Neglected FB parasitic zoonoses

DIAGNOSTICS: Basic considerations for mapping and monitoring and evaluation

David Jenkins Hong Sung-Tae Meritxell Donadeu

Luang Prabang, 18th October 2018 Purpose (use case) of diagnostic tests

• Disease diagnostic INDIVIDUAL level

• Disease diagnostic POPULATION level

• MONITOR control intervention Focus of this presentation is on 2 use cases:

1. Mapping: Identification of endemic or risk areas

2. Monitoring and evaluation of a control program Diagnostics T. solium

Definitive (adult tapeworm)

Accidental host (larval stages) Neurocysticercosis Intermediate host (larval stages)

Porcine Taeniasis by T. solium

Sensitivity Specificity Comments

No No • Does not differentiate eggs Microscopy • Does not detect immature tapeworms Guezala 2009 published sps Copro-antigen Yes No ELISA specific. Not commercially available.

Copro-DNA Yes Yes Mayta 2008, Nkouawa (Copro-PCR, 2016. Independent LAMP) validation needed.

Serology Yes 5% CE Detects exposure, not 14-17% only active infection (depending on format)

Considerations: 1. Very low prevalence (typically 1-2%). Implications for sample size and test requirements. 2. Copro-analysis: Use of infectious material. Precautions should be taken at laboratory level, but more importantly when collecting samples in areas where sanitation is not adequate Taeniasis by T. solium

1. Mapping: • Tests with low sensitivity and specificity can be used to screen the areas, but MUST be confirmed. Example: coproantigen or KK followed by copro-DNA of the positive samples or detection in pigs.

2. Monitoring & evaluation of a control program: • Needs high specificity tests: copro-DNA methods. Neurocysticercosis

• tests • Antigen tests • Imaging

Considerations: 1. Antibody and antigen tests: • Antibody tests do not differentiate patients with viable and non- viable • Presence of transient positives (in the absence of clinically relevant cysticercosis) in endemic areas – in both Ab and Ag test 2. Imaging is critical before treatment of patients with viable cysts. Neurocysticercosis

1. Mapping: • NCC cases might not be recent. • Consider if they are referral cases. • Can be useful by providing additional information.

2. Monitoring & evaluation of a control program: • Not recommended, only very long term Pig cysticercosis

• Antibody tests • Antigen tests • Tongue inspection and slaughter check • Necropsy and carcass dissection

Considerations: 1. Porcine cysticercosis has a higher expected prevalence than taeniasis (implications for sample size and specificity). 2. Ab and Ag tests: sensitivity and specificity are a problem: • Current commercial Ag tests cross react with T. hydatigena, T. asiatica. • Many positive pigs have no cysts at necropsy (several reports >50% false positives) • EITB: ≥3 bands best compromise between sensitivity (77%) and specificity (76%), possible cross-reactivity of T. hydatigena to GP50 Ag. • Transient positives observed with both Ab and Ag test Pig cysticercosis Considerations (cont): 3. Tongue inspection and slaughter check can be highly specific but have low sensitivity: • Tongue inspection: # cysts Sensitivity <100 cysts 0 – 3 % > 100 cysts ~ 35% >1000 cysts >95%

• Slaughterhouse: Se 38% (Phiri 2006. Including special meat cuts) Viable Pig cysticercosis Considerations (cont): 4. Necropsies and carcass dissection are labour intensive, but is the definite method.

Viable cyst Pig cysticercosis

1. Mapping: • Methods with low sensitivity (tongue inspection) can be used. • Tests with low specificity can be used as screening tests (serology), but MUST be confirmed (necropsies) before starting a project.

2. Monitoring & evaluation of a control program: • Necropsies. • Serology and confirmation of positives by necropsy. Summary diagnosis T. solium

Se/Sp Advantage Limitation Mapping M & E Taeniasis by T. solium Microscopy L/L Inexpensive Not specific Screen Screen Copro-Ag H/L Not specific. Not Screen Screen available. Copro-DNA H/H High Se, high Sp Expensive. Not available. Yes Yes Serology H/M Serum samples Detects exposure, not Screen No only active infection Porcine cysticercosis Antigen H/L Commercial tests Low specificity Screen Screen Antibodies H/L Low specificity Screen No Tongue insp L/H Inexpensive Low sensitivity Yes No Slaughter L/H Routinely done in Many pigs are home Yes No inspection some places slaughtered. Low Se. Necropsies H/H High Se and Sp Laborious. (pre-selection Yes Yes can be done by serology) Summary diagnosis T. solium

Se/Sp Advantage Limitation Mapping M & E Taeniasis by T. solium Microscopy L/L Inexpensive Not specific Screen Screen Copro-Ag H/L Not specific. Not Screen Screen available. Copro-DNA H/H High Se, high Sp Expensive. Not available. Yes Yes Serology H/M Serum samples Detects exposure, not Screen No only active infection Porcine cysticercosis Antigen H/L Commercial tests Low specificity Screen Screen Antibodies H/L Low specificity Screen No Tongue insp L/H Inexpensive Low sensitivity Yes No Slaughter L/H Routinely done in Many pigs are home Supportive No inspection some places slaughtered. Low Se. data Necropsies H/H High Se and Sp Laborious (pre-selection Yes Yes can be done by serology) Diagnostics of granulosus Echinococcosis

Definitive host (adult tapeworm)

Accidental host (larval stages) Hydatid disease Intermediate host (larval stages)

Echinococcosis sheep Echinococcosis detection in Dogs • Examination post mortem • Purging with arecoline hydrobromide • Direct microscopy & faecal egg floats • Copro-ELISA • Copro-PCR

Considerations: 1. Killing dogs and examining them post-mortem not popular with owners!...... but it is the gold standard. 2. Arecoline purging is difficult to implement. Highly specific. Not to be used on pregnant females, puppies or old dogs, some dogs do not purge. Also, presents infection risks to people handling and examining the faeces. Dog purging

Purge sample

Adult E. granulosus tapeworms Echinococcosis detection in Dogs

Considerations (cont): 3. Microscopy detection of eggs in faeces: • Not specific: The eggs of all taeniid cestodes (Taenia spp & Echinococcus) are morphologically identical, therefore not useful. • Pre-patent period 4. Copro-Ag: detectable 3 weeks after infection. Specificity??, some level of cross-reactivity with Taenia spp. Proper validation, local controls. In house tests. 5. Copro-PCR: in-house tests, need proper validation 6. Lack of commercial tests COPRO-ANTIGEN Echinococcosis detection in dogs

1. Mapping: • Tests useful for screening: microscopy, copro-Ag. Need to be confirmed copro-PCR.

2. Monitoring & evaluation of a control program: • Copro-Ag and Copro-PCR properly validated. Echinococcosis detection in Humans

• Serology • Imaging: X-Ray (lungs), ultra-sound (). • Patient history

Considerations: 1. Most patients are serologically positive: IHA, ELISA, WB (but some can be negative). Suggestion is to use 2 tests together. 2. Serology is used as an adjunct to imaging. 3. Serology is valuable for post-treatment follow-up, differentiating those with continuing viable infection (post-surgical recurrences). 4. Ultra-sound in children <5 yrs is indicative of new infections and active transmission. DETECTION OF CYSTS IN HUMANS Mobile ultrasound monitoring in remote areas (Turkana, Kenya) Echinococcosis (hydatid cyst) detection in humans

1. Mapping: • Ultrasound in children.

2. Monitoring & evaluation of a control program: • Ultrasound in children. Echinococcosis detection in Livestock

• Ultrasound detection • Necropsy and lung & liver inspection • Meat inspection at abattoirs

Considerations: 1. Ubiquitous co-infections with related taeniid cestode species (T. hydatigena, T. ovis, T. multiceps) 2. Weak serological response to Eg infection in livestock 3. Ab ELISA: Many reports of excellent results. None validated independently. • Typically flawed by non-infected controls not matched for exposure to taeniid cestodes other than Eg. 4. Necropsy: cysts take long time to develop (1 year) 5. Meat inspection: many animals are home slaughtered ABATTOIR MONITORING

Image: AHBD Beef & Lamb

Image: The Land Echinococcosis detection in livestock Useful for….

1. Mapping: • Slaughter data • Post-mortem examination

2. Monitoring & evaluation of a control program: • Slaughter data • Post-mortem examination Summary diagnosis E. granulosus Se/Sp Advantage Limitation Mapping M & E Dogs Post mortem H/H High Sp Dog owners don’t like it Yes No Arecoline M/H High Sp Some dogs don’t purge Yes Yes Microscopy L/L Inexpensive Low sensitivity Screen No Copro-Ag H/M Practical Lack of availability. Yes Yes Validation Copro-PCR H/H High Se/High Sp Needs validation Yes Yes Humans Imaging ?/H Well accepted, In children indicates Yes Yes non invasive active transmission Serology M/M Widely available Indicates exposure Screen No Livestock Necropsy H/H High Sp Slow cyst development No Yes Abattoir M/H Routinely done Home slaughter Yes Yes Summary diagnosis E. granulosus Se/Sp Advantage Limitation Mapping M & E Dogs Post mortem H/H High Sp Dog owners don’t like it Yes No Arecoline M/H High Sp Some dogs don’t purge Yes Yes Microscopy L/L Inexpensive Low sensitivity Screen No Copro-Ag H/M Practical Lack of availability. Yes Yes Validation Copro-PCR H/H High Se/High Sp Needs validation Yes Yes Humans Imaging ?/H Well accepted, In children indicates Yes Yes non invasive active transmission Serology M/M Widely available Indicates exposure Screen No Livestock Necropsy H/H High Sp Slow cyst development No Yes Abattoir M/H Routinely done Home slaughter Yes Yes Echinococcus multilocularis • Detect tapeworms in gut contents post mortem Adult tapeworm • Detect tapeworms in faeces following arecoline purge • Copro-antigen detection • Copro-PCR

Definitive host (adult tapeworm)

Accidental host (larval stages) Alveolar echinococcosis • Detect liver lesions with Intermediate host ultrasound (larval stages) • Detect lung lesions with X-ray Multilocular cysts • Detect circulating • Post mortem observation of antibodies in serum lesions in the liver Diagnostics FBT FBTs of public health importance

• : East Asia (China, Korea, Taiwan, Northern Vietnam) • : Mekong delta (Lao, Thailand, Cambodia, Vietnam) • , F. gigantica: Mediterranean, Latin America, Asia • Paragonimus spp.: Focally spread • Metagonimus or other intestinal trematodes: Asia, Mediterranean

Considerations: 1. Zoonotic 2. Area specific distribution 3. Liver flukes are commonly mixed prevalent with intestinal flukes Clonorchis sinensis Opisthorchis viverrini Clonorchis sinensis and Opisthorchis viverrini

• Recovery of parasite or parasite fragment: limited • Stool microscopy to identify eggs: difficult • Molecular techniques: proteins, DNA • Serology: Ag, Ab

Considerations: 1. Area specific for Cs & Ov 2. Mixed infection by fish-borne trematodes

Cs eggs on KK smear

Cs egg on wet smear

Metagonimus eggs on wet smear

Metagonimus yokogawai Cs & Ov distribution map in Asia (2011). Presented at RNAS+ Annual Meeting, Bogor, 2014 From: Clonorchis sinensis and Opisthorchis spp. in Vietnam: current status and prospects by Doanh & Nawa Trans R Soc Trop Med Hyg. 2016;110(1):13-20. doi:10.1093/trstmh/trv103 Trans R Soc Trop Med Hyg | © The Author 2015. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: [email protected]. Clonorchis sinensis and Opisthorchis viverrini

1. Mapping: • Stool microscopy to find eggs is standard, low sensitivity in light burden infections. • Eggs are morphological unidentifiable • Egg differentiation with heterophyid eggs • ELISA and ultrasonography supplement case detection.

2. Monitoring & evaluation of a control program: • Stool microscopy by 2 KK smears • ELISA negative conversion over one year after cure

• Recovery of parasite or parasite fragment • Stool microscopy • Sputum microscopy • Image diagnosis • Serology

Considerations: 1. Stool or sputum microscopy: low sensitivity 2. Serology for Ab is sensitive, commercial kit? 3. Some cross reactions with other trematode antigens 4. Images similar with those of tuberculosis or cancer 5. Unknown cross reactivity between species in the genus Paragonimus Paragonimiasis

1. Mapping: • Serology for Ab” high Se & Sp • Environmental and sociocultural parameters

2. Monitoring & evaluation of a control program: • ELISA: cross reaction with other trematodes • High ELISA absorbance suggests active infection Fascioliasis

• Recovery of parasite or parasite fragment: endoscopy, • Stool microscopy: differentiation of Fh & Fg • Serology Ab: low specificity

Considerations: 1. Ectopic fascioliasis is common in humans 2. Stool microscopy: low sensitivity, low specificity 3. ELISA for Ab is sensitive but cross reacts with Cs, Ov, Pw 4. No commercial serology kit: setting reference ELISA laboratory Sah et al. BMC Res Notes (2017) 10:439 DOI 10.1186/s13104-017- 2761-z. Gross worm of F. hepatica recovered from the common bile duct of a woman in Nepal by ERCP. Fasciolasis

1. Mapping: • Stool microscopy • Ab serology by ELISA

2. Monitoring & evaluation of a control program: • Stool microscopy • Ab ELISA: decreased absorbance Summary diagnosis Clonorchis sinensis & Opistorchis viverrini

Se/Sp Advantage Limitation Mapping M & E Kato-Katz H/H Cheap, mass Sp identification, well Screen Screen screen, EPGs trained expert Yes Yes FECT H/H Sens for light inf, Complicated procedure, Yes Yes easy identification limited for mass screen Direct smear L/H Simple, cheap Less sens for light inf Low Se Low se Copro PCR H/H Sens 95% DNA ext & complicated No No procedure, limited for research LAMP H/H Sens 97% DNA ext & complicated No No procedure, research Real time H/H Diff diagnosis Complicated, expensive No No PCR Summary diagnosis Clonorchis sinensis & Opistorchis viverrini

Se/Sp Advantage Limitation Mapping M & E ELISA IgG M/M Cheap, mass Low se in light infection, Screen Screen screen, sens 88% cross reaction ELISA IgM L/L Not acceptable No No ELISA Ag M/H Active infection Less sens for light inf No No Ag in urine M/M Sampling Research Screen No (Ov) Summary diagnosis Paragonimus

Se/Sp Advantage Limitation Mapping M & E Kato-Katz L/M Cheap, mass Limited se, many false Screen Screen screen negatives YES YES FECT L/H Sp identification Limited se, limited for mass screen YES YES ELISA IgG H/M Cheap, mass No kit, YES YES screen, se 95% cross reaction Summary diagnosis Fasciola

Se/Sp Advantage Limitation Mapping M & E Kato-Katz L/L Cheap, mass Limited se, many false Screen Screen screen negatives, diff diagnosis YES YES FECT L/M Sp identification Limited se, limited for mass screen YES YES ELISA IgG H/M Cheap, mass No kit, unknown se & sp, No No screen cross reaction