Advantages of microscopy, serology, antigen tests and molecular techniques for diagnosis of

parasitic infections

Tom van Gool, MD Section Clinical Parasitology Academic Medical Center, Amsterdam

Clinical parasitology in western hospitals: the essentials

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Importance (frequency of use) of techniques in routine clinical diagnosis of parasitic infections

Microscopy

Serology Mol diagnosis

M

Important frquent use

Infrequent use

S

Less frequent

Culture

Antigen detection Ag

CU

Ag

S

M

MD

CU

M

Ag

S

MD

CU

MD

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Different parasitic infections different diagnostic methods!

Malaria Intestinal helminths Intestinal protozoa Toxoplasmosis Leishmaniasis, cutaneous Leishmaniasis, visceral Trypanosomiasis, African Trypanosomiasis South American Acanthamoebiasis Echinococcosis

M Ag MD

S M MD

MD M Ag

CU M MD

S M MD

S M

M S MD

CU MD

S M MD

MD S M

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M Ag MD

Microscopy and antigen tests

Malaria

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Malaria M Ag MD

Severe illness, potential lethal Effective treatment available when early diagnosed What do we need for diagnosis: Speed (result < 1h after having received bloodsample)

Species determination: P. falciparum, P. vivax, P. ovale, P. malariae, P. knowlesi Parasitaemia of P. falciparum

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Malaria Basic techniques routine lab western hospital

M Ag MD

Thick smear

Thin smear

Antigen test

QBC (Quantitative Buffy Coat) (≥ 4 - 6 examinations / week)

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Recommendations for proper diagnosis:

• ICT Now good test: beware of lower sensitivity of P. vivax, P. ovale, P. malariae!

• Before reporting negative thick smear: study 200 – 400 (1000x) fields • Thin smear stand alone reasonable/good sensitivity! (5 min. sens 93 %)

• Diagnosis at night shifts: make thick smear, thin smear and antigen test Study: antigen test and thin smear (5-10 min): combined good sensitivity and no major clinical mistakes. Study, as good as possible, thick smear. Repeat exam in early morning by experienced technicians!!

M Ag MD

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Malaria: PCR

• Suspicion mixed infections (start malarone..)

• Low parasitemia: no microscopical discrimination possible. (start malarone..)

• P. malariae and P. knowlesi morphologically similar

M Ag MD

L. Link, A. Bart, N. Verhaar, T. van Gool, M. Pronk, V. Scharnhorst (submitted) Case-report: Molecular diagnosis of Plasmodium knowlesi in a Dutch traveler by real-time PCR.

For special cases:

(start chloroquine)

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S

Microscopy, serology, molecular tests, antigen tests

Intestinal Parasites

MD M Ag CU

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Diagnosis of intestinal parasites

Helminths: adults, eggs, larvae

Protozoa: vegetative stages, (oo)cysts

Clinical: abdominal complaints, varying degrees, symptoms most often nonspecific

Main groups of intestinal parasites:

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Different stages of intestinal parasites: distinctive morphology and relative large easy to recognize under a light microscope!

cysts of protozoa eggs of worms

Diagnosis of intestinal parasites

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S. mansoni

Ascaris

T. trichiura

C. cayetanensis

E. histolytica/ dispar hookworm

G. lamblia

S. stercoralis

A 10 min search in a slide after i.e. Ridley concentration Both effective for helminths and protozoa!!

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Diagnosis of intestinal helminths

…..is there still a need for diagnosis of helminth infections in patients in western hospitals?

S M MD CU

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Outside the western world it is (still) a “wormy world”!

Ascaris Trichuris

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No. holidays / year Dutchmen outside Europe

0

1

2

3 Others

Caribbean

Far East

Egypt

Turkey

USA

Hol

iday

s (x

1,0

00,0

00)

Contacts with a “wormy world” ?

3 million

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http://www.palet.nl

Can we precisely predict who was in contact with a “wormy world”?

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Intestinal helminths of humans: there are many…..

• Ascaris lumbricoides • Trichuris trichiura • Hookworms • Enterobius vermicularis • Hymenolepis nana • Hymenolepis dimunita • Diphyllobothrium latum • Taenia saginata • Taenia solium • Dipylidium caninum • Clonorchis sinensis* • Parogonimus westermani* • Opisthorchis viverrini/felineus* • Heterophyes heterophyes • Fasciola buski

• Strongyloides stercoralis • Schistosoma mansoni* • Schistosoma japonicum* • Fasciola hepatica*

But: all relative easy to recognize with basic microscopy:

Specificty: high Sensitivity: good (after i.e.Ridley concentration, scotch tape), repeat examination can be necessary

Cost of examination: low

* Eggs can be found in stools, adult worms not located in intestine

S M MD CU

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Intestinal helminths

• Strongyloides stercoralis • Schistosoma mansoni

• Fasciola hepatica

Helminth infections were serodiagnosis is preferred for diagnosis in travellers*:

* combined with microscopy when indicated

frequently imported!!

eggs can be found in stools, adults not in intestine

S M MD CU

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Intestinal helminths

Molecular diagnosis • Limited number of molecular targets • Ongoing research, especially in tropics • Little data evaluation in travellers • Replacement microscopy by multiple PCR’s for all

helminth infections most likely not cost effective

S M MD CU

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MD M Ag

Microscopy, molecular diagnosis, serology*

Intestinal protozoa

* for amoebic abcess

S*

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Intestinal protozoa

• Many developments:

• Improved microscopic diagnosis. Triple Feces Test TFT), multiple sampling, use of fixative: strongly improved sensitvity of microscopy, compared to exam. of one, non fixed sample.

• Antigen test for giardia and cryptosporidium: fast and useful

MD M Ag

Triple-Feces-Test (TFT)

Increaded yield TFT* • G. lamblia, E. histolytica / dispar : 25 - 30% • Dientamoeba fragilis: 100% (10% of all patients pos) • Blastocystis spp: 98% (25% of all patients pos) * compared to microscopy of Ridley concentrate of one, non fixed sample

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Intestinal protozoa

Molecular diagnosis • Multiplex PCRs with Giardia, Cryptosporidium, E. histolytica and D. fragilis

• High sensitivity and specificity

• Easy to implement with other molecular tests in lab

MD M Ag

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Intestinal protozoa

Some matters in ongoing debate • T: are all positive PCRs, especially also the low positive ones, of clinical significance?

• T: DNA persists long in stools, difficult for monitoring effect treatment?

• P: Should PCR’s be regarded good screening methods and used in combination with microscopy or is /should it be the aim (multiple) multiplex PCR’s replace all microscopy (also for helminths)?

MD M Ag

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MD S M

F: Serology and molecular diagnosis

Toxoplasmosis

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Toxoplasmosis MD S M

Clinical problems • Immunocompetent persons

• Primary infection in pregnancy, congenitial toxoplasmosis

• Toxoplasmosis in patients with immunosuppression

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Toxoplasmosis

• Immunocompetent persons: serology • IgG quantitiative, IgM semi-quantitative, avidity IgG

MD S M

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Toxoplasmosis

• Moment of infection in pregancy: IgG, IgM, avidity IgG

• Congenital infection in pregnancy ? B529 real-time PCR,10 ml amniotic fluid: sensitivity 92.2%, NPV 98.1% (Wallon et al 2010)

• Evaluation child: IgG, IgM, IgA, Westernblotting,(PCR)

MD

S M

Congenital toxoplasmosis

S

S

MD

MD

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Toxoplasmosis MD S M

• In immunosuppressed persons

• In AIDS: major role “clinical suspicion”, serology (IgG pos/neg), radiography (CT, MRI), and effectiveness treatment. PCR of little importance • In transplant patients: serology (repeats!) and molecular diagnosis both of value, but importance of findings strongly dependant of

clinical findings at the time. Microscopy of i.e. CSF or BAL can be very useful !

Cerebral Toxo in AIDS

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Serology, microscopy and molecular diagnosis

S M MD

Visceral leishmaniasis

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Visceral leishmaniasis

Typical (non specific) lab findings: Anemia Neutropenia Thrombocytopenia Eosinopenia Hypergammaglobulinemia

Severe, fatal disease Fever Enlarged liver and spleen Visit endemic area

Recent: Dutch child 6 weeks ill, three hospitals diagnosis: “malignancy” Visit: Nothern Italy, one day coast Med

S M MD

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Visceral leishmaniasis S M MD

DiaMed-IT Leish dipstick: antibodies to rk39 antigen

Molecular diagnosis: PCR and sequencing (when microscopy is negative)

Reliable result:15 min !

Direct Agglutination Test (DAT) : 12 h

Bonemarrow punction

Culture (1-4 weeks)

Microscopy 30-60 min

Visceral leishmaniasis

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Molecular diagnosis and microscopy

CU M MD

Cutaneous leishmaniasis

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Cutaneous leishmaniasis

6y: 471 CL >10 5-10 1-5

CU M MD

Frequently observed Often not recognised as CL From all over world, also Mediterranean area Military personnel frequent South American infections potentially spread to mucous membranes

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Cutaneous leishmaniasis

Biopsy taken from lesion Diagnostic method Sensitivity Microscopy 69% Culture 84% Microscopy + culture 90% Mini Exon Repeat-PCR 98% fast typing possible

CU M MD

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Cutaneous leishmaniasis

L. major 48%

L. tropica 5% L. donovani/infantum complex

3%

L. infantum 15%

L. mexicana 7%

L. braziliensis 10% L. guyanensis

5% L. panamensis 4%

L. naiffi 2%

Leishmania spp. 1%

CU M MD

Different Leishmania species in skin lesions: species directed therapy! No. positive patients: 196

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Different parasitic infections different diagnostic methods!

Malaria Intestinal helminths Intestinal protozoa Toxoplasmosis Leishmaniasis, cutaneous Leishmaniasis, visceral Trypanosomiasis, African Trypanosomiasis South American Acanthamoebiasis Echinococcosis

M Ag MD

S M MD

MD M Ag

CU M MD

S M MD

S M

M S MD

CU MD

S M MD

MD S M

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Conclusions, recommendations

• Microscopy, molecular methods, serology, antigen tests and culture all have a place in diagnosis of parasitic infections in western hospitals

• Don’t think dogmatic about techniques: not one is the best, combinations which combine strengths of different techniques make good diagnosis!

• Think over carefully before what you want to do yourself in your own lab.

• Diagnosis of parasitic infections is an art, as with bacteriology and virology It is not performing one test !

• Outsourcing tests which are difficult or less frequently done is not a shame.

• When decided to do proper parasitic diagnosis, select a motivated small (er) team. Do give them proper training and place them in an high level quality control scheme.

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Acknowledgements Dr. A. Bart, Dept. Clinical Parasitology, AMC Dr. P. van Thiel, Dept. Infectious Diseases, AMC Technicians, Dept. Clinical Parasitology, AMC Dr. J. van Hellemond, Harbour Hospital, Rotterdam R. Koelewijn, Harbour Hospital, Rotterdam

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