indicator of poor standards ofhygiene and public health control.

• T. solium causes human cysticercosis and neurocysticercosis.• Expanding through travel to and from endemic areas• Reciprocal models

Tenia solium neurocysticercosis

• WHO 50,000 human deaths/annum but the morbidity is highWHO 50,000 human deaths/annum but the morbidity is high• Endemic countries (Peru) over 10% acute care admissions to

neurological wards – neurocysticercosis and c30% late onsetepileptic seizures are T. solium associated

• A strong indicator of poor standards of hygiene and publichealth controlhealth control.

• Non‐endemic countries up to 2% neurological admissionsSouth California due to neurocysticercosis (over 1000y (cases/year in the USA. Causes include infected immigrants,travel to endemic countries, contact with infected people)

Taenia solium neurocysticercosis

MRI longitudinal section              Transverse section

C t l f C ti iControl of Cysticercosis

• High prevalence of worms is an objective indicator of poorpublic health and hygiene.C t l f th di th h h i l t d d• Control of the disease through hygiene, clean water and coveredsewage, as in Europe, is expensive .

• Meat inspections do not detect mild infections• Meat inspections do not detect mild infections .• Low cost control in the short term in rural communities at risk is

feasible through reliable sensitive diagnosis and ruralfeasible. through reliable, sensitive diagnosis and ruraleducation programs. These will bring colateral benefits, forexample, reduced levels of soil and water transmitted diseases.

• In the long term, pig vaccination .

Diagnosis and protection in cysticercosis

• Diagnosis before, during and after control– Reproducibility

Cloned antigens, synthetic peptides, Mabs, PCRs.Parasite detection– Parasite detection 

Viable metacestodes through secreted HP10.All stages through DNA by PCR.Adults through “coproantigens”.

– Detection of AntibodiesRecombinant proteins and synthetic peptidesRecombinant proteins and synthetic peptides.

As immunity is antibody mediated, some antigensmay have utility in both diagnosis and protectionmay have utility in both diagnosis and protection.

• Protection– “Functional” surface and secreted oncosphere proteins.

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p p– Pehaps immature cestodes proteins.

Ante mortem diagnosis of viable metacestodes in manAnte mortem diagnosis of viable metacestodes in man, pigs and cattle

• Detection of secreted metacestode glycoprotein by MAb HP10 ELISA

• Levels in cerebrospinal fluid and serum provide a useful indicator and a monitor for drug treatmentindicator and a monitor for drug treatment

• “Pending confirmation from a large clinical trial serumPending confirmation from a large clinical trial, serum antigen detection might help to monitor treatment efficacy in basal subarachnoid neurocysticercosis and reduce the need for frequent imaging” (Garcia HH, (2007) Nature, Neurology)

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Mab HP10 antigen detection ELISAMab HP10 antigen detection ELISA

ELISA – low backgrounds

Mab HP10 recognises a repeating epitope

HP10 Ag‐ELISA 

Serum HP10 antigen detection in neurocysticercosis

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Sensitive differential diagnostic PCR protocolsSensitive differential diagnostic PCR protocols

• Multiplex protocols to identify and distinguish T. saginata, T.solium T asiatica T hydatigena Echinococcus spp andsolium, T. asiatica, T. hydatigena, Echinococcus spp andSarcocystis spp. (González LM, 2006)

• Identification of cysts (eg. abbatoir, clinic)

E i t l it i b f d i d ft t l• Environmental monitoring before, during and after controlprogrammes

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Detection of Taenias to interrupt transmissionDetection of Taenias to interrupt transmission

M h l• Morphology– Not possible until 3 months after infection– Expulsion of proglottids and eggs is intermittent– Rapid breakdown and deterioration of proglottids

• “Coproantigen” detection by capture ELISACoproantigen  detection by capture ELISA – Specific for Taenia – Detects prepatent tapeworm

Stable antigen liberated in absence of proglottids– Stable antigen liberated, in absence of proglottids– Permits monitoring of treatment

iff i i f li d i• Differentiation of T. solium and T. saginata– Uterine branches, but frequently deteriorated– PCR very sensitive

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y

Detection of antibodies in cysticercosis

• Demonstrates exposure not necessarily a viable infection

y

• Demonstrates exposure, not necessarily a viable infection.• Useful for establishing prevalence before, during and aftercontrol programmes.p g

• Reproducibility through recombinant proteins and syntheticpeptides, principally of the oncosphere and metacestode

• Increased specificity focusing on IgG4• Recent infections focusing on IgM

diff i i ibl• May differentiate resistant vs. Susceptible• As immunity is antibody mediated, some antigens mayhave utility in both diagnosis and protectionhave utility in both diagnosis and protection

• What is a “Gold standard?”

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What is a Gold standard?

Bovine antibodies to synthetic peptides ofT. saginata oncosphere protein HP6

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2,0nm

C t fl id

1,0

1,5

2,0

D 4

50

n Cyst fluid

PeptidesHP6‐3

0,0

0,5

1,0

LIS

A O

Peptides

0,0-1 0 1 2 3 4 5 6 7 8 9 10 11 12

Weeks Post Infection

E

Synthetic peptides of Synthetic peptides of T. saginataT. saginata HP6 also applicable to HP6 also applicable to T. soliumT. solium

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Serum and cerebrospinal fluid based ELISA analysis of clinically defined*Serum and cerebrospinal fluid based ELISA analysis of clinically defined  Venezuelan cases of active (26) and inactive (23) neurocysticercosis (NCC)

Sensitivity (%) Specificity (%)ELISA assays

Sensitivity (%) Specificity (%)

Active NCC Inactive NCC Other infections$

Ab ELISA’Ab-ELISA’s

HP6** (oncosphere) 77 (77) 65 (48) 95

Ts8B2** (metacestode) 97 (65) 70 (39) 95

Peptides*** 85 (ND) 85 (ND) 84

Cyst Fluid**** 100 (100) 100 (100) 67

Ag-ELISA

MAb HP10 71 (81) 13 (9) 98

*Del Brutto et al 1996,2001 and Garcia et al 2005**recombinant antigen expressed in the eukaryotic BACULOVIRUS system**recombinant antigen expressed in the eukaryotic BACULOVIRUS system***Peptides HP6‐3, Ts45W‐1, Ts45W‐5 used in combination, ****Taenia solium cyst fluid used as antigen$ based on data from 87 cases of hydatidosis,schistosomiasis, fascioliasis, toxocariosis or healthy individuals 

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Distribución de personas con antígeno circulante y anticuerpos anti‐metacéstodo en

SECTOR IIIcirculante y anticuerpos anti metacéstodo en 

suero. Valle del Río 2005‐2006

SECTOR IV

Ag+/Ac+Ag+/Ac‐

SECTOR IV

Ag‐/Ac‐Ag‐/Ac+

SECTOR II

SECTOR I

SECTOR II

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Obtención de los gusanos adultos de T. soliumi d i di id i f dprovenientes de un individuo infectado. 

Tratamiento con “Praziquantel”y purgas salinasy purgas salinas

3232

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ProtectionProtection in in cysticercosiscysticercosisbb HP6HP6 f ti lf ti l dh idh i l ll lbyby HP6, a HP6, a functionalfunctional adhesionadhesion moleculemolecule

(Subseqently named TSA18 by Lightowlers)

• Mediated by antibody to infective oncospheres.  

• HP6,  a rational vaccine candidate.‐ An oncosphere surface/secreted molecule,                  ‐ Homology to adhesion molecule fibronectin type III.‐ Mediates cellular adhesion (inhibited by Mab HP6)‐ Confers 100% protection to cattle against T. saginatap g g

•Function‐ Helminth molecule evolved for host manipulationPerhaps facilitating tissue invasion of oncospheres

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HP6HP6‐‐Mab inhibition of HP6Mab inhibition of HP6‐‐Tsag Tsag –– mediated cell adhesionmediated cell adhesion

Binding Inhibition

i il l d d f l35

Now similarly demonstrated for HP6‐Tsol

HP6HP6 Tsag rec protects cattle againstTsag rec protects cattle against T saginataT saginataHP6HP6‐‐Tsag rec protects cattle against Tsag rec protects cattle against T. saginataT. saginata

2000

2500

Live

Dead

1500

2000Total

1000

500

0TSE - 1 TSE - 2 HP6 - 1 HP6 - 2 PeP - 1 PeP - 2 Shm - 1 Shm - 2

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Vaccination Group

Diagnosis and protection in cysticercosis

• Diagnosis before, during and after control– Reproducibility

Cloned antigens, synthetic peptides, Mabs, PCRs.Parasite detection– Parasite detection 

Viable metacestodes through secreted HP10.All stages through DNA by PCR.Adults through “coproantigens”.

– Detection of AntibodiesRecombinant proteins and synthetic peptidesRecombinant proteins and synthetic peptides.

As immunity is antibody mediated, some antigensmay have utility in both diagnosis and protectionmay have utility in both diagnosis and protection.

• Protection– “Functional” surface and secreted oncosphere proteins.

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p p– Pehaps immature cestodes proteins.

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