الرحمن الله بسمالرحیم

DIAGNOSIS OF MALARIA

By Shervin Ghaffari Hoseini

MD. PhD

Malaria is the world's most important parasitic disease, and kills more people than any other communicable disease except tuberculosis.

Each year 350-500 million cases of malaria occur worldwide, and over one million people die

Laboratory diagnosis of malaria Microscopic Diagnosis

Blood smear Fluorescent Microscopy Quantitative Buffy Coat (QBC ®)

Antigen Detection Immunochromatographic Dipstick: RDT

Serology IFA ELISA

Molecular Diagnosis PCR Real time PCR

Malaria Blood Smear

Remains the gold standard for diagnosis

Blood sample from vein or Puncture from finger pulp

New and clean slide

Both thin and thick films for all patients

Malaria Blood Smear

Thick films: Dry Do not fix but

dehemoglobinate

Stain

Thin films: Dry Fix Stain

Staining methods:Giemsa stainLeishman's stainField’s stain

Malaria Blood Smear

Thick films:

first step: Examination of a thick blood film

20 fold more concentrated species identification difficult minimum of 200 oil immersion fields about 15 minutes for an

experienced observer

Malaria Blood Smear

Thin films:

determine the species entire thin film should be

examined about 20-40 minutes for an

experienced observer

Interpreting Thick and Thin Films

THICK FILM

lysed RBCs larger volume 0.25 μl blood/100

fields more difficult to

diagnose species good screening test

THIN FILM

fixed RBCs, single layer smaller volume 0.005 μl blood/100 fields good species

differentiation requires more time to

read low density infections

can be missed

Calculating Parasite Density - 1

Count the number of parasitized and nonparasitized RBCs in the same fields on thin smear

Count 500-2000 RBCs

% parasitemia = # parasitized RBCs total # of RBCs

X 100

Calculating Parasite Density -2

Count ≥ 200 WBCs on thick film Assume WBC is 8000/l (or count it)

parasites/l =parasites counted WBC counted

X WBC count/l

Estimating Parasite DensityAlternate Method

Count the number of asexual parasites per high-power field (HPF) on a thick blood filmParasites observed Percentage of red cells

parasitized

10-20 per field 1

1-2 per field 0.1

1-2 per 10 fields 0.01

1-2 per 100 fields 0.001

1-2 per 1000 fields 0.0001

1-10 parasites per 100 HPF +

11-100 parasites per 100 HPF ++

1-10 parasites per each HPF +++

> 10 parasites per each HPF ++++

Malaria Blood Smear Prepare smears as soon as possible

Don not fix thick smear

time-honoured peripheral smear study

Identify P. falciparum in a dual infection with P. vivax

Mixed infections are not uncommon.

Malaria Blood Smear

negative test DOES NOT rule out malaria Repeat tests

partial antimalarial treatment sequestration of parasitised cells in

deep vascular beds

malarial pigment in circulating neutrophils and monocytes is

useful

Malaria Blood Smear Advantages Distinguishes between species and

life cycle stages

Parasitemia is quantifiable

Threshold of detection thin film: 100 parasites/ 1 μ lit thick film: 5 -20 parasites/ 1 μ lit

Simple and inexpensive

Malaria Blood Smear

Disadvantages labor-intensive

equipment, training and supervision needed

Fluorescent Microscopy

Fluorescent dyes detect RNA and DNA that is contained in parasites

Nucleic material not normally in mature RBCs

Stain thin film with acridine orange (AO)

Requires special equipment – fluorescent microscope

Staining itself is cheap Sensitivities around 90%

Quantitative Buffy Coat (QBC ®)

Quantitative Buffy Coat (QBC ®)

Advantages Useful for screening large numbers of

samples

Quick, saves time

Due to larger volume of blood observed, method is more sensitive

diagnosis of other diseases such as Babesiosis, Trypanosomiasis and Filariasis is possible

Quantitative Buffy Coat (QBC ®)

Disadvantages Species identification and

quantification difficult: thick/thin films on QBC-positive samples is required

High cost of capillaries and

equipment

Can’t store capillaries for later reference

An adapted light microscope for the viewing of QBC tubes.

Trophozoites of P. falciparum.

Malaria Antigen Detection

Immunologic 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)

Mode of action of common malaria RDT format

Malaria Antigen Detection - RDTs

Feature PfHRP-2 tests pLDH tests

Sensitivity/Specificity*

Sensitivity 92-100%Specificity 85- 100%

Sensitivity P.f. 88-98% P.v. 89-94%Specificity P.f. 93-99% P.v. 99-100%

Commercialcost/test**

Approximately US$ 0.60 –1.00 Approximately US$ 2.50

Commercial products

1) PATH falciparum Malaria IC Strip test – Program for Appropriate Technology in Health

2) MAKROmed™3) Orchid ®

1) OptiMAL® - Flow, Inc.2) Binax NOW ®ICT

Malaria - Binax, Inc.

* Compared to microscopy, results from multiple studies** Varies by size of order and vendor

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)

Antigen DetectionMalaria Immunochromatographic Dipstick

OptiMAL Assay

ControlPlasmodium pan specificmonoclonal antibody

P. falciparum specificmonoclonal antibody

Malaria Antigen Detection - RDTs

Disadvantages The use of the RDT does not

eliminate the need for malaria microscopy Cannot detect mixed infections may not be able to detect infections

with lower parasitemia Cannot detect P. ovale and P. malariae microscopy is needed to quantify

parasitemia

Application of RDTs

Potential uses Epidemics and emergencies Inadequate or absent lab services,

unskilled staff Mobile clinics self-diagnosis by travelers entering

endemic areas outbreak investigation and surveys of

parasite prevalence

Para Sight F test

Malaria Serology – antibody detection

Methods IFA ELISA

Not practical for routine diagnosis of acute malaria because: Delaied development of antibody persistence of antibodies

Serology does not detect current infection but rather measures past experience

Malaria Serology

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 Retrospective confirmation of empirically-

treated non-immunes

Indirect fluorescent antibody (IFA) test. The fluorescence indicates that the patient serum being tested contains antibodies that are reacting with the antigen preparation (here, Plasmodium falciparum parasites).

Polymerase Chain Reaction (PCR)

Molecular technique to identify parasite genetic material

Uses whole blood collected in anticoagulated tube (200 µl) or directly onto filter paper (5 µl)

Polymerase Chain Reaction (PCR)

Advantages PCR is a reference method. It is at least

10-fold more sensitive than microscopy. Threshold of detection

0.1 parasite/µl if whole blood in tube 2 parasites/µl if using filter paper

more reliable for determining species in a mixed infection.

Can identify mutations – try to correlate to drug resistance

May have use in epidemiologic studies

Polymerase Chain Reaction (PCR)

Disadvantages

Parasitemia not quantifiable

Requires specialized equipment, reagents, and training

analysis of a PCR diagnostic test for species-specific detection of Plasmodium DNA.  PCR was performed using nested primers

Real-Time PCR

potential to quantify parasitemia,

may detect multiple wavelengths in same tube identifying multiple species in one run

Needs further research and validation for malaria

Real-Time PCR

Quantitative Real-Time PCR

BCSH Guidelines for Quality Control

All malaria films should be examined by two observers

All new batches of Giemsa stain should be tested with a known P. vivax infection ensure that Schüffner’s dots are stained parasitised cells are decolourised. Blood films for this purpose can be

sealed in plastic slide boxes and frozen

BCSH Guidelines for Quality Control continue

All laboratories must ensure that new staff are adequately trained and maintain their skills: Sets of mixed positive and negative

thick and thin films should be available for examination

reference laboratories can often provide spare films for training purposes.

High quality photographs of malaria parasites should be available for reference

BCSH Guidelines for Quality Control continue

Websites can be used for on-going training. www.dpd.cdc.gov/dpdx/HTML/

Image_Library.htm (Centres for Disease Control and Prevention, USA)

www.rph.wa.gov.au (Royal Perth Hospital, West Australia, click on malaria information for learn and test yourself site)

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

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)

“malariae - like parasite in vivax - like erythrocyte”

Rings

Trophozoites: compact

Schizonts: 6-14 merozoites; dark pigment; (“rosettes”)

Gametocytes: round-oval

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

Plasmodium malariae

Trophozoite:compact

Trophozoite:typical band form

Schizont:6-12 merozoites;coarse, dark pigment

Gametocyte:round; coarse,dark pigment