HAEMOPARASITES

Presenter -: Dr. Subhash Kumawat, JR 1

Central Lab, Department of Pathology,

SMS Medical college, Jaipur

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
• Roman Fever

• Roman physician Francisco Torti- 

Italian=malaria
Mal+aria or “bad air”

• French surgeon Alphonse Laveran-

Parasite moving within a red blood cell

• Sir Ronald Ross-

Malarial Transmission
EPIDEMIOLOGY

 World wide

 Death rate :1.5 – 2.7 million

 In India P.vivax & P.falciparum are common

LIFE CYCLE
1:-Asexual division (schizogony )
Humen (intermediate host)

2:-Sexual development (sporogony)

Female Anopheline mosquito(definitive host)

CYCLE IN MAN COMPRISES

 Pre erythrocytic schizogony
 Erythrocytic schizogony
 Gametogony
 Exo erythrocytic schizogony
LIFE CYCLE OF MALARIA
TRANSMISSION ALSO OCCUR THROUGH

 Blood transfusion
 Bone marrow transplants
 Transplacentaly
 Drug addicts
PATHOGENICITY

Infection with the plasmodium causes

intermittent fever –malaria

Incubation period
(10-14days) in
p.vivax, p.falciparum
& p.ovale

P.malariae 28-30days
COMPARATIVE FEATURE OF MALARIAL PARASITES
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
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
Preparing thick and thin films
THICK SMEAR
Thick smear is based on the principle that during preparation of the
smear, 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
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 features, 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
Exflagellated microgametocyte of P. vivax.
P. falciparum rings
P. falciparum gametocytes
50-year-old patient with severe malaria, haemoglobinuria, jaundice, and renal failure.
P. falciparum rings and schizonts
A and B. P. falciparum infection showing late trophozoites, Maurer’s dots, and rings
 A-D. Extreme P. falciparum parasitaemia (35%) with numerous small rings

 presence of toxic neutrophils in panel C and the banana-shaped female gametocyte in panel D
gametocytes of P. falciparum
chüffner’s dots, amoeboid trophozoites, and rings in enlarged red cells consistent with P. vivax
Schüffner’s dots, amoeboid trophozoites, gametocytes, and ring forms consistent
with P. vivax malaria
P. vivax rings, amoeboid trophozoites, schizonts
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.
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
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%
 Filarial Nematodes
Nematodes which infect the diff. tissues are called tissue or somatic
nematodes.

The Filarial nematodes are the major group of tissue nematodes

Morphology
Adult worms:-
 Whitish, thread &smooth surface
 Lives in lympatics,connective tissue, &muscle
 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

It Observed in lymph node aspirates

Microfilariae may occasionally be observed in bone marrow aspirates,

more commonly in immunocompromised hosts
e.g. patients with HIV, haematological malignancies, and solid tumours
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 by
- sheath pattern
- nuclei distribution &size
All the diagnostic features can be seen in Giemsa stained
films, so occasionally special stains such as Delafield’s
haematoxylin or diluted haematoxylin must be used to
demonstrate them

Use a 10× objective to locate microfilariae (search the entire

blood film systematically)
and switch to 40× and 100× (oil immersion) objectives to
examine microfilariae for specific identification
Identification of microfilariae
Fibres, fibrin, threads, hair, and other artefacts found on blood films are often
confused with microfilariae especially if the patient has eosinophilia
Absence of nuclei rules out identification of these structures
Characteristic basic structure: sheath—head—body—tail;
They do not contain vacuoles and they are not refractile or septated.

Note: spores of helicosporous fungi (e.g. Ηelicosporium, Helicomyces) that

get on the blood film while it is drying, may be mistaken for microfilaria
Spores of helicosporous fungi may be mistaken for microfilariae
Brugiamalayi lymphatic filariasis.
FILARIASIS
A 50-year-old man with eosinophilia and lymphadenopathy. Lymph node biopsy showed
marked infiltration with eosinophils and eosinophilic abscesses. Night thick blood film showing
Wuchereria bancrofti
 Sheath
(pale pink)

Thick film

Eosinophil
Blood tests showed eosinophilia.Capillary blood film showing microfilariae
Concentration method

A concentration method may be used to increase sensitivity

(dilute blood 1:2 in distilled water or saponin saline solution or 2%
formalin to lyse red cells, spin at high speed and examine deposit

Knott method:-
 1 ml blood + 9 ml formalin
 centrifuge at 2000 rpm for 20 mts
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
Microfilaraemia is calculated with the use of a thick film (as described
for P. falciparum).
This method is based on the estimation that about 100 fields, using a
100× (oil immersion) objective is the equivalent of 0.25 μl of blood.

If Loa loa is seen, counts of microfilariae per unit quantity of blood

are necessary before the administration of diethylcarbamazine (DEC)
or ivermectin because high grade L. loa microfilaraemia
(≥8000 microfilariae/ml) is associated with serious complications such
as high fever, encephalopathy (headache and confusion, progressing to
stupor and coma), glomerulonephritis, systemic allergic reaction
(Mazzotti reaction) and excess mortality due to the rapid death of
microfilariae and massive degranulation of eosinophils causing
inflammation
Molecular methods:-

 PCR –detect as low as 1 pg of filarial DNA.

TREATMENT
 DEC(Diethylcarbamazine)
 Leishmaniasis
Leishmaniasis is caused by infection with the flagellate
protozoan parasites of the genus Leishmania. 

Visceral leishmaniasis (kala-azar), caused by 

L. donovanni 

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  Amastigote (Leishman-Donovan bodies)

 Spindle shaped  Round or oaval

 Flagellar  Vertebrate(Humans)stage
 Insect(sand flies)  Non-motile
 Motile  Intracellular
 Aflagellar stage
 In macrophage
Amastigotes (Leishman-Donovan bodies) are recognised by their characteristic basic structure:
they appear as oval or round structures, measuring 2-4 μm in length. Each amastigote has a
dark nucleus and a characteristic perinuclear rod-shaped or dot-shaped organelle called
kinetoplast within a pale bluish-reddish cytoplasm. The kinetoplast stains red purple.
Vector-phlebotomus fly(Sand fly)
Life cycle
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

Leishmania amastigotes are seen within the phagocytes of the

reticuloendothelial system (bone marrow, lymph nodes, spleen, and
liver)
INDIRECT EVIDENCE
Blood examination- pancytopenia ,
mainly neutropenia
↓erythrocyte count
leucopenia, thrombocytopenia.
A:G ratio reversed
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.

Amastigotes are typically found inside macrophages (rarely inside neutrophils or
metamyelocytes, too). Isolated extracellular amastigotes are also commonly seen in aspirates
 A 23-year-old man who had undergone
allogeneic stem cell transplant for AML.
Presented with fever and pancytopenia

Visceral leishmaniasis in
immunocompromised host.
CULTURE
• N.N.N(Novy-MacNeal-Nicolle)
medium is used for culture
• Incubated at 22-24c.
• Promastigote forms can be
demonstrated
GRADING
6+  = >100 parasites/field
5+  = 10-100 parasites/field
4+  = 1-10 parasites/field
3+  = 1-10 parasites/10 fields
2+  = 1-10 parasites/100 fields
1+  = 1-10 parasites/1000 fields (These cases are very difficult.
Careful examination and a high index of suspicion are required)
0= 0 PARASITES/1000FIELD
BONE MARROW ASPIRATION

 From sternum / iliac crest

 Amastigote forms demonstrated in macrophages and monocytes
 Promastigote –demonstrated in N.N.N medium
NOTE:-
Platelets, phagocytosed material, haemosiderin
granules, cellular debris, dust, unidentified objects
or artefacts may be mistaken for leishmania.

These objects however, do not have the basic

structure of amastigotes (nucleus-kinetoplast).
IMMUNOLOGICAL TEST NON SPECIFIC TEST
 Aldehyde test
 Antimony test
 CFT with WKKantigen
(witebsky,kleingenstein&kuhn)

SPECIFIC TEST
 DAT,IHA
 IFAT
 ELISA
TREATMENT

PENTAVALENT ANTIMONIAL SODIUM

STIBOGLUCONATE
THANK YOU……