Diagnostic procedures of Schistosomiasis in high and low endemic areas/immigrants

1. COLLEGE OF MEDICINE AND HEALTH SCIENCES
SCHOOL OF HEALTH SCIENCES
BLS Dpt
Year 4
PARASITOLOGY IV
ASSIGNMENT
TOPIC: Diagnostic procedures of Schistosomiasis
in high and low endemic areas/immigrants
GROUP 10 MEMBERS
215040646 NSENGIYUMVA Emmanuel
215040593 NSENGIMANA Bernard
215040715 NSENGIYUMVA Prosper

2. INTRODUCTION
• Schistosomiasis is parasitic disease caused by schistosoma species.
affect more than 200 million people worldwide, and has been
classified as the second most common tropical disease following
Malaria.
• The schistosomes are a group of closely related flukes that inhabit the
portal vascular system of a number of animals(blood flukes).
• Species which infect human: S. mansoni,, S. haematobium, and S.
japonicum, S. Intercalatum, and s. mekongi.
• Transmission: cercariae penetration of human host skin
• Schistosomiasis manifestation: Schistosomiasis manifested in three
stages, The first is initiated by the penetration and migration of the
schistosomula, the second or intermediate begins with oviposition and
is associated with a complex of clinical manifestations (fever and
chills, patients experience cough, urticaria, arthralgia,
lymphadenopathy, splenomegaly, abdominal pain, and diarrhea )and
The third or chronic stage is characterized by granuloma formation
and scarring around retained eggs.

3. • katayama syndrome: syndrome characterized by
leukocytosis; marked peripheral eosinophilia; and
elevated levels of IgM, IgG, and IgE
immunoglobulins
• Infective stage: Cercariae
• Definitive host: Human
• Intermediate host: snails, bulinus spp , oncomelania
spp, and biomphalaria spp for schistosoma
haematoboum, s. japonicum,and s. mansoni
respectively
• Reproduction: the schistosomes are dioecious,
sexual dimorphism; the male surrounds the female
and encloses her within his gynacophoric canal for
the entire adult lives of the worms.
• Treatment: Praziquantel (PZQ) is the drug of first
choice for treatment of all types of schistosomiasis

4. LIFE CYCLE

5. DIAGNOSTIC PROCEDURES OF SCHISTOSOMIASIS IN
HIGH AND LOW ENDEMIC AREAS/IMMIGRANTS
• Schistosomiasis diagnostic procedures in low endemic
area use tests or procedures with high sensitity and
specifity than in high endemic area in order to find even
light infections
• Laboratory procedures includes direct methods and
indirect methods.
• Direct methods are used for detection of eggs, while
indirect methods are used for detecting specific
antibodies and antigens.
• Sample : urine for urinary schsitosomiasis (S.
hematobium), blood and/or serum, stool for intestinal
schistosomiasis, biopsy, and lavages

6. MICROSCOPIC EXAMINATION
• For the diagnosis of schistosomiasis,
microscopic detection of excreted eggs in stool
or in urine remains the gold standard.
• In direct examination a small quantity of the
faeces, is placed on glass slide along and drops
of physiological or normal saline are added.
• The faecal sample is thoroughly emulsified
,evenly spread over the slide, covered with
cover slide and examined under the low power
of microscope.

7. Cont’
• This method is easy and useful in heavy
infection.
• In case no egg is found, does not mean human
host is worm free.
• So, Varieties of techniques are available such as;
direct concentration, sedimentation concentration,
concentration floatation and filtration assays to
increase sensitivity.
• The principle of these methods is to concentrate
all the eggs in a given amount of faeces.

8. Direct concentration(Kato katz )
• In the Kato-Katz technique faeces are pressed
through a mesh screen to remove large particles.
• A portion of sieved sample is then transferred to the
hole of a template on a slide. After filling the hole,
the template is removed and the remaining sample
is covered with a piece of cellophane soaked in
glycerol.
• The glycerol clears the faecal material from around
the eggs. The eggs are then counted and the number
calculated per gram of faeces.

9. Sedimentation concentation
• The majority of trematode eggs are too
large and heavy to float reliably in the
flotation fluids normally used for
nematode eggs.
• They do however sink rapidly to the
bottom of a faecal/water suspension and
this is the basis of the faecal
sedimentation technique

10. Principle of Formal ether (Formalin-Ethyl
Aceatate) sedimentation technique
• Sedimentation techniques use solution of
lower specific gravity than the parasitic
organisms, thus concentrating the latter
in the sediment.
• Ethyl acetate is used as an extractor of
debris and fat from the feces and leaves
the parasites at the bottom of the
suspension.

11. Floatation Concentration technique
• Eggs are separated from faecal material and
concentrated by a flotation fluid of an appropriate
specific gravity. The principle is to use an emulsifying
fluid of a greater specific gravity than that of the parasite
eggs, which result in flotation of eggs in the solution.
Thereby mere examination of the solution in the top
most layer will clearly indicate the presence of egg. The
faecal material and fiber settle at the bottom
Filtration assays
• Urine filtration is recommended by the WHO for S.
haematobium. A standard 10-ml of the urine to be tested
is forced through the device with a syringe. If eggs are
present, they are unable to pass through a Millipore filter
and can be observed and counted under the microscope

12. McMaster counting technique
• This is a quantitative technique to determine the
number of eggs present per gram of faeces
(e.p.g.). A flotation fluid is used to separate eggs
from faecal material in a counting chamber
(McMaster) with two compartments.
• Multiply the total of eggs in 2 chamber by 50 .
This gives the e.p.g. of faeces. (Example: 12
eggs seen in chamber 1 and 15 eggs seen in
chamber 2 = (12 + 15) x 50 = 1350 e.p.g.)

13. Eggs of schistosoma spp
SCHISTOSOMA HEMATOBIUM SCHISTOSOMA JAPONICUM
SCHISTOSOMA MANSONI
SCHISTOSOMA INTERCALATUM

14. Diagnostic procedures of Schistosomiasis low
endemic areas/immigrants
• Diagnosis of schistosomiasis in low
endemic area and in immigrants is
done with serological tests and
molecular biology tests to measure
even less infection that can not be
seen on fecal or urine microscopic
exam.

15. Serological tests
• Serological tests done by detecting
Antibody and/or antigen in sample of
infected patients who have traveled in
schistosomiasis endemic areas and in
whom eggs cannot be demonstrated in
fecal or urine specimens .

16. Antibody detection
• This performed with Detection of antibodies to
schistosoma adult worm microsomal antigen
including mansoni adult worm microsomal
antigen [MAMA], haematobium adult worm
microsomal antigen [HAMA], japonicum adult
worm microsomal antigen [JAMA]) using
Falcon assay screening test (FAST), enzyme-
linked immunoassay (ELISA), and immunoblot
assays.
• In acute presentation of katayama syndrome,
Antibody detections are generally negative, even
though serology often becomes positive before
eggs become detectable. Seroconversion
generally occurs 4-8 weeks after infection.

17. Antigen detection
• In infected persons, Schistosome antigens are present in
serum and urine, these antigens are referred as CAA
(circulating anodic antigen) and CCA (circulating
cathodic antigens) according to their migratory behaviour
in immunoelectrophoresis.
• These two circulating adult worm antigens are the basis
of antigen capture immunoassays. Measurement of CAA
in the blood, serum, and urine by ELISA-based assays is
sensitive, specific and much less variable than egg
counts. The CCA assay has been further developed as a
point of care (POC) urine ELISA dipstick.
• These tests can differentiate between active and past
infections, as the circulating antigens are probably
present only when there is active infection

18. Test used in schistosoma antigens and
antibodies detection
Test used are:
• Indirect immunofluorescence (IIF) and
• Enzyme-linked immunosorbent assay (ELISA)
• Circumoral precipitin test (COPT),
• the cercaria-Hullen reaction (CHR), and
• Total and fractionated soluble adult worm antigens
(SWAP),
• Bilharziose Fumouze IHA,
• Schistosoma mansoni IgG-ELISA,
• Schistosoma serology microwell ELISA and
• West blotting for confirmation of ELISA test.

19. Schistosoma ELISA Test Principle:
• This test use microwells that are coated with Schistosoma
recombinant antigen.
• Firstly diluted patient sample is incubated in microwells
and containing antibodies which are reactive to antigen
bind to coated microwells and unbounded antibodies are
washed away by washing step,
• secondly, enzyme conjugate anibodies are added and react
with these antibodies, then unbound conjugate antibodies
are washed away,
• thirdly, substrate is added to react with enzymes
conjugated with antibodies, the substrate color will turn
blue.
• Finally the reaction is ended with the stop solution, and
what appears is a yellow color instead of the blue
(Automation/Cortez & Diagnostics 2015).

20. Schistosoma Indirect Fluorescent-
Antibody Tests(IFA-Test)
• Indirect fluorescent-antibody tests are used to
demonstrate the presence of antibodies against a
Schistosoma specific antigen in serum, urine and
stool.
• Antigen is incubated with the patient's serum,
Excess serum is washed away, leaving only
antibodies specific for the antigen present in the
patient's serum bound.
• Then, antibodies labeled with fluorescent dye that
are specific for human antibodies is added. The
sample is viewed with a fluorescence viewer.

21. Indirect Haemagglutination Test (IHT)
• The IHT detects reactivity between antibodies in
the serum of an infected individual and
schistosome antigen-coated red blood cells
which is indicated by agglutination.
• Due to its simplicity and relatively high
sensitivity, this test has been widely used in
large scale community surveys and for
surveillance tool studies in schistosome-
endemic areas.

23. Circumoval precipitin test (COPT)
• The circumoval precipitin test (COPT) is based on
patient serum precipitation with lyophilized eggs or
purified live eggs identified under microscope.
• This method is useful for the diagnosis of S.
mansoni and S. japonicum.
• One drop, about 0.025 mI, of the suspension
containing living larvae, eggs or 6-8 specimens of
immature adults is put into the well of a slide, and 3
drops, about 0.075 ml, of serum is added.
• A cover slip is placed over the well. The slide is
incubated at 34 DC. After 24 hr, the slide is
examined by microscope for the appearance of
precipitates attached to the worms or eggs.

24. COPT

25. Cercarian hullen reaction(CHR)
• The test is simple rapid and sensitive.
• A positive reaction is indicated by formation
of an envelope or a preceracrial sheath around
the ceracariae when incubated in the positive
sera.

26. Fractionated soluble adult worm antigens
(SWAP)
• Soluble adult worm antigens (SWAP) of
Schistosoma mansoni are fractionated by fast
protein liquid chromatography (FPLC) system,
using Q-Sepharose anion-exchange resin, in order to
characterize antigenic fractions that may elicit cell
responses in human schistosomiasis.
• SWAP fractions eluted. The FPLC system resolves
6 fractions, enumerated I to VI, according to the
NaCl gradient.
• The analysis of each fraction on SDS-PAGE showed
that fractions I to IV are constituted by multiple
protein bands with M, ranging from 21 to > 200
kDa.

27. • Large amounts of nucleic acids evidenced in
fractions V and VI, as revealed by ethidium
bromide staining of agarose electrophoresis
gels. Using ELISA, it shows that sera from
chronic schistosomiasis patients contained
antibodies that recognized antigens in
practically all fractions.

28. Western blot
• Western blot :schistosomula antigens preparation
(SAP) and adult worm antigens proteins are
separated denaturing polyacrylamide gel by
electrophoresis according to size, charges and
molecular weight. From the gel, the proteins are
transferred to nitrocellulose membranes.
• The nitrocellulose membranes is blocked with 5% dry
milk
• Appy primary antibody against these proteins.
• After three washes, the membranes are incubated anti-
mouse IgG-alkaline phosphatase conjugate.
• After three washes, the membranes are treated with
alkaline phosphatase reaction (Carvalho et al. 2011) .

29. Western blot cont’
• Results are measured looking for bands
with colors.
• Ex. Recognition of proteins with
approximately 200 kDa, 100 kDa, 43 kDa
and 18 kDa by sera from infected mice in
AWP similar recognition pattern of proteins
from SAP with approximately molecular
weight of 100 kDa, 43 kDa and 18 kDa by
sera of infected mice was observed for
diagnosis of S.mansoni (Carvalho et al.
2011).

31. Specific bands for schistosomiasis-positive
sera.

32. Reagent strip test
Reagent strip for antigens
• Reagent strip used by detecting parasite antigen in urine
of infected individuals. The principle of test is based
lateral flow through a nitrocellulose strip of the sample
mixed with a colloidal carbon conjugate of a monoclonal
antibody specific for Schistosoma circulating cathodic
antigen (CCA).
• The presence of the analyte (CCA) is made visible by
capture of the immune complex of antigen and
carbon-labeled antibody by the anti-CCA monoclonal
antibody that is immobilized on the strip as a test line.
• In addition, a line of immobilized polyclonal anti-mouse
antibodies is used to capture the excess carbon-labeled
antibodies to act as a positive control line.

33. Results

34. Point of care (POC) – CCA dipstick.
• The urine POC- CCA test is a lateral flow
immuno-chromatographic urine dipstick assay
that uses a nitrocellulose strip with a
monoclonal antibody coated test line to detect
the presence of Schistosoma-specific CCA
antigen in urine.
• When urine from an infected individual flows
through the strip, the antigen will bind to the
test line, which becomes visible with the
binding of added labelled monoclonal
antibodies.

35. Results

36. Reagent strip test for blood in urine
(heamaturia)
• This test is based on the pseudoperoxidase
action of hemoglobin and erythrocytes which
catalyzes the reaction of 3, 3’, 5, 5’-
tetramethyl-benzidine and buffered organic
peroxide.
• The resulting colors range from orange to
yellow-green and dark green. Very high blood
concentration may cause the color
development to continue to dark blue
(Diagnostics 2008).

38. Intradermal test
• The test is made intradermally injection of antigen
from adult worm, cercaria and snail, the aim being
to inject 0.01 cc. only. It is read in 10, 15 and 20
minutes, and a positive result is indicated by a
raised, button-like weal, which may have
pseudopodia.
• Intradermal test has widely been used in screening
human population for presence of schistosomiasis
in endemic countries with good results, both
immediate and delayed reactions are recorded
after 15 min and 24 hour of application
respectively

40. Molecular biology tests
• Molecular approaches can be used to detect DNA of
different life cycle stages of S. mansoni by analyzing
different biological samples: feces, snail tissues and
infested water bodies, resulting in diagnosis of vertebrate
and invertebrate host infections and identification of
transmission sites
• The application of PCR as a technique for the detection
of schistosomiasis has been explored for S. mansoni and
S. japonicum in human faeces and urine in areas of
medium and low intensity of infection.
• PCR
• DNA for amplification can be extracted from adult
worm, eggs, and cercaria stages, these can be ribosomal,
mitochondria and nuclear DNA.

41. PCR Amplification
Materials: PCR primers (forward and reverse) ,The reaction
volume of 25 μL consisted of 1.25 units Taq DNA polymerase,
2.5 μL 10× buffer, 1.5 mM MgCl2, 200 μM (each) of dATP,
dCTP, dGTP, and dTTP, 1 μM of each of the amplification
primers and 5 μL (3 ng) of template DNA.
Steps of PCR
• Activation /denaturing step of 15 min at 95°C
• Annealing temperature of 53°C for 1.5 min, and expansion at
72°C for 1 min
• Final extension step at 60°C for 5 min.
• Amplification is conducted in 0.2 mL PCR tubes in a thermal
cycler. The products are analyzed on a 2% agarose gel stained
with ethidium bromide (10 mg/μL) and visualized with UV
light. Bp ladder are used to estimate band size (Ibironke et al.
2011).
• Types of PCR used are real PCR, nested PCR,

42. PCR STEPS
2. ANNEALING
3. EXTENSION
1. DENATURATION

43. PCR REPEATS

44. Loop-mediated isothermal amplification
(LAMP)
• Loop-mediated isothermal amplification
(LAMP) is a unique amplification method with
extremely high specificity and sensitivity able
to discriminate between a single nucleotide
difference.
• It is characterized by the use of six different
primers specifically designed to recognize
eight distinct regions on a target gene, with
amplification only occurring if all primers bind
and form a product

45. Histological tests
• Biopsy is helpful when stool sample findings are
negative or in light infection.
• Mucosal biopsy is effective for visualizing eggs.
• Rectal or bladder biopsy.
• Sigmoidoscopy/proctoscopy: to obtain mucosal
biopsies for diagnosis and to identify
complications such as pedunculated and sessile
polyps
• Cystoscopy : is useful in schistosomiasis with
primary bladder involvement for ulcers, polyps ,
hematuria, dysuria.

46. histology
• Surgical biopsy: used to identify ectopic
schistosomiasis
• Bronchoscopic washings or transbronchial
biopsies : for determining Eggs individuals with
pulmonary involvement.
• Lumbar puncture : for Eosinophils present in
the cerebrospinal fluid (CSF) of individuals with
neurologic involvement.
• Upper endoscopy : to assess for esophageal
varices; treat upper intestinal bleeding with
endoscopic sclerotherapy

47. AA, S Mansoni, B: S. mansoni, C: S. Japonicum, D: S. hematobium in urinary bladder
biopsy

48. Adults worms(cross sectional cuts)
Adults of Schistosoma spp. in lung tissue, stained with
H&E. Images courtesy of Harvard Medical School,
Cambridge

49. Imaging tests
• Ultrasonography: for assessing hepatosplenic disease
with periportal fibrosis or urinary obstruction. It can
demonstrate periportal fibrosis, splenomegaly, portal
collaterals, periportal adenopathy, ureteral obstruction,
and obstructive nephropathy.
• Echocardiography and/or invasive hemodynamic
studies can demonstrate pulmonary hypertension and
cor pulmonale, if present.
• Chest radiographs may show patchy infiltrates in
acute schistosomiasis and can indicate pulmonary
hypertension and cor pulmonale in end-stage chronic
infection, if present.
• Computed tomography (CT) or magnetic resonance
imaging (MRI) scanning may be useful in the
evaluation of CNS disease or in the detection of
periportal fibrosis.

50. A.calcified bladder in a chronic form of schistosomiasis, B. Liver
fibrosis in hepatosplenic schistosomiasis (white areas around the
vessels). There is a thrombus inside the portal vein (red arrow)
A B

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