Laboratory diagnosis of Malaria

MODERATOR - DR. A. PANCHONIA

Malaria and Man

Ancient History of Malaria

• Malaria parasites have been with us since the dawn of time. They probably originated in Africa (along with mankind), and fossils of mosquitoes up to 30 million years old, show that the malaria vector, the malaria mosquito, was present well before the earliest history.

Hippocrates and Malaria• Hippocrates, a physician

born in ancient Greece, today regarded as the "Father of Medicine", was the first to describe the manifestations of the disease, and relate them to the time of year and to where the patients lived.

History – Events on Malaria• 1753- name of malaria was given.• 1847-Meckel observed presence of pigment in organs.• 1880 - Charles Louis Alphose Lavern discovered malarial

parasite in wet mount. wins Nobel Prize in 1907• 1883 - Methylene blue stain - Marchafava• 1891 - Polychrome stain- Romanowsky• 1898 - Roland Ross - Life cycle of parasite transmission,

wins Nobel Prize in 1902• 1948 - Site of Exoerythrocytic development in Liver by

Shortt and Garnham

Major Developments in 20th Century

• 1955 - WHO starts world wide malaria eradication programme using DDT

• 1970 – Mosquitos develop resistance to DDT Programme fails

• 1976 – Trager and Jensen in vitro cultivation of parasite

Lavern and Ronald RossPioneered the Events on Malaria

Introduction• Malaria is probably one of the oldest diseases known to

mankind that has had profound impact on our history.

• It is a huge social, economical and health problem, particularly in the tropical countries.

• Malaria is a vector-borne infectious disease caused by single-celled protozoan parasites of the genus Plasmodium.

• Malaria is transmitted from person to person by the bite of female mosquitoes.

Global problem

What causes Malaria

• Malaria is caused by a parasite called Plasmodium, which is transmitted via the bites of infected mosquitoes. In the human body, the parasites multiply in the liver, and then infect red blood cells.

• Transmission of Malaria do not occur <160c and >330c

• Do not occur > 2000 meters altitude.

Etiology of Malaria

• Five Species known to infect Man1 Plasmodium vivax – Benign Tertian, Tertian

Malaria(Grassi and Feletti)2 P.ovale - Ovale tertian Malaria(Stephens)3 P.malariae – Quartan malaria (Laveran)4 P.falciparum – Falciparum malaria or

Malignant Tertian malaria(Welch)5 P. knowlesi (Sinton and Muller)

Exo-erythrocytic (hepatic) cycle

Hypnozoites

Sporozoites

Salivary Gland

LIFE CYCLE OF MALARIALIFE CYCLE OF MALARIA

Gametocytes

Erythrocytic Cycle

Zygote

Adapted from:

Oocyst

Stomach Wall

Pre-erythrocytic (hepatic) cycle

LIFE CYCLE OF MALARISA PARASITE

IN MAN• PRE ERYTHROCYTIC

SCHIZOGONY-CRYPTOZOITES.

• ERYTHROCYTIC CYCLE- MEROZOITES.

• GAMETOGONY-GAMETOCYTES

• EXOERYTHROCYTIC CYCLE-PHANEROZOITES.

IN MOSQUITO• GAMETOCYTE• ZYGOTE• OOKINETE• OOCYST• SPOROZOITE

Period of Pre erythrocytic cycle

• 1 P.vivax 8 days• 2 P.falciparum – 6 days• 3 P.malariae - 13 – 16 days,• 4 P.ovale 9 days On maturation Liver cells ruputure Liberate Merozoites into blood stream

Affinity of Parasite to Erythrocytes

• P.vivax Young red blood cells• P.malariae Old red blood cells• P.ovale Young red blood cells

• P.falciparum Infects all age groups Also adhere to the endothelial lining of Blood

vessesl Causes the obstruction, Thrombosis and Local

Ischemias

Erythrocytic Schizogony

• Liberated Merozoites penetrate RBC

• Three stages occur 1 Trophozoites 2 Schizont 3 Merozoite

Trophozoites

• After invasion grow and feed on hemoglobin

• Blue cytoplasm and red nucleus, Called as Signet ring appearance

• Hence called ring form

Schizont

• When the Trophozoite is fully developed becomes compact.

• Malarial pigments are scattered through the cytoplasm

• The Nucleus is large and lies at the periphery starts dividing.

• Becomes Schizont

Exo Erythrocytic Schizogony

• Some Sprozoites do not undergo sporogony in the first instance

• But go into resting stage called as Hypnozoites,( hibernation )

• Within 2 years reactivate to form Schizonts release Merozoites and attack red cell and produce relapses

• Absent in P falciparum

Gametogony• Merozoites differentiate into Male and female

gametocytes• Macrogametocytes also called female gametocytes• Microgametocyte also called as male gametocytes• They develop in the red cells• Found in the peripheral blood smears• Microgametocyte of all species are similar in size• Macro gametocytes are larger in size.

Mosquito cycleA definitive Host – Mosquito

Clinical Features ofMalaria

http://en.wikipedia.org/wiki/File:Symptoms_of_Malaria.png

Malaria Diagnosis

Clinical DiagnosisMalaria Blood SmearFluorescent microscopyAntigen DetectionSerologyPolymerase Chain Reaction

Malaria Blood Smear• Prepare smears as soon as possible after

collecting venous blood to avoid• Changes in parasite morphology• Staining characteristics

• Take care to avoid fixing the thick smear • Risk of fixing thick when thin is fixed with

methanol if both smears on same slide• Let alcohol on finger dry to avoid fixing

thick• Be careful if drying with heat

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 45degree angle.

Recognizing Malaria Parasites

Inside a red blood cell

One or more red chromatin dots

Blue cytoplasm

RING TROPHOZOITE

SCHIZONT GAMETOCYTE

BlueCytoplasm

RedChromatin

BrownPigment

Recognizing Erythrocytic Stages:Schematic Morphology

Malaria Parasite Erythrocytic Stages

Ring form

TrophozoiteSchizont

Gametocytes

Plasmodium falciparum

Rings: double chromatin dots; accole forms;multiple infections in same red cell

Gametocytes: mature (M)andimmature (I) forms (I is rarelyseen in peripheral blood)

Trophozoites: compact(rarely seen in

peripheral blood)

Schizonts: 18-32 merozoites(rarely seen in peripheral blood)

Infected erythrocytes: normal size

M I

Plasmodium vivax

Trophozoites: ameboid; deforms the erythrocyte

Gametocytes: round-oval Schizonts: 12-24 merozoites

Rings

Infected erythrocytes: enlarged up to 2X; deformed; (Schüffner’s dots)

Plasmodium ovaleInfected erythrocytes: moderately enlarged (11/4 X); fimbriated; oval; (Schüffner’s dots)

RingsTrophozoites: compact

Schizonts: 6-14 merozoites; dark pigment;

Gametocytes: round-oval

Infected erythrocytes: size normal to decreased (3/4X)

Plasmodium malariae

Trophozoite:compact

Trophozoite:typical band form

Schizont:6-12 merozoites(rosette like);coarse, dark pigment

Species Differentiation on Thin FilmsFeature P. falciparum P. vivax P. ovale P. malariae

Enlarged infected RBC + +

Infected RBC shape round round, distorted

oval, fimbriated

round

Stippling infected RBC Maurer dots

Schuffner dots

Schuffner dots

Ziemann dots

Trophozoite shape Small ring, accoleform

large ring, amoeboid

large ring, compact

small ring, compact

Chromatin dot often double single large single

Mature schizont rare, 18-32 merozoites

12-24 merozoites

8-12 merozoites

6-12 merzoites

Gametocyte crescent shape large, round

large, round

compact, round

Species Differentiation on Thin Films

P. falciparum P. vivax P. ovale P. malariae

Rings

Trophozoites

Schizonts

Gametocytes

Species Differentiation on Thick FilmsFeature P. falciparum P. vivax P. ovale P. malariae

Uniform trophozoites +

Fragmented trophozoites ++ +

Compact trophozoites + +

Pigmented trophozoites +

Irregular cytoplasm + +

Stippling (“RBC ghosts”) + +

Schizonts visible very rarely often often often

Gametocytes visible occasionally usually usually usually

Calculating Parasite Density - 1• Using 100X oil immersion lens, select area

with 10-20 WBCs/field on Thick smear• Count the number of asexual parasites

and white blood cells in the same fields on thick smear

• Count ≥ 200 WBCs• Assume WBC is 8000/l (or count it)

parasites/l = parasites counted WBC counted

X WBC count/l

Calculating Parasite Density - 2• Count the number of parasitized and

nonparasitized red blood cells (RBCs) in the same fields on thin smear

• Count 1000 RBCs (fewer RBCs if parasitemia is high)

Number of parasite in 1 l Of blood = RBC IN million/cmm X Parasite %

Estimating Parasite DensityAlternate Method

• Count the number of asexual parasites per high-power field (HPF) on a thick blood film

+ 1-10 parasites per 100 HPF++ 11-100 parasites per 100 HPF+++ 1-10 parasites per each HPF++++ > 10 parasites per each HPF

Differentiating Babesia from Malaria

High Power

• Ring shaped trophozites• The intraerythrocytic

trophozoites multiply by binary fission or schizogony, forming two to four separate merozoites. .

• White eccentric “food vacuole” in a ring form.

• Very transient stage in Malaria. Very rarely seen.

the famous Maltese Cross

• Presence of 4 daughter merozoites in a tetrad is pathomnemonic. • However, rarely seen. • Never seen in malaria.

Fluorescent Microscopy• Modification of light microscopy • Fluorescent dyes detect RNA and DNA that is contained in

parasites• Nucleic material not normally in mature RBCs• Kawamoto technique

– Stain thin film with acridine orange (AO)– Requires special equipment – fluorescent microscope– Nuclei of malaria parasites floresce bright green and

cytoplasm red.– Staining itself is cheap– Sensitivities around 90%

Quantitative Buffy Coat (QBC ®)• Useful for screening large numbers of samples• Quick, saves time• Requires centrifuge, special stains• Malaria parasite floresce green yellow against

dark red –black background.

• 3 main disadvantages– Species identification and quantification difficult– High cost of capillaries and equipment– Can’t store capillaries for later reference

Principle of QBC System

Malaria Serology – antibody detection

• Immunologic assays to detect host response

• Antibodies to asexual parasites appear some days after invasion of RBCs and may persist for months

• Positive test indicates past infection• Not useful for treatment decisions

Malaria Serology – antibody detection• Valuable epidemiologic tool in some settings• Useful for

– Identifying infective donor in transfusion-transmitted malaria

– Investigating congenital malaria, esp. if mom’s smear is negative

– Diagnosing, or ruling out, tropical splenomegaly syndrome

– Retrospective confirmation of empirically-treated non-immunes

Target antigens for malaria RDT

pLDH

Pf and pan-specific bands

Closely reflects parasite viability

Asexual and sexual stages

? Potential for monitoring treatment efficacy

Pv, Po, Pm-specific Mabs developed

HRP2Pf-only

Persists after parasite death

AldolasePan-specific

Closely reflects parasite viability

RDTs : test formats

Current RDT formats

• Card / cassette / dipstick

• HRP2• HRP2 & aldolase• pLDH Pf & pan • pLDH Pf & Pv• HRP2, pLDH pan• HRP2, pLDH pan & pLDH Pv• aldolase

"COMBO" tests

RDT procedure

Plastic cassette format of RDT

Card format of RDT

Dipstick format

of RDT

Potential applications for RDTs.

Diagnosis in remote areas

Laboratory-based screening / diagnosis

Rapid outbreak investigation

and surveillance

Confirmation of dubious

microscopy diagnosis

Polymerase Chain Reaction (PCR)

• Molecular technique to identify parasite genetic material

• Uses whole blood collected in anticoagulated tube or directly onto filter paper

Polymerase Chain Reaction (PCR)• Threshold of detection

– 5 parasites/µl • Definitive species-specific diagnosis now

possible• Can identify mutations – try to correlate to drug

resistance• Parasitemia not quantifiable• May have use in epidemiologic studies• Requires specialized equipment, reagents, and

training

Lane S: Molecular base pair standard (50-bp ladder).  Black arrows :size of standard bands. Lane 1: P. vivax (size: 120 bp). Lane 2: P. malariae (size: 144 bp). Lane 3: P. falciparum (size: 205 bp). Lane 4: P. ovale (size: 800 bp).

PCR: identification of malaria species

Comparison of methods for diagnosing Plasmodium infection in blood

PARAMETER MICROSCOPY PCR FLUORESCENCE Dipstick HRP-2 Dipstick pLDH, ICT-Pf/Pv

Sensitivity (parasites/micol) 50 5 50 >100 >100

Specificity All species All species P.f good, others difficult P. falciparum P. falciparum and P.vivax good P.o

and P.m only Pldh

prarasite density or parasitemia Yes No No crude

estimation crude estimation

time for result 30-60 min 24 hr 30-60 min 20 min 20 min

skill level High High Moderate Low Low

equipment Microcsope PCR appratus

QBC apparatus or direct fluorescence microscope

Kit only Kit only

cost /test Low High moderate/low Moderate Moderate

Hame jindgi apni kamiyo ko door karne ke bajay.

Bhagwan ne jo hame khubiya di hai unka upyog karne me gujarni chahiye.

SPEAKER-DR. NARMADA PRASAD TIWARI

a consequence was natural selection for sickle-cell disease, thalassaemias, glucose-6-phosphate dehydrogenase deficiency, ovalocytosis, elliptocytosis and loss of the Gerbich antigen (glycophorin C) and the Duffy antigen on the erythrocytes because such blood disorders confer a selective advantage against malaria infection (balancing selection).[7] The three major types of inherited genetic resistance (sickle-cell disease, thalassaemias, and glucose-6-phosphate dehydrogenase deficiency) were present in the Mediterranean world by the time of the Roman Empire, about 2000 years ago.[8]

The term 'miasma' was coined by Hippocrates of Kos who used it to describe dangerous fumes from the ground that are transported by winds and can cause serious illnesses.[13] The name malaria, derived from ‘mal’aria’ (bad air in Medieval Italian). This idea came from the Ancient Romans who thought that this disease came from the horrible fumes from the swamps