Post on 15-Apr-2017
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
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