rhsi 2015, plasmodium species (malaria)

Plasmodium speciesMathew Tut MosesRHSI 2015

Related Tasks:By the end of this session, students are expected to be able to:Describe Morphological Characteristics of Plasmodium species.Explain the mode of transmission of Plasmodium species Explain the effects of Plasmodium species on the hostPerform the laboratory diagnosis of plasmodium species

Introduction to MalariaA serious and sometimes fatal disease caused

by a parasite transmitted by a mosquitoPatients with malaria are typically very sick

with high fever, shaking chills, and flu-like illness

Four kinds of malaria parasites can infect humansPlasmodium Falciparum (deadly)Plasmodium VivaxPlasmodium OvalePlasmodium Malarie

Malaria• Malaria is typically found in warmer regions of the world. In tropical and subtropical climates.

• Malaria parasites which grow and develop inside the mosquito need warmth to complete their growth before they are mature enough to be transmitted to humans.

TransmissionPeople get bitten by an infected female anopheles mosquito (only

anopheles mosquitoes can transmit malaria)When the mosquito bites, a small amount of blood is taken which

contains a small amount of microscopic parasitesThe parasite grows and matures in the mosquito’s gut for about 7

days and then travels to the mosquito’s salivary glandsWhen the mosquito takes its next blood meal, these parasites are

mixed with the saliva and injected into the biteOnce in the blood of the human the parasites travel to the liver and

multiplyAfter 8 days or more the parasites leave the liver and enter red blood

cells where they continue to multiplyMalaria can be transmitted through blood transfusions, organ

transplants, or the shared use of needles or syringes, and to a mother to her fetus before or during delivery (MTCT)

Malaria life cycle• Refer to L.C diagram!

Who is at risk for malaria?• Anyone can get malaria• Most cases occur in residents of countries with malaria transmission and travelers to those countries

• In non-endemic countries, cases can occur in non-travelers as congenital malaria, introduced malaria, or transfusional malaria

Symptoms of Malaria• Fever• Flu-like illness including: shaking, chills. Headache, muscle ache, and tiredness

• Nausea, vomiting and diarrhea may also occur.• Anemia and jaundice may occur due to the loss of red blood cells

• Plasmodium falciparum may cause kidney failure, seizures, mental confusion, coma and death

Symptoms (cont.)

Symptoms begin 10 days to 4 weeks after infection although a person may feel ill as early as 7 days or as late as 1 year later

Plasmodium vivax and plasmodium ovale can relapse

In plasmodium vivax and plasmodium ovale infections some parasites can remain dormant in the liver for several months for up to 4 years after a person has been bitten by an infected mosquito

If an individual has symptoms after traveling in an malaria risk area they should seek medical help immediately

Lab. Diagnosis of malaria• Clinical Diagnosis• Malaria Blood Smear• Fluorescent microscopy• Quantitative Buffy coat • Antigen Detection• Serology• Other tests

Clinical diagnosis of Malaria• Hyperendemic and holoendemic areas• Laboratory resources not needed• Fever or history of fever• Sensitivity ranges from poor to high• Often has poor specificity and predictive values• Overlap with other syndromes

Cont. …• Clinical case description: Fever case with any of the following:

Chills, Sweating, Jaundice, Splenomegaly Convulsions, Coma, Shock, Pulmonary edema & Death (in severe cases)

• Case classification: SUSPECT : Any case of fever PROBABLE : Case that meets the clinicalcase definition CONFIRMED: A suspected/probable case that is laboratory

confirmed

Malaria blood smear• Remains the gold standard for diagnosis

• Giemsa stain• distinguishes between species and life cycle stages• parasitemia is quantifiable

• Threshold of detection• thin film: 100 parasites/Field• thick film: 5 -20 parasites/Field

• Requirements: equipment, training, reagents, supervision

• Simple, inexpensive yet labor-intensive• Accuracy depends on laboratorian skill

Interpreting Thick and Thin FilmsTHICK FILM

lysed RBCs larger volume0.25 μl blood/100 fieldsblood elements more

concentratedgood screening testpositive or negativeparasite densitymore difficult to diagnose

species

THIN FILM fixed RBCs, single layersmaller volume0.005 μl blood/100 fieldsgood species differentiation requires more time to read low density infections can be

missed

Malaria blood smear Cont. • Prepare smears as soon as possible after collecting capillary

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

Diagnostic Points for Plasmodium falciparum• Red Cells are not enlarged. • Rings appear fine and delicate and there may be several in one

cell. • Some rings may have two chromatin dots. • Presence of marginal or applique forms. • It is unusual to see developing forms in peripheral blood

films. • Gametocytes have a characteristic crescent shape appearance.

However, they do not usually appear in the blood for the first four weeks of infection.

• Maurer's dots may be present.

Plasmodium falciparum

Rings: double chromatin dots; appliqué 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: 8-24 merozoites(rarely seen in peripheral blood)

Infected erythrocytes: normal size

M I

Diagnostic Points for P. vivax• Red cells containing parasites are usually enlarged. • Schuffner's dots are frequently present in the red cells • The mature ring forms tend to be large and coarse. • Developing forms are frequently present.

Diagnostic Points for P. malariae• Ring forms may have a squarish appearance. • Band forms are a characteristic of this species. • Mature schizonts may have a typical daisy head appearance

with up to ten merozoites. • Red cells are not enlarged. • Chromatin dot may be on the inner surface of the ring.

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)

Diagnostic Points for P. ovale• Red cells enlarged. • Comet forms common .• Rings large and coarse. • Schuffner's dots, when present, may be prominent. • Mature schizonts similar to those of P. malariae but

larger and more coarse.• Quantifying parasites:

% parasitemia = (parasitized RBCs/total RBCs) × 100

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 Mauer clefts Schuffner spots

Schuffner spots

none

Trophozoite shape small ring, appliqu

large ring, amoeboid

large ring, compact

small ring, compact

Chromatin dot often double single large single

Mature schizont rare, 12-30 merozoites

12-24 merozoites

4-12 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

Parasitemia and clinical correlates

Parasitemia Parasites /l Remarks

0.0001-0.0004% 5-20 Sensitivity of thick blood film

0.002% 100 Patients may have symptoms below this level, where malaria is seasonal

0.2% 10,000 Level above which immunes show symptoms

2% 100,000 Maximum parasitemia of P.v. and P.o.

Parasitemia and clinical correlates

Parasitemia Parasites/l Remarks

2-5% 100,000-250,00

Hyperparasitemia/severe malaria*, increased mortality

10% 500,000 Exchange transfusion may be considered/ high mortality

*WHO criteria for severe malaria are parasitemia > 10,000 /l andsevere anaemia (haemaglobin < 5 g/l).

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

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 microscopeStaining itself is cheapSensitivities around 90%

Quantitative Buffy Coat (QBC)

Fluorescent microscopy after centrifugation

AO-coated capillary is filled with 50-100 µl blood

Parasites concentrate below the granulocyte layer in tube

May be slightly more sensitive than light microscopy but some reports of 55-84%

Quantitative Buffy Coat Cont. …

Useful for screening large numbers of samples

Quick, saves time

Requires centrifuge, special stains

Three main disadvantagesSpecies identification and quantification difficultHigh cost of capillaries and equipmentCan’t store capillaries for later reference

QBC

The QBC TestThe QBC Test

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 forIdentifying infective donor in transfusion-transmitted

malariaInvestigating congenital malaria, esp. if mom’s smear

is negativeDiagnosing, or ruling out, tropical splenomegaly

syndromeRetrospective confirmation of empirically-treated

non-immunes

Malaria Antigen DetectionImmunologic assays to detect specific antigens

Commercial kits now available as immunochromatographic rapid diagnostic tests (RDTs), used with blood

P. falciparum histidine-rich protein 2 (PfHRP-2)parasite LDH (pLDH)

Monoclonal and polyclonal antibodies used in antigen (Ag) capture test

Species- and pan-specific Ab

Cannot detect mixed infections

Cross reactivity with rheumatoid factor reportedly corrected

RDT Test FormatRDT Test Format

Detection of Plasmodium antigens

A: HRP-2 (histidine-rich protein 2) (ICT) B: pLDH (parasite lactate dehydrogenase)(Flow)C: HRP-2 (histidine-rich protein 2) (PATH)

Reporting result of Malaria parasite• Blood film for malaria/(BFFM) or Blood smear for malaria

(BSFM):• Malaria parasite seen (+ve)• No malaria parasite seen (-ve)

• For RDTs or ICT report:• RDTs/ICT for malaria is Negative or,• RDTs/ICT for malaria is Positive.

OTHER TESTS

Polymerase chain reaction (PCR)

Detection of Anti-malarial antibodies

Intraleucocytic malaria pigment

Flowcytometry

Mass spectrometry

Preventing Malaria

Keep mosquitoes from biting you especially at night

Take anti-malaria drugs to kill the parasitesEliminate places around your home where mosquitoes breed

Spray insecticides on your home’s walls to kill adult mosquitoes that come inside

Sleep under mosquito nets – especially effective if they have been treated with insecticides

Wear insect repellant and long sleeve clothing when outdoors at night

Currently there is no vaccine for malaria

TTHHAANNKKSS

Recap Questions• Which kind of malaria parasite is most deadly?

a. Plasmodium falciparumb. Plasmodium vivaxc. Plasmodium ovaled. Plasmodium malariae

Questions• Malaria is typically found in which climate?

a. Subtropicalb. Tundrac. Tropicald. A and C

Questions• Which of the following is not a symptom of malaria?

a. Feverb. Rashc. Nausead. Headache

Questions• Which two kinds of malaria can relapse?

a. Plasmodium falciparum and plasmodium ovaleb. Plasmodium ovale and plasmodium malariaec. Plasmodium malariae and plasmodium vivaxd. Plasmodium vivax and plasmodium ovale

Questions• There is no vaccine for malaria?

a. Trueb. False

Questions• Name 3 ways to prevent malaria: