Haemoparasites....

13 October 2018 1SUNIL KUMAR.P

DEFINITION
 Haemoparasites are those parasites that lives within its
host bloodstream.
 The parasites which are found in blood are
Malarial parasites
Filaria
Leishmania
Trypanosoma
Babesia

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

EPIDEMIOLOGY
 World wide
 Death rate :1.5 – 2.7 million
 In India P.vivax & P.falciparum are common

LIFE CYCLE
 1:-Asexual division (schizogony )
man (intermediate host)
 2:-sexual development (sporogony)
female Anopheline mosquito(definitive host)
CYCLE IN MAN COMPRISES
 Pre erythrocytic schizogony
 Erythrocytic schizogony
 Gametogony
 Exo erythrocytic schizogony

TRANSMISSION ALSO OCCUR THROUGH
 Blood transfusion
 Bone marrow transplants
 Transplacentaly
 Drug addicts

LIFE CYCLE OF MALARIA

 PATHOGENICITY
 Infection with the plasmodium causes
intermittent fever –malaria
 Incubation period (10-14days) in p.vivax,
p.falciparum & p.ovale
 P.malariae28-30days

Contin……
 P.vivax:-benign tertian malaria
 P.malariae:-quartan malaria
 P.ovale:-tertian malaria
 P.falciparum:-malignant tertian malaria &responsible
for black water fever& pernicious anaemia

COMPARATIVE FEATURE OF
MALARIAL PARASITES
FEATURE P.VIVAX P.FALCIPARU
M
P.OVALE P. MALARIAE
schizogony 48hrs 48hrs 48hrs 72 hrs
Forms in P.S Trophozoites
schizont
gametocytes
Rings
gametocytes
Trophozoites
schizont
gametocytes
Trophozoites
schizont
gametocytes
Ring stage 2.5Large
prominent
single
chromatin dot
1.25-1.5Small
delicate double
chromatin dots
multiple rings
Similar to
p.vivax
Similar to
p.vivax
trophozoite Irregular,
amaeboid,vacu
ole present
Compact form
rarely
amaeboid
,pigments
collect into a
single mass
band shaped,
slightly
amoeboid,vacu
ole
inconspicuous
Compact ,not
amoeboid,
vacuole
inconspicuous

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

Clinical feature
 typical features - Febrile paroxysm, anaemia and spleenomegaly
 Febrile paroxysm
comprises 3 successive days.
cold stage:-20 – 60 mts
hot stage:-1 – 4 hrs
sweating stage:- 2 – 3 hrs
• high risk group
pregnancy
children

• Anaemia- microcytic or normocytic hypochromic
• Splenomegaly- enlarged and palpable.
-No relapses in p.falciparum infection but relapses occur in
p.vivax
Complications of p.falciparum infections include
- Pernicious malaria
- Black water fever

Pernicious malaria
Life threatning occur in acute falciparum malaria
Due to heavy parasitization
Various manifestations of PM
1. Cerebral malaria: characterised by
hyperpyrexia,coma and paralysis.Brain is congested
2. Algid malaria:cold clammy skin leading to peripheral
circulatory failure.
3. Septicaemic malaria:high continuous fever ,
involvement of various organs

Black water fever
 It is the manifestation of repeated infections Pl.
falciparum, which were inadequately treated with
quinine
 Clinical condition:
Intravascular haemolysis
High fever
vomiting
haemoglobinuria

IMMUNITY
 Species-specific,
 Stage –specific and lasts only till malarial parasite
infection remains active. (premunition immunity)

DIAGNOSIS
 Specimen: blood
- febrile paroxysm
-Before starting treatment.
METHODS OF EXAMINATION
1)Light microscopy
2)Fluorescence microscopy
3) QBC

Light microscopy
 conventional light microscopy of stained blood smear
is the gold standard for confirmation of malaria
 Ring forms and gametocytes are most commonly seen
in the PBS
THICK & THIN smears are prepared from the capillary
blood
Stained with Giemsa or Leishman stain
Examined under oil immersion lens

Collection of Blood Smears
5.
Touch the drop of
blood to the slide
from below.
4.
Slide must always
be grasped by its
edges.
2.
Puncture at the
side of the ball of
the finger.
3.
Gently squeeze
toward the
puncture site.
1.
The second or third
finger is usually
selected and
cleaned.

Preparing thick and thin films
1.
Touch one drop of
blood to a clean
slide.
2.
Spread the first
drop to make a 1
cm circle.
3.
Touch a fresh drop
of blood to the
edge of another
slide.
6.
Wait for both to
dry before fixing
and staining.
5.
Pull the drop of blood
across the first slide in
one motion.
4.
Carry the drop of blood
to the first slide and
hold at 45 degree angle.

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.
USES(THICK SMEAR)
i. Detecting parasites
ii. quantitating parasitemia and
iii. Demonstrating malarial pigment
not used for species diagnosis
Not used for Species diagnosis

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.

THIN SMEAR
i. Detecting parasites and
ii. for determining the species of the infecting parasite
The major diagnostic feature s, which suggest
P.falciparum in a stained blood smear are
 Occurrence of ring forms alone or along with
gametocytes
 the tendency for multiple rings in an individual RBC
with ‘accole’ forms.

 Presence of maurer’s clefts in the RBC’s containing
large rings, and
 Banana –shaped gametocytes
The diagnosis of malaria is ruled out by obtaining
negative thick blood smears on at least 3 different
occasions

Plasmodium falciparum
Rings: double chromatin dots; appliqué forms;
multiple infections in same red cell
Gametocytes: mature (M)and
immature (I) forms (I is rarely
seen in peripheral blood)
Trophozoites: compact
(rarely seen in
peripheral blood)
Schizonts: 8-24 merozoites
(rarely seen in peripheral blood)
Infected erythrocytes: normal size
M I

Plasmodium vivax
Trophozoites: ameboid; deforms the erythrocyte
Gametocytes: round-ovalSchizonts: 12-24 merozoites
Rings
Infected erythrocytes: enlarged up to 2X; deformed; (Schüffner’s dots)

Species Differentiation on Thin Films
P. falciparum P. vivax P. ovale P. malariae
Rings
Trophozoite
s
Schizonts
Gametocytes

GRADING
 1 – 10 PARASITES/100 FIELD- +
 11 – 100 PARASITES/100 FIELD + +
 1 – 10 PARASITES /FIELD + + +
 >10 PARASITES /FIELD + + + +

QBC
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.

Reagents
Each QBC capillary blood tube is internally coated with
Acridine Orange , Potassium oxalate, Sodium Heparin
and K2EDTA.
Prepare and centrifuge blood tube
1. Fill the QBC capillary blood tube, from end nearest
the two blue lines, directly from a finger(or
heel)puncture or a collection tube of well-mixed
venous blood
- fill the tube by capillary action to a level between the
two blue lines .
-wipe off any blood on the outside of the tube

2. Keep the tube nearly horizontal and roll between the
fingers several times to mix the blood with the
anticoagulant coating.
3.Turn the tube around and tilt, allowing the blood to
flow to the end with the orange-coated stain.roll the
tube between the fingers 5 times to mix the blood with
the staining agent.

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.

QBC MICROSCOPY

FLUORESCENCE MICROSCOPY
Kawamoto technique
 blood smears are stained with acridine orange
 This result in a differential staining of the malarial
parasites.
 Nuclear DNA is green & cytoplasmic RNA is red
 The stained slide is examined with a flourescent
microscope
 Sensitivity 90%

SEROLOGICAL DIAGNOSIS
 IHA
 IFA
 ELISA
-To identify the infected donors incase of transfusion
malaria.
-To confirm past malaria in patients.
-Epidemiological survey in malaria.

Rapid diagnostic tests (DIPSTICK
METHOD)
 Principle
Enzyme Immunoassay.
• Which detects HRP-2
 Dipstick containing monoclonal antibodies directed
against the parasitic antigens ,is used
 Takes 5mnts
 Test has High sensitivity & specificity.
 Useful in detecting only P.falciparum

Molecular diagnosis
 DNA probe: high sensitive & specific.
 Detect <10/µl of blood.
 PCR
TREATMENT:
 Chloroquine –acute malaria
 Mefloquine active against chloroquine resistant strains
 Artemisinin & its derivatives newer drugs

Filarial nematodes
Nematodes which infect the diff tissues are called tissue
or somatic nematodes.
The Filarial nematodes are the major group of tissue
nematodes
Family filarioidea

FILARIASIS
PARASITE ADULT MICROFILARIA CHARACTERISTI
CS
1.LYMPHATIC
FILARIASIS
Wu.bancrofti lymphatics blood sheathed
Brugia malayi lymphatics blood sheathed
Subcutaneous
Loa loa Connective tissue blood sheathed
Onchocerca
volvulus subcutaneous skin unsheathed
Serous cavity
Mansonella ozardi Body cavities
blood unsheathed
Mansonella
perstans
Body cavities blood

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 PB, hydrocele fluid,& chylous urine
 Covered by hyaline sheath

Life cycle
 Definitive host:-human
 Intermediate host:-female culex anopheles & aedes
mosquito
 Infective form:-third stage larva (mosquito)

Clinical presentation
 Fever
 Lymphangitis
 Hydrocele
 Chyluria
 elephantasis

Lab diagnosis
 Blood collection:-
 Mid night (nocturnaly)
 Other specimens-chylous urine,hydrocele fluid.
 15 -20 mts after administration of DEC drug
 Detection wet preparation
 Thick & thin smear examination
 Microfilaria differentiated
- sheath pattern
- nuclei distribution &size

Concentration method
Knott method:-
 1 ml blood + 9 ml formalin
 centrifuge at 2000 rpm for 20 mts
Survey work:-
 Counter chamber technique
 Membrane filter conc. technique

DEC provocation test
 2-8mg/kgBW, DEC orally administrated
 After 30 mts capillary blood collected
 By wet mount & stained smear
 QBC TEST
 Urine microscopy
SEROLOGICAL TEST
 IHA; IFA
 ELISA

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

LESHMANIA
 World wide
 Only the visceral form(kala azar)is associated with
organism in haemopoeitic tissue
 Indian visceral leishmaniasis is caused by L.donovani

MORPHOLOGICAL FORMS
 Promasitogte
 Spindle shaped
 Flagellar
 Insect(sand flies)
 Motile
 Amastigote
 Round or oaval
 Vertebrate stage
 Non-motile
 Intracellular
 Aflagellar stage
 In macrophage
Digenetic Life Cycle

Vector-phlebotomus fly(Sand fly)

LIFE CYCLE13 October 2018 54SUNIL KUMAR.P

Clinical features
produces KALA AZAR(black disease)
 Visceral leishmaniasis is a serious & pottentially fatal
systemic disease caused by L .donovani
 Incubation period:-3 – 6 months high fever
 Pyrexia
 Spleenomegaly
 Hepatomegaly
 Lymphadenopathy

Haematological
 Anaemia
 Leucopenia
 Thrombocytopenia
LAB DIAGNOSIS
 Specimen :-
-Blood
-B.M aspiration & splenic puncture

cs
LYMPH
NODE
PUNCTURE
BONE
MARROW
PUNCTURE
SPLEEN
PUNCTURE13 October 2018 58SUNIL KUMAR.P

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.

CULTURE
• N.N.N medium is used for culture
• Incubated at 22-24c.
• Promastigote forms can be demonstrated

PROMASTIGOTES

culture
 N.Nnmedium
 GRADING
 >100 PARASITES/FIELD 6+
 10 -100 PARASITES/FIELD 5+
 1 -10 PARASITES/ FIELD 4+
 1 – 10 PARASITES/10 FIELD 3+
 1 -10PARASITES/100FIELD 2+
 1- 10 PARASITES/1000 FIELD 1+
 OPARASITES/1000FIELD 0

BONE MARROW ASPIRATION
 From sternum / iliac crest
 Amastigote forms demonstrated in macrophages and
monocytes
 Promastigote –demonstrated in N.N.N medium

INDIRECT EVIDENCE
 Blood examination- pancytopenia , mainly
neutropenia
 ↓erythrocyte count
 leucopenia, thrombocytopenia.
 A:G ratio reversed

IMMUNOLOGICAL TEST
NON SPECIFIC TEST
 Aldehyde test
 Antimony test
 CFT with WKKantigen(Ag prepared from TB by
witebsky,kleingenstein&kuhn
SPECIFIC TEST
 DAT,IHA
 IFAT
 ELISA

TREATMENT
 PENTAVALENT ANTIMONIAL SODIUM
STIBOGLUCONATE

BABESIA
Named after Babes – in blood of cattle & sheep
 Tick borne disease;(USA&Europe)
 3 species B.bovis , B.divergens , B microti.
 Habitat :-inside RBC
MORPHOLOGY
INFECTIVE FORM:
 sporozoite (ticks)
Trophozoites
Oval & spindle shaped
 size 1 – 5 µm
 Small chromatin dot and scanty cytoplasm

Merozoite:-oval or round present as pyriform bodies
arranged in pairs or multiples of 2
LIFE CYCLE
 Definitive host:-ticks of ixodes,boophilus
 Intermediate host:-man & domestic animals

Severe complications
 Acute tubular necrosis
 Pulmonary oedema
 Respiratory failure

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

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

Lab diagnosis
 Thin & thick smear
 Stain:-leishman,wrights,giemsa
 Only ring form seen
 Feature:-
 Haemozoin & gametocyte are absent
 Merozoite arranged in tetrads or maltese cross form

Serodiagnosis
- IFA
MOLECULAR DIAGNOSIS;--PCR
OTHER TEST:-
 Serum protein electrophoresis:-poly
clonalgammapathy
 Urine:-haemoglobinuria & proteinuria
 Haptoglobin:-decreased

treatment
 Combination of clindamicin (600mg 3times daily) &
oral quinine20mg/kg body wt daily for children;
650mg 3-4times daily for adults)
 Period 7-10 days.

TRYPANOSOMA
 These are haemoflagellates that live in blood & tissue
of their hosts.
 They cause two important diseases.
 1:-sleeping diseases
 2:-chagas diseases.
CHAGAS DISEASES (American trypanosomiasis)
 it is caused by protozoan parasites T.cruzi .
 Indurated inflammatory lesion called chagoma

Life cycle
 Definitive host:-man
 Intermediate host:-reduviid bugs
Clinical features
 Malaise
 Fever
 Anorexia
 Oedema of face
 Hepatosplenomegaly
 lymphadenopathy
 Development of subcutaneous inflammatory nodule

Laboratory diagnosis
 Wet,thin &thick blood film
 Conc .method
 Serodiagnosis
-IHA
-IFA
-ELISA
 Molecular diagnosis
 Blood culture- NNN MEDIUM
 -LIT MEDIUM
 Animal inoculation(mice)

Sleeping sickness(african
trypanosomiasis)
 Caused by
-T.brucei
 Clinical features
 Head ache
 Arthralgia
 Malaise
 Wtg loss
 Oedema
 Hepatosplenomegaly

Life cycle
 Defenitive host-man
 Intermediate host-tse tse fly
Hematological abnormalities
 Anemia
 Luekocytosis
Treatment
 Pentamidine & Arsenical

THANK YOU……

Haemoparasites....

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Haemoparasites....