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Strongyloides

A Neglected Tropical Disease

A Review of Diagnosis

Dr Harsha Sheorey

Clinical Microbiologist St Vincent’s Hospital, Melbourne

Tropical Disease A Review of Diagnosis Dr Harsha Sheorey Clinical Microbiologist St Vincent’s Hospital, Melbourne

Clinical presentations

Asymptomatic carriers (majority)

Reactivation with immunosuppression

Acute infection

Cutaneous – larva currens Abdominal – GI symptoms (uncommon)

Chronic infection with vague abdo symptoms

Hyperinfection

Life threatening GN sepsis/meningitis

When is diagnosis/screening indicated

Acute/recent infection

Larva currens or unexplained eosinophilia, urticarial or serpiginous skin lesions

Diarrhoea/GI symptoms/Pulmonary symptoms in people from endemic areas

Unexpected/unexplained invasive GN sepsis/meningitis

Chronic infection

Refugees/migrants/residents* of endemic areas

PoW, past exposure in endemic areas, sanitation workers

Immunosuppressed/HTLV-1/undergoing

immunosuppression/transplant/chemotherapy

Follow up of treatment

* Including Indigenous Australians

Acute infection Chronic infection

Acute infection

Acute infection Chronic infection
Chronic infection
Chronic infection

What’s available for diagnosis

Eosinophilia

Microscopy (faeces, other)

diagnosis  Eosinophilia  Microscopy (faeces, other)  Culture (faeces, other)  Histology  Endoscopy
diagnosis  Eosinophilia  Microscopy (faeces, other)  Culture (faeces, other)  Histology  Endoscopy

Culture (faeces, other)

Histology

Endoscopy

Radiology

(faeces, other)  Histology  Endoscopy  Radiology  Serology  Intradermal skin tests  Copro-antigen

Serology

Intradermal skin tests

Copro-antigen

Molecular diagnosis

Not

routinely

available

Requenq-Mendez et al. PLOS NTD Jan 2013; 7 (1) e200

What are the issues in diagnosis

Most methods are insensitive

Variable excretion

Eggs not present in faeces

Inexperience

Fresh specimen usually not available

Use of Serology is not well ‘standardised’ Many other methods are non-

Eosinophilia (or IgE levels)

Non-specific – other parasites Transient – only in migratory phase Low PPV for intestinal parasites in travellers from endemic areas (~15%) and ~40% in pediatric refugees If positive, could be a potential marker of Strongyloidiasis in the right groups

Eosinophilia

Parasites that cause Eosinophilia

Overview

Eosinophilia is a reaction to tissue invasive stages of helminth (worm) infection.

Two protozoans, Cystisospora, and Sarcocystis, rarely cause a mild eosinophilia.

The following helminth infections cause eosinophilia, in travelers or otherwise.

Specific Infections

Frequent and Often Intense (>5,000 eos/μL):

Strongyloides (absent in compromised hosts)

Lymphatic filariasis (especially tropical pulmonary eosinophilia)

Toxocara (visceral

Moderate During Larval Migration; Absent or Mild During Chronic Infections:

Ascaris

Hookworm

Fasciola

Clonorchus

Paragonimus

Opisthorchis

Occurs at Various Stages of infection:

Schistosoma

Cysticercosis

Echinococcus (especially if cyst rupture occurs)

Trichuris

Angiostrongylus

Onchocerca

Loa

Gnathastoma

Capillaria

Fasciolopsis

Trichostrongylus

Baylisascaris

Other human and non-human filarias (Dirofilaria, Mansonella)

Anisakis

Specimen for Dx of Strongyloidis

Faeces

Fresh (not refrigerated)

Several (sensitivity increases with numbers)

Respiratory spec: sputum, BAL Duodenal aspirate: Entero-string test, endoscopy CSF - rare

Microscopy

Look for rabditiform larvae; eggs not excreted Highly specific (d/d from Hookworm larvae) Low sensitivity

intermittent shedding

technical experience

Easier if motile

fresh specimen, not refrigerated

without preservative

Wet Prep of duodenal contents Rhabditiform larvae Wet Prep of plate culture or in hyperinfection

Wet Prep of duodenal contents

Rhabditiform larvae

Wet Prep of duodenal contents Rhabditiform larvae Wet Prep of plate culture or in hyperinfection syndrome

Wet Prep of plate culture or in hyperinfection syndrome

Filariform larvae

Culture (concentration)

Fresh specimen required Various techniques

Baermann’s

Harada-Mori’s Agar plate

Water emergence method

Charcoal culture

Baermann’s – Harada-Mori’s – Agar plate – Water emergence method – Charcoal culture Not done routinely

Not done routinely

Baermann’s technique

Baermann’s technique  Most sensitive (cf APC)  Labour intensive  “messy”  Chances of lab

Most sensitive (cf APC)

Labour intensive

“messy”

Chances of lab infection

Harada-Mori culture

Falcon tube Filter paper Faeces specimen Larvae migrate towards water Water
Falcon tube
Filter paper
Faeces specimen
Larvae migrate
towards water
Water

Closed system

Relatively easy to set up

Less sensitive

Superseded by APC

Agar plate culture (APC):

now the preferred technique

Agar plate culture (APC): now the preferred technique

Endoscopy

Any segment of GI involved Non-specific unless larvae/adult seen Ulceration (apthoid, erythematous, serpigenous), bleeding, pustule like lesions (? larvae burying) mucosal oedema, duodenal spasms, thickened folds, brown discoloration, yellowish nodules

Histology

Highly specific Sections of larvae or adults (occasionally eggs) Mainly in duodenum or gastric crypts Eosinophilic infiltrates in lamina propia – directly related to intensity

Histology (H&E)

Histology (H&E)

Radiology

Non-specific findings

Radiology  Non-specific findings  Highly variable – Normal appearance of GIT – Mild edema with

Highly variable

Normal appearance of GIT

Mild edema with thickened folds oh mucosa

Significant dilatation

Stricture (best seen with Barium Swallow)

Intra-dermal skin test

Using somatic and ES antigens Cross reactions with other nematodes Persists after successful treatment Lower sensitivity in HTLV-1 and other immunosuppression Difficult to perform and read

Serology

Various methods used: IFAT, IHA, EIA GPAT, EIA, WB, LIPS

EIA: An optic density (O.D.) ≤ 0.2 is considered negative, while O.D ≥ 0.5 is considered positive; intermediate values are recorded as indeterminate

Various antigens used – variable sensitivity and specificity

Cross reacts with other nematodes

Highly variable response

Exact time of sero-conversion not clearly defined

Varies with stage/severity of infection – wide variation

Generally higher in pts from endemic areas Only IgG tests – difficult to distinguish past/present and Rx

Table 1. Characteristics of the main serological tests for strongyloidiasis.

of the main serological tests for strongyloidiasis. Requena-Méndez A, Chiodini P, Bisoffi Z, Buonfrate D, et

Requena-Méndez A, Chiodini P, Bisoffi Z, Buonfrate D, et al. (2013) The Laboratory Diagnosis and Follow Up of Strongyloidiasis: A Systematic Review. PLoS Negl Trop Dis 7(1): e2002. doi:10.1371/journal.pntd.0002002

Dis 7(1): e2002. doi:10.1371/journal.pntd.0002002 http://www.plosntd.org/article/info:doi/10.1371/journal.pntd.0002002

Luciferase Immuno- Precipitation System: LIPS

LIPS assay was developed based on immunoglobulin Ig G antibody to a 31kD recombinant Strongyloides antigen (NIE) and was compared with an NIE enzyme-linked immunosorbent assay (ELISA). A second antigen, S. stercoralis immunoreactive antigen (SsIR), was tested alone and in combination with NIE.

purified easily and produced in large amounts

LIPS involves fusion of a protein antigen to the enzyme reporter Renilla luciferase (Ruc), expression of the Ruc- antigen fusion in mammalian COS cells, immobilization of the Ruc-antigen fusion on protein beads, and quantitation of antigen-specific antibody by the addition of a coelenterazine substrate and the measurement of light production

generates values with substantial separation between negative and positive antibody responses

QLIPS (quick/rapid) has been designed

Copro-antigen

ELISA on faeces Little cross reaction Easy and inexpensive Better with formalin-treated faeces More human studies required

Molecular diagnosis

Several RT-PCRs designed Targets – 18s rRNA and 28s rRNA Directly on faeces Specificity and sensitivity depends on number of larvae (severity of infection) (cf APC)

~100% in endemic areas

Role in low incidence countries unclear

Multiplex (up to 7 parasites) designed

Follow up of treatment

Faeces micro not reliable

negative does not = cure

excreted intermittently

APC better – multiple over one year

Serology most suitable currently

Abs fall takes between 1-2 years (mini 6 m):

depends on initial titre (endemic > non-endemic)

Should be done in same lab with same method, preferably in parallel

No uniform international criteria/No reliable cut-off

Ratio <0.6 (divide postRx titre by preRx titre)

Current Screening & Issues

Serology (+eosinophilia)

No standardisation (antigen, kits)

Both NOT sensitive/specific enough

EIA has no titre and very narrow range

Fresh faeces for APC

Fresh specimen difficult to get (distances)

Multiple required

4 = ~80% sensitivity

7 = ~100% sensitivity

Combination – ideal

Loa & NC should be ruled out Fresh Multiple samples may be necessary Fresh Multiple
Loa & NC
should be
ruled out
Fresh
Multiple
samples may
be necessary
Fresh
Multiple
samples may
be necessary
Loa & NC
should be
ruled out
BEWARE ! may Intermediate /indeterminat e may be an issue BEWARE take 12 mths !
BEWARE
! may
Intermediate
/indeterminat
e may be an
issue
BEWARE take 12
mths
!
Variable
? APC x4
BEWARE
!
Variable
(EIA:

OD)

Expect definite drop
Expect
definite
drop

yper-infection Syndrome in a Vietnamese man on steroids for Giant cell arteritis

N Engl J M 2013; 368:e15 March 21, 2013ed Since is it associated is critical
N Engl J M
2013; 368:e15 March 21, 2013ed
Since
is
it associated
is critical
with
mortality,
to
the parasite
this
for areas
patients
where
high before from
it in is of
endemic
initiation
a screen hyperinfection immunosuppression.

DRAFT

Before immunosuppression
Before immunosuppression
Everyone PPV ↓ Serology + APCx4 Serology Both Either POSITIV EQUIVOCAL POSITIV E E E
Everyone
PPV ↓
Serology + APCx4
Serology
Both
Either
POSITIV
EQUIVOCAL
POSITIV
E
E
E
E
Rule
out Loa
Get more
loa* and
history
APCx4
pregnancy
Treat with
Ivermecti
POSITIV
NEGATIV
n
E
E
pregnancy Treat with Ivermecti POSITIV NEGATIV n E E Continue with immunosuppression From/been in endemic area
pregnancy Treat with Ivermecti POSITIV NEGATIV n E E Continue with immunosuppression From/been in endemic area

Continue with immunosuppression

From/been in endemic area or doubtful history

From non-endemic area or no history of exposure

NEGATIV

NEGATIV

non-endemic area or no history of exposure NEGATIV NEGATIV * Loa Loa endemic in Western and

* Loa Loa endemic in Western and Central Africa

Repeat testing after immunosuppression: ?

APC= Agar Plate Culture on fresh faece

every 3 mths

Possible Future Screening/tests

LIPS with NIE (in place of EIA)

More sensitive/specific than EIA/IFAT/IHA

PCR (possibly quantitative)

Multiplexed with other parasites

Copro-antigen in faeces (in place of APC)

Can be done on preserved faeces

Combination - ideal

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THANK
YOU!
Don’t forget to
check out