Estimated incidence of malaria episodes ( any species)

resulting from local transmission, country level averages, 2004

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

Stages of malaria infectionStages of malaria infection

Mosquito Vector

Human Host

Sporogonic cycle

Infective Period

Mosquito bitesgametocytemic person

Mosquito bitesuninfected person

PrepatentPeriod

Incubation Period

Clinical Illness

Parasites visible

Recovery

Symptom onset

CLINICAL CLINICAL SIGNS & SIGNS &

SYMPTOMSYMPTOMS OF S OF

MALARIAMALARIAFever

ChillsSweating

Chronic Disease

Chronic Asymptomatic

Infection

PlacentalMalariaAnemia

InfectionDuring

Pregnancy

Developmental Disorders;

Transfusions;Death

LowBirth weight

IncreasedInfant

Mortality

Acute DiseaseAcute Disease

Non-severeAcute Febrile

disease

CerebralMalaria

Death

Outcomes of Malaria InfectionOutcomes of Malaria Infection

Plasmodium falciparum (trophozoite

stage in thick smear)

CCMOVBD

Plasmodium falciparum (trophozoite stage in thin smear)

MORPHOLOGY OF MALARIA PARASITES

CCMOVBDPlasmodium falciparum

CCMOVBDPlasmodium vivax

CCMOVBDPlasmodium malariae

Malaria Tutorials, Wellcome Trust Plasmodium ovale

Features of Plasmodium

CCMOVBD

Nucleus/chromatin dot

Cytoplasm

Stippling

Vacuole

3. Gametocyte

The Malaria Parasite

Three developmental

stages seen in blood

films:

1. Trophozoite

2. Schizont

CCMOVBD CCMOVBD

Trophozoites

CCMOVBD

GametocyteSchizont

CCMOVBD

The two methods common in use :

1: Light microscopy 2: Rapid diagnostic tests (RDTs).

Common methods for parasitological diagnosis of malaria

Goverment recommendation on malaria diagnosis

• Parasitological confirmation (microscopy or RDT) before treatment

• Exceptions:– children under 5 years of age in areas of high

transmission - treatment should be based on clinical diagnosis

– suspected severe malaria, if parasitological confirmation is not immediately possible

Clinical diagnosis

~ Few symptoms, non-life-threatening

+

–

FP

TP

TN

FN

Patient population

FP: False pos.FN: False neg.TP: True pos.TN: True neg.

Advantages of Laboratory DiagnosisLaboratory diagnosis

TN

FP

TP

FN

Drugs saved,

other diseases considered

Cost in health to the patient

Potential costs later to Health Service.

GAIN / LOSS

Anti-malarial drugs

Parasitologic diagnosis of malaria:Advantages

• Improved patient care in parasite-positive patients• Identification of parasite-negative patients in whom

another diagnosis must be sought

• Prevention of unnecessary exposure to antimalarials.

• Improved health information

• Confirmation of treatment failures.

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

At least 2 other pan-specific target antigens commercially available

HRP2 pLDH Aldolase

P.falciparum -specific antigen + +Pan-specific antigen + +P.vivax -specific antigen +

Target antigens of commercially available malaria rapid diagnostic tests

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

• Price range: – Up from $0.50 for dipstick, $0.60 for cassette.

"COMBO" tests

RDT procedure

RDT procedure

RDT procedure

RDT procedure

RDT procedure

RDT procedure

Plastic cassette format of RDT

Card format of RDT

Card format of RDT

Dipstick 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

Special issues on RDTs for acute diseases

Use in remote areas

Limited temperature controlProlonged exposure to tropical temperatures

Poor re-supplyRequire long shelf life

Limited supervision

Potentially rapidly-fatal diseaseHigh sensitivity or limited reliance on negative result essential

Advantages of RDTs

• Lower capital costs and infrastructure

• Less training required

• Lower maintenance costs

• Rapid, (accurate) diagnosis in remote area:

– Reduced patient travel costs / improved access• Improvement in treatment-seeking…

– Improved confidence /esteem of local health worker• Effects on HW morale…

– Improved confidence of patient in diagnosis• Effects on treatment-seeking, compliance with treatment…

Vulnerabilities of RDTs in the field

Cautions:• Detect antigen, not parasites.

• (-) May reflect recent, not current, parasitaemia.

• (+) May reflect total parasite load better than microscopy.

• Degraded by excessive heat.• Limited shelf life.• Accuracy is dependent on technique used.

Current situation ….

• Increased demand for accurate remote diagnosis (drug costs etc.)

• Increasing number of manufacturers entering market

• Widely varying results in field trials

• Impossibility of storage / use within manufacturers temperature specifications.

• Little data on cost-benefits / public health benefit

– When and where to use?

• User-dependent ++

Product ~ Manufacturer / Distributor Palutop Alldiag dBest Malaria Rapid Test Ameritek Rapimal Cellabs Immu-Sure Malaria (?) Malaria Test

Brittney Bio-Analytics C.A.

ICT Malaria Pf ICT Malaria cc Malar-Check Pf Cumberland Hexagon Malaria Human Gmbh KatQuick KAT Medical MakroMAL Makro Medical Paracheck Orchid Biomedical Systems ParaHIT Span Diagnostics Uni-Gold Malaria Pf Trinity Biotech Malaria Pf Vision Biotech Malaria Pf/Pv Vision Biotech Smart Check Malaria World Diagnostics

Pf (HRP2)

NOW-ICT Binax ICT Malaria Pf/pan-malaria ICT Malaria cc Malaria Pf/Pv Rapid Diakey (ShinJin Medic) MalaQuick (~NOW-ICT) r-Biopharm Malaria P.f. RapiCard InstaTest Pf/Pv Cortez Smart Check Malaria Pf/Pv World Diagnostics

Pf HRP2 /pan

OptiMal IT OptiMal 48

Diamed AG

Malaria Test Card International Immunodiagnostics SD Bioline Malaria Pf/Pv SD Bioline

pLDH

First Response® Malaria Antigen Test Premier Medical Corporation

Known commercially available malaria RDT products

Variation in results in published trials… e.g. ICT Malaria, ‘OptiMAL’:

Sensitivity >90% in multiple trials, but…...

Sensitivity (%) for P. falciparum (vs. microscopy)

ICT Malaria Pf, Pf/Pv

82-95 Iqbal et al 2001

82.6 Huong et al 2002 80.8 Stow et al 1999 74-90 Wongsrichanalai 1999 89 Gaye et al 1998 86.2 Mason et al 2002 66.1 Rubio et al 2001 84 Leke 1999

Sensitivity (%) for P. falciparum (vs. microscopy) 'OptiMAL' 80% Hernandez et al 2001 88.5 Jelinek et al. 1999 70.7 Mankhambo et al 2002 83 Ricci et al 2000 49.7 Huong et al 2002 42.6 Mason et al 2002 81.3 Rubio et al 2001

Field trials…

All had poor sensitivity below 100 parasite per microlitre.

Rapidly increasing range of malaria RDT

products

Estimated global productionat 28.2 million RDTs in 2005

Product ~ Manufacturer / Distributor Pf (HRP2) Malaria Pf Rapid Test Device ACON laboratories Palutop Alldiag dBest Malaria Rapid Test Ameritek Malaria Pf-only Rapid Test Biotech Trading Partners Rapimal Cellabs Immu-Sure Malaria Brittney Malaria Test Bio-Analytics C.A. Core Malaria Pf Core Diagnostics Malar-Check Pf Cumberland Assure Malaria Pf Genelabs Smart Check Malaria GlobaleMed Hexagon Malaria Human Gmbh Advantage Malaria Pf J Mitra KatQuick KAT Medical MakroMAL Makro Medical Malaria Pf test Merlin Labs Visitect malaria Pf Omega Diagnostics Paracheck Orchid Biomedical Systems ICT Malaria Pf R&R Market ing ParaHIT Span Diagnostics Uni-Gold Malaria Pf Trin ity Biotech FirstSign Malaria Pf Unimed Malaria Pf Vision Biotech Pf HRP2 /pan NOW-ICT Binax One Step malaria Rapid Test Bio-Quant Inc. Malaria Group Rapid Test with Diff'

Pf Biotech Trading Partners

ICT Malaria Pf/pan-malaria R&R Market ing MalaQuick (~NOW-ICT) r-Biopharm Malaria P.f. RapiCard InstaTest Pf/Pv Cortez Smart Check Malaria Pf/Pv GlobaleMed Malaria Combo Test Merlin Labs ParaHit Total Span Diagnostics Malaria Pf/Pv Vision Biotech Aldolase-only Malaria Group Rapid Test Biotech Trading Partners Malaria Fever Test Merlin Labs pLDH Carestart Malaria Access Bio Core Malaria Pv/Pf Core Diagnostics OptiMal IT

OptiMal 48 Diamed AG

Malaria Test Card International Immunodiagnostics SD Bioline Malaria Pf/Pv SD Bioline Falcivax Zephyr Biomedical Systems First Response Malaria Antigen Test Premier Medical Corporation FirstSign Paraview 2 (Pv/Pf) Unimed HRP2/pLDH Core Malaria Pan/Pv/Pf

Core Malaria Pan/Pf Core Diagnostics

Advantage Malaria J Mitra Visitect Malaria Omega Diagnostics FirstSign Paraview Unimed Parascreen Zephyr Biomedical Systems ?Ag Combo Malaria Home Test Kit Sanitoets (Sallamander concepts) Malaria Test Fortress Diagnostics Bioline Malaria Pacific Biotech

Pf (HRP2)

? Antigen combo

HRP2 / pLDH

pLDH

Aldolse only

Pf HRP2 / aldolase

Similar examples of poor sensitivity exist for other widely-tested RDTs.Inadequate information available to determine reasons for variation

– ? Variation in RDT quality– ? Technique– ? Damaged RDTs

• age of RDTs• storage and transport conditions• exposure to humidity after opening

– ? Poor microscopy as comparison– ? Variation in parasite antigen

Field trials…

Goverment initiative for product testing and quality assurance

• First – product testing to demostrate performance of Ag detection and specificity, stability and ease of use

• Second – post-purchase lot testing to ensure delivered product is consistent with established performance characteristics

• Third – means for end-user to ensure that delivered and stored product has retained these qualities

Minimum requirements of RDT program:

1. Purchase carefully (is RDT useful? Type?)

2. Test repeatedly (QC labs, peripherally)

3. Cool chain for transport and storage

4. Take health worker training and monitoring seriously

5. Have clear policy of action on results

6. Monitor effects on treatment-seeking, morbidity, mortality

….. and allow for this in RDT budget.

Purchasing RDTs

• Species to be detected (P. falciparum / pan-specific)

• Stability in intended conditions of storage and use– demand temperature stability data from manufacturer

– Shelf-life 18+ months

• Ease of use– cassette vs. dipstick

– product instructions

• Packaging (box size, individual packaging)

• Requirement for post-treatment testing

• Viability and quality of manufacturer

• Return /replacement of product

• Cost

• Sensitivity and accuracy required

MICROSCOPY RDTs

Equipment Microscope NoneElectricity Preferred, not necessary None NoneSupplies Blood collection,

staining reagents Blood collection(supplied in some kits

Training Trained microscopist Only minimal training required

Test duration s Usual minimum 60 15–20 minutesLabour-intensiveness High LowSubjectivity High LowRobustness Average High

Cost per test US$ 0.12–0.40 US$ 0.60–2.50

Detection threshold 50 parasites/µl 40–100 parasites/µl bloodDetection of all four species Yes Some RDTsQuantification Possible Not possibleDifferentiation between P. vivax, P. ovale and P.malariae

Possible Not possible

Differentiation between sexulal stages and asexual stages

Possible Not possible

Detection of (P. falciparum) sequestered parasites No Yes

Antigen persistence Not applicable Some RDTs

TECHNICAL SPECIFICATIONS

PERFORMANCE

REQUIREMENTS

DIRECT COSTS

Comparison of RDTs and Microscopy

Microscopy is the gold standard for

diagnosis of malaria

• Parasite density

• Species diagnosis

• Monitoring response to treatment

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 speciesP.f good, others difficult

P. falciparumP. falciparum and P.vivax good P.o and P.m only Pldh

prarasite density or parasitemia

Yes No Nocrude 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 MicrocsopePCR appratus

QBC apparatus or direct fluorescence microscope

Kit only Kit only

cost /test Low High moderate/low Moderate Moderate