HAEMOPARASITES

• Dr. Ashok Wiselin

DCP
SUNIL KUMAR.P 13October 2018
1
DEFINITION
Haemoparasites are those parasites that lives within its
host bloodstream.
The parasites which are found in human
blood are
• Malarial parasite
• Filarial parasite
• Leishmania species
• Trypanosoma species
• Babesia species

2
MALARIA
It is a protozoan disease transmitted by the bite of
infected female anopheles mosquito
4 species
P.vivax
P.falciparum
P.malariae
P.ovale

3
LIFE CYCLE
-:1Asexual division (schizogony )

man (intermediate host)

-:2sexual development (sporogony)

female Anophelus mosquito(definitive host)

CYCLE IN MAN COMPRISES

Pre erythrocytic schizogony( hepatic )

Erythrocytic schizogony
Gametogony
Exo erythrocytic schizogony(hypnozoites)

4
TRANSMISSION ALSO OCCUR THROUGH
(rare modes)
Blood transfusion
Bone marrow transplants
Transplacentaly
Drug addicts-contaminated syringes or
needles)

5
LIFE CYCLE OF MALARIAL PARASITE
7
Clinicalfeature

• Febrile paroxysm, anaemia and splenomegaly

• Three stages
• cold stage:-20 – 60 mts
• hot stage:-1 – 4 hrs
• sweating stage:- 2 – 3hrs
• Recur for every 48hrs(p.vivax,p.ovale and p.falciparum)-tertian
malaria
• 72 hrs-p.malariae(quartian malaria)

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• Anaemia- microcytic or normocytic hypochromic

• Splenomegaly

-No relapses in p.falciparum infection but relapses occur in

p.vivax and p.ovale

❖ Complications of p.falciparum infections include

- cerebral malaria (CYTOADHESION)
- Black water fever

9
METHODS OF DIAGNOSIS
• 1.Clinical diagnosis

• 2.Microscopic examination of blood smears

• 3.Rapid diagnostic tests(detection of malarial parasite

antigen or enzyme)

• 4.Fluorescence microscopy

• 5.Serologic techniques

• 6.Detection of nucleic acid of parasite by PCR

MICROSCOPIC EXAMINATIONOF BLOODSMEARS
-before next febrile paroxysm or onset of fever and
chills.
-Before starting antimalarial treatment.
METHODS OF EXAMINATION
)1Light microscopy of thick and thin blood smear
)2Fluorescence microscopy
)3QBC

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Light microscopy
▪ gold standard for confirmation of malaria

➢ THICK & THIN smears are prepared from the capillary

blood or anticoagulated blood

➢ Stained with Giemsa or Leishman stain

➢ Examined under oil immersion lens

➢ Ring forms and gametocytes are most commonly seen

in the peripheral blood smear

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Collection of BloodSmears
1. 4.
The second or third Slide must always
finger is usually be grasped by its
selected and edges.
cleaned.

2. 5.
Puncture at the Touch the drop of
side of the ball of blood to the slide
the finger. from below.

3.
Gently squeeze
toward the
puncture site.

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Preparing thick and thin films
1. • 4.
Touch one drop of • Carry the drop of
blood to a clean
slide.
blood
• to the first slide
2. and
Spread the first • hold at 45 degree
drop to make a 1 angle.
cm circle.

3. • 5.
Touch a fresh drop
• Pull the drop of
of blood to the
edge of another blood across the
slide. first slide in one
motion.
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 THICK SMEAR
Thick smear is based on the principle that during
preparation of the smear, conc red blood cells are lysed
with distilled water, showing intact parasites. The smear
is dried thoroughly and stained.
not fixed
USES(THICK SMEAR)
i. Detecting parasites
ii. quantitating parasitemia (p.falciparum)
iii. not used for species diagnosis

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PLUSSYSTEM GRADING

1– 10 PARASITES/100 oil immersion thick film field - +

11– 100 PARASITES/100 oil immersion thick film field-++

1– 10 PARASITES/single oil immersion thick film field-+++

>10 PARASITES /single oil immersion thick film field -++++

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 Quantitation of parasitaemia is of prognostic value

➢ determine whether parasitaemia is increasing or

decreasing during antimalarial treatment

➢ At least 100-200 fields, each containing 20WBCs

should be examined before a thick smear is reported
as negative for malaria.

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PARASITE DENSITY(P.FALCIPARUM)
• Gametocytes are non pathogenic they should be
excluded while counting parasites
• Two methods- 1.thick smear
• 2.thin smear
• THICK SMEAR
• Parasites are counted for 200 white blood cells
• - parasites per µl of blood is calculated from
•
• total leucocyte count * number of parasites
•
• number of WBC counted
 THIN SMEAR

i. Detecting parasites and

ii. for determining the species of the infecting parasite

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PARASITE DENSITY

• THIN SMEAR

• PERCENT INFECTED RBC=

• ( number of infected RBC÷Total number of RBC counted)*100
•
• Parasites among 1000 red cells is counted and reported as percentage

• >10% parasitemia –indication of exchange transfusion

• -antimalarial treatment –daily,till no parasite is detectable

•
 COMPARATIVEFEATUREOFMALARIALPARASITES

FEATURE P.VIVAX P.FALCIPARU P.OVALE P. MALARIAE

M
fever 48hrs 48hrs 48hrs 72 hrs
Forms seen in Trophozoites Rings Trophozoites Trophozoites
peripheral smear schizont gametocytes schizont schizont
gametocytes gametocytes gametocytes
Ring stage 2.5Large 1.25-1.5Small Similar to Similar to
prominent delicate double p.vivax p.vivax
single chromatin dots
chromatin dot multiple rings
trophozoite Irregular, Compact form band shaped, Compact ,not
amaeboid,vacu rarely slightly amoeboid,
ole present amaeboid amoeboid,vacu vacuole
,pigments ole inconspicuous
collect into a inconspicuous
single mass

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 p.vivax p.falciparum p.ovale p.malariae
schizont 9-10μm almost 4.5-5μm fills 6.2μm fills 6.5-7μm
completely two-third of a about three almost fills a
fills an normal RBC quarters of a normal sized
enlarged rbc slightly RBC
enlarged RBC
merozoite 12 – 24, 18 - 24 6 - 12 6 - 12
gametocytes spherical cresentic round round
Malarial Yellowish- Dark brown- Dark yellowish Dark brown
pigment brown;fine black;coarse brown;coarser
granules than p.vivax

Infected Enlarged, Normal size, Enlarged oval Normal size

erythrocyte Schuffners
dots

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 Plasmodium vivax
Infected erythrocytes: deformed; (Schüffner’s dots)

Rings Trophozoites: ameboid; deforms the erythrocyte

Schizonts: 12-24 merozoites Gametocytes: round-oval

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 Plasmodium falciparum
Infected erythrocytes: normal size

Rings: double chromatin dots; appliqué forms;

M I

Gametocytes: mature (M)and

immature (I) forms (I is rarely
seen in peripheral blood)
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FLUORESCENCE MICROSCOPY
1.QUANTITATIVE BUFFY COAT
PRINCIPLE
Ability of acridine orange to stain nucleic acid
containing parasites.
blood is collected in a capillary tube coated with
fluorescent dye .
After centrifugation the buffy coat in the centrifuged
capillary tube is examined directly under the
fluorescent microscope.
Acridine orange stained malaria parasites appear
brillant green.
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4.Tilt the tube slightly so that blood flows away from the
orange –coated end by at least ¼ to allow space for
installing the closure;then place the index finger over the
end of the tube nearest the blue fill lines.

5.Press a plastic closure onto the unsealed end of the tube.

Then manually twist and press the closure to form a tight
seal.

6.With a clean forceps, pick up a float and insert it into the

unsealed end of the tube.Tap with the forceps until the float
is inside the tube.

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QBC
FLUORESCENCEMICROSCOPY
Kawamoto technique
blood smears are stained with acridine orange
This result in a differential staining of the malarial
parasites.
-nuclei of malarial parasite is green & cytoplasm
is red
The stained slide is examined with a flourescent
microscope

34
Rapid diagnostic tests(DIPSTICKMETHOD)

Principle
Enzyme Immunochromatographic method.
• Which detects HRP-2(histidine rich protein)-asexual
stages and young gametocytes –P.Falciparum
• Parasite LDH-parasite density
Dipstick containing monoclonal antibodies directed
against the parasitic antigens ,is used
rapid
Test has High sensitivity & specificity-p.falciparum
>90%
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SEROLOGICALDIAGNOSIS
Indirect immune fluorescence
Indirect hemagglutination
ELISA
-To identify the infected donors incase of transfusion
malaria.
-To confirm past malaria in patients.
-Epidemiological survey in endemic areas.

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Molecular diagnosis
DNA probe: high sensitive & specific.
Detect <5parasites/µl of blood.
PCR

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 FILARIA

nematodes-slender thread
like worms

Lymphatic filariasis is caused

by Wuchereria bancrofti,brugia
malayi and brugia timori

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FILARIASIS
PARASITE ADULT MICROFILARIA CHARACTERISTI
CS
1.LYMPHATIC
FILARIASIS
Wu.bancrofti lymphatics blood sheathed
Brugia malayi lymphatics blood sheathed
2.SUBCUTANEOUS
Loa loa Connective tissue blood sheathed
Onchocerca
volvulus subcutaneous skin unsheathed
3.SEROUS CAVITY
Mansonella ozardi Body cavities
blood unsheathed
Mansonella Body cavities blood
perstans
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41
Life cycle
Definitive host:-human

Intermediate host:-female culex anopheles &aedes

mosquito

Infective form:- larva (mosquito)

42
Life cycle of filaria
morphology
Adult worms:-
Whitish, thread &smooth surface
Lives in lympatics,connective tissue, &muscle
in males 4 – 6cm,in females 8 – 10 cm
Microfilaria:-
Found in Peripheral blood, hydrocele fluid,&
chylous urine
Covered by hyaline sheath

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 Clinical presentation
Fever
Lymphangitis (filarial fever)
Inflammation of lymphatics of limbs, genitals and
breasts
Axillary or inguinal lymphadenopathy
Lymphedema-fibrosis and obstruction
Late manifestations
Hydrocele
Elephantasis
Chyluria (rupture of urogenital lymphatics)
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Labdiagnosis
• Microscopy- microfilariae
• Filarial antigen in blood –rapid
immunochromatographic test
• Microfilariae –hydrocele fluid or chylous
urine
• Adult worms-lymph node biopsy
• Detection of parasite DNA by PCR

46
MICROSCOPIC EXAMINATION OF BLOOD
• DEMONSTRATION OF MICROFILARIA

• Nocturnal periodicity-10pm to 4am

• Greater in number in capillary blood(skin puncture)
• Scanty in peripheral blood
• Following methods are used
• *thick blood smear
• *concentration techniques
CONCENTERATION TECHNIQUE
• 1.MEMBRANE FILTRATION METHOD

• sensitive method

• Expensive

• 10ml of anticoagulated venous blood –polycarbonate

filter- 3µm or 5µm pore size

• 10ml of methylene blue saline solution –staining

• Viewed in microscope
• 2.MICROHEMATOCRIT TUBE OR CAPILLARY TUBE METHOD
• two capillary tubes filled with anticoagulated blood
• centrifuge for 5 minutes
• capillary tubes are placed in glass tube fixed with adhesive tape
• buffy coat layer is examined for microfilaria
• RAPID IMMUNOCHROMATOGRAPHIC TEST
• -FILARIAL ANTIGEN IN BLOOD( CARD TEST)

• Highly sensitive and specific

• Finger prick blood sample-to detect filarial antigens
• ADVANTAGES
- blood can be collected at any time of the day
- Time -10 to 15 minutes
- No cross reactivity
- Positive result is obtained even if microfilaria is not
circulating in blood
- Remains positive even after 18 months of treatment
- Prevalence of infection in population
DECprovocation test
2mg/kg body weight, DEC orally administrated
It induces microfilaria to appear in peripheral
blood
After 20 to 50 min capillary blood is collected
even in day time for surveys
stained smear
QBC TEST
Urine microscopy

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Molecular methods:-pcr
PCR –detect as low as 1pg of filarial DNA.

TREATMENT
Diethyl ethyl carbamazine

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LEISHMANIA
Obligate intracellular protozoa-GENUS
LEISHMANIA
Bite of infected sand fly-GENUS phlebotomus
Endemic in tropical and sub tropical regions
Three clinical forms-visceral,cutaneous,mucocutaneous
Indian visceral leishmaniasis is caused by L.donovani
(KALA AZAR or BLACK SICKNESS)

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Vector-phlebotomus fly(Sandfly)

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Morphological forms
• Promastigote
• spindle shaped
• flagellar
• sand flies
• motile

• Amastigote
• round or oval
• vertebrate stage
• non motile
• intracellular
• aflagellar 56
• inside macrophages
l
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Clinical features
KALA AZAR (black sickness)
greyish discoloration of skin develops during
course of disease
serious & potentially FATAL systemic disease
caused by L .donovani
Incubation period:-3 – 6 months
high fever
Splenomegaly
Hepatomegaly
pancytopenia
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LAB DIAGNOSIS
• 1.EXAMINATION OF -BUFFY COAT PREPARATION OF PERIPHERAL
BLOOD(67-99%) FOR AMASTIGOTE FORMS(LD bodies)
• -most commonly used
•

• 2.SPLENIC ASPIRATE(95-98%)

• 3.BONE MARROW ASPIRATE(60-85%)

•
 Peripheral Blood smear
➢ Thick blood film-demonstration of
amastigote form

➢ LD bodies –monocytes and

neutrophils

in stained PBS

➢ Smears stained by leishman, giemsa or

wright stain.

➢ It should be differentiated from

histoplasma capsulatum(shows budding
,no kinetoplast)
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SPLENIC ASPIRATE
• Highest sensitivity to detect tissue forms

• DISADVANTAGES
• -fatal haemorrhage

• contraindicated if - platelet count <40000/µl

• prothrombin time >5sec
BONE MARROW ASPIRATION
From sternum / iliac crest

Amastigote forms demonstrated in macrophages and

monocytes

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BONE MARROW ASPIRATION
 • CULTURE

• NOVY-McNEAL NICOLLE(N.N.N )medium is

used for culture

• Incubated at 22-28c.

• Promastigote forms can be demonstrated

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culture
N.N.Nmedium
GRADING
>100 PARASITES/FIELD 6+
10 -100 PARASITES/FIELD 5+
1-10 PARASITES/ FIELD 4+
1– 10 PARASITES/10FIELD 3+
1-10PARASITES/100FIELD 2+
1-10 PARASITES/1000 FIELD 1+
OPARASITES/1000FIELD 0
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SKIN TEST
• MONTENEGRO (LEISHMANIN TEST)

• -delayed type of hypersensitivity reaction

• -0.5ml of killed promastigotes injected intradermally
• -read after 72 hrs,>5mm is positive
• -positive-recovered from kala azar
• -negative – active infection
IMMUNOLOGICALTEST
1.ALDEHYDE TEST
Detect raised non specific immunoglobins(IgM
and IgG)
1ml patient serum +2 drops of 40% formalin
Positive test-turns milky white on standing
Disease >3 months duration

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• 2.DETECT SPECIFIC ANTI-LEISHMANIA ANTIBODIES
• -complement fixation
• -indirect immunofluorescent assay
• -ELISA

• DNA DIAGNOSIS
• polymerase chain reaction
TRYPANOSOMIA
These are haemoflagellates that live in blood & tissue
of their hosts.
They cause two important diseases.
1:- sleeping sickness (African trypanosomiasis)-
T.brucei
2:-chagas disease(American trypanosomiasis)-T.cruzi

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Life cycle-sleeping sickness
Defenitive host-man
Intermediate host-tse tse fly
Bite site-transient chancre
Clinical features: rapidly progessive acute febrile illness
Lymphadenopathy-posterior cervical
CNS-somnolescence,confusion ,stupor ,coma and
death

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Life cycle –Chagas disease
Definitive host:-man
Intermediate host:-reduvid bugs
Clinical features
Malaise
Fever
Swelling of tissues around eyes-romano sign
myocarditis
lymphadenopathy
Development of subcutaneous inflammatory
nodule-CHAGOMA

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Laboratory diagnosis

Wet,thin &thick blood film- peripheral blood

Buffy coat preparation
Lymph node aspiration
CSF
Serodiagnosis
• -IHA
• -IFA
• -ELISA

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74
BABESIA (Piroplasmosis)
• Named after Babes(1883) – intra erythrocytic
parasite in blood of cattle & sheep

Tick borne disease;(USA&Europe)

3 species B.bovis , B.divergens , Bmicroti.

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LIFE CYCLE
Definitive host:-ixodid ticks(sexual cycle)
Intermediate host:-man & domestic animals

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Clinical features
Incubation period :-1 – 4 weeks
Fever
Mild splenomegaly
Anaemia
Jaundice
haemoglobinuria

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haematological
Haemolytic anaemia
WBC count;-normal or decreased
Reticulocyte count:-increased
Thrombocytopenia
ESR:-increased

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Labdiagnosis
Thin & thick smear
Stain:-Leishman
Only ring form seen( mistaken for P.falciparum)
Feature:-
Haemozoin & gametocyte are absent
Merozoite arranged in tetrads or maltese cross form

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80
➢ MOLECULAR DIAGNOSIS;--PCR
➢ OTHER TEST:-
Blood from suspected cases inoculated into
hamsters
After a month blood smear from inoculated
animal show parasites in large numbers

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 BIBLIOGRAPHY
• HENRY’S CLINICAL DIAGNOSIS AND MANAGEMENT BY
LABORATORY METHODS

• APURBA SASTRY – ESSENTIALS OF MEDICAL MICROBIOLOGY

• PANICKERS TEXT BOOK OF MEDICAL PARASITOLOGY

• ROBBINS PATHOLOGICAL BASIS OF DISEASE