World Health Organization ctd/mal/97. 20 Rev. 2 2001 Organisation Mondiale de la Santé distr.: Limited original: english

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World Health Organization

CTD/MAL/97.20 Rev. 2 2001

Organisation Mondiale de la Santé

DISTR.: LIMITED

ORIGINAL: ENGLISH

IN VITRO MICRO-TEST (MARK III) FOR THE ASSESSMENT

OF THE RESPONSE OF Plasmodium falciparum TO

CHLOROQUINE, MEFLOQUINE, QUININE,

AMODIAQUINE, SULFADOXINE/PYRIMETHAMINE AND

ARTEMISININ

Instructions for use of the in vitro micro-test kit (Mark III)

World Health Organisation

Division of Control of Tropical Diseases

This document is not issued to the general public, and all

rights are reserved by the World Health Organization

(WHO). The document may not be reviewed, abstracted,

quoted, reproduced or translated, in part or in whole,

without the prior written permission of WHO. No part of

this document may be stored in a retrieval system or

transmitted in any form or by any means - electronic,

mechanical or other - without prior written permission of

WHO.

Ce document n

=est pas destiné à être distribué au grand

public et tous les droits y afférents sont réservés par

=Organisation Mondiale de la Santé (OMS). Il ne peut être

commenté, résumé, cité, reproduit ou traduit, partiellement ou

en totalité, sans une autorisation préalable écrite de l

=OMS.

Aucune partie ne doit être chargée dans un système de

recherche documentaire ou diffusée sous quelque forme ou

par quelque moyen que ce soit - électronique, mécanique, ou

autre - sans une autorisation préalable écrite de l

=OMS

The views expressed in documents by named authors are

solely the responsibility of those authors.

Les opinions exprimées dans les documents par des auteurs

cités nommément n

=engagent que lesdits auteurs.

CTD/MAL/97.20

Page 3

1. Content of the Micro-test Kit

1.1 Micro-test kit A (basic kit)

Quantity

Tissue culture plate, 12 x 8 wells, predosed with:

Chloroquine: 1 - 64

pmol per well

Mefloquine: 2 - 128 pmol per well

Quinine:

4 - 256 pmol per well

Amodiaquine: 0.25 - 16.0 pmol per well

Artemisinin: 0.15 - 150.0 pmol per well

Sulfadoxine/pyrimethamine: SDX 10 - 10 000 pmol per well

PYR 0.125 - 125.0 pmol per well

NOTE: 12 plates in any combination of 6 types of plates as required

Eppendorf pipette, 50 µl

Eppendorf pipette tip, sterile

400

Stock bottle containing RPMI 1640 LPLF, sterile, 20 ml

Tube, Falcon, presscap, sterile, 6 ml

100

Syringe, sterile, disposable, 5 ml

Needle, 1

2" x 20 gauge, sterile

Microscope slide, frosted edge (box of 72 or 50)

Scalpel holder

Scalpel blade

Forceps

Aluminium foil (roll of 30 m x 30 cm)

Capillary tube, heparin-treated, sterile, 100 µl (tube of 20)

Bulb for capillary tube

Rack, plastic covered wire

Label, round (sheet of 77)

Alcohol swabs

Autolet pricker

Lancet for autolet

100

Platform for autolet

100

Glass writing pencil

Candle, pure paraffin

Buffer for staining, vial with powder sufficient for 1 litre solution

Romanowsky stain A, plastic dropping bottle, 60 ml

Romanowsky stain B, plastic dropping bottle, 60 ml

Curved staining plate

Instructions for use of the Mark III micro-test kit

Document WHO/MAL/83.1000, Rev.1

Record forms (as appropriate to plates)

Photograph of pre- and post-culture parasites (Plasmodium falciparum) 1

Grease, silicone, for candle jar (tube)

Gloves, protective, rewashable, size medium/large (5 pairs each), pairs

CTD/MAL/97.20

Page 4

1.2 Micro-test kit B (replenishment) Quantity

Tissue culture plate, 12 x 8 wells, predosed with:

Chloroquine: 1 - 64

pmol per well

Mefloquine: 2 - 128 pmol per well

Quinine:

4 - 256 pmol per well

Amodiaquine: 0.25 - 16.0 pmol per well

Artemisinin: 0.15 - 150.0 pmol per well

Sulfadoxine/pyrimethamine:

SDX 10 - 10000 pmol per well

PYR 0.125 - 125 pmol per well

NOTE: 6 plates in any combination as required

Eppendorf pipette tip, sterile

200

Stock bottle containing RPMI 1640 LPLF, sterile, 20 ml

Tube, Falcon, presscap, sterile, 6 ml

Syringe, sterile, disposable, 5 ml

Needle, 1

2" x 20 gauge, sterile

Microscope slide, frosted edge (box of 72 or 50)

Scalpel blade

Label, round (sheet of 77)

Alcohol swabs

Lancet for autolet

Platform for autolet

Capillary tube, heparin treated, sterile, 100 µl (tube of 20)

Bulb for capillary tube

Glass writing pencil

Candle, pure paraffin

Buffer for staining, vial with powder sufficient for 1 litre solution

Romanowsky stain A, plastic dropping bottle, 60 ml

Romanowsky stain B, plastic dropping bottle, 60 ml

Instructions for the use of the Mark III micro-test kit

Document WHO/MAL/83.1000, Rev.1

Sets of record forms for the Mark III micro-test kit (as appropriate to plates)

Gloves, protective, rewashable, size medium/large (3 pairs each), pairs

2. Important Notes

2.1 The in vitro micro-test provides information on the quantitative drug response of

P. falciparum irrespective of the patient

=s immune status. It is an epidemiological tool for

assessing baseline sensitivity and for monitoring the drug response of P. falciparum over time

and place and therefore can provide background information for the development and evaluation

of drug policies. Changes in parasite drug sensitivity in vitro can be an indicator of future

therapeutic failure. Unlike the in vivo tests, the results of an in vitro test are not disturbed by on-

going malaria transmission. The in vitro test may be carried out with several drugs

CTD/MAL/97.20

Page 5

simultaneously and is independent of the patient

=s clinical condition. It also permits the

monitoring of drug response with compounds for which an in vivo test is not yet available. In

vitro tests are not, under normal circumstances, employed for the guidance of individual

treatment. In the context of drugs with widely varying absorption (e.g. halofantrine) and in

conjunction with in vivo tests, they permit the differentiation between true resistance and

treatment failure due to pharmacokinetic factors.

2.2 Tissue culture plates predosed with halofantrine (range 0.015 - 15.0 pmol per well),

pyrimethamine (range 0.125 - 125 pmol per well) and pyronaridine (range 0.1 - 6.4 pmol per

well) are available on request, but not routinely provided as part of test kits A and B.

2.3 There are risks associated with taking human blood for the tests. Hepatitis B virus, human

immunodeficiency virus (HIV) and other infectious pathogens can be transmitted by the repeated

use of lancets, needles and other instruments. All materials supplied with this micro-test kit to

handle human blood are disposable. They should be used once only and great care must be taken

with its proper disposal after use. The correct procedures are detailed in document

WHO/MAL/83.1000, Rev.1, Biosafety in in vivo and in vitro studies of human malaria, by

D. Payne.

UNDER NO CIRCUMSTANCES

SHOULD DISPOSABLE MATERIAL BE RE-USED

2.4 In the selection of material used in the WHO standard (Mark III) in vitro micro-test kits,

every effort has been made to specify material that has a long shelf-life at normal ambient

temperatures. However, the RPMI 1640 LPLF liquid medium and the artemisinin-dosed

plates (ART) have a limited shelf-life which is considerably reduced when the material is

stored at normal ambient temperature. Therefore, these items should always be stored at

1C (normal refrigerator temperature) and transported at this temperature.

2.5 Additionally and whenever possible, the other pre-dosed plates (CHL, MEF, QNN, HAL,

AMO, PYR, SDX/PYR, PND) should also be stored at +4

1C.

DO NOT PUT ANY OF THE COMPONENTS OF THE TEST KIT

IN THE DEEP FREEZER

2.6 The most convenient way of transporting temperature-sensitive material is in a thermos or

cool box with appropriate quantities of cooling blocks. If ice is used, it should be sealed in

waterproof bags.

ALWAYS PROTECT TEST MATERIAL FROM

DIRECT CONTACT WITH COOLANT

2.7 On receipt of the micro-test kits from the supplier, the recipient should immediately

separate the components with a limited shelf-life and store them appropriately.

CTD/MAL/97.20

Page 6

3. Layout of Micro-culture Plates

3.1 Chloroquine

Well

(1)

(2)

(3)

(4)

(5)

(6)

(7)

(8)

(9)

(10)

(11)

(12)

pmol/

well

Well A is the control.

Wells B - H represent chloroquine concentrations of 0.2; 0.4; 0.8; 1.6; 3.2; 6.4 and 12.8 µmol per l blood. The

concentrations are expressed in

µmol / l blood as the malaria parasite shows selective uptake of chloroquine.

3.2 Mefloquine (MEF)

Well

(1)

(2)

(3)

(4)

(5)

(6)

(7)

(8)

(9)

(10)

(11)

(12)

Pmol/

well

128

Well A is the control.

Wells B - H represent mefloquine concentrations of 0.4; 0.8; 1.6; 3.2; 6.4; 12.8 and 25.6 µmol per l blood. The

concentrations are expressed in

µmol / l blood as the malaria parasite shows selective uptake of mefloquine.

CTD/MAL/97.20

Page 7

3.3 Quinine

Well

(1)

(2)

(3)

(4)

(5)

(6)

(7)

(8)

(9)

(10)

(11)

(12)

pmol/

well

128

256

Well A is the control.

Wells B - H represent quinine concentrations of 0.08; 0.16; 0.32; 0.64; 1.28; 2.56 and 5.12 µmol per l blood-medium

mixture (BMM). The concentrations are expressed in

µmol / l BMM as the malaria parasite does not show selective

uptake of quinine.

3.4 Halofantrine

(available on request)

Well

(1)

(2)

(3)

(4)

(5)

(6)

(7)

(8)

(9)

(10)

(11)

(12)

pmol/

well

0.015

0.05

0.15

.50

1.50

5.00

15.00

Well A is the control

Wells B - H represent halofantrine concentrations of 0.3; 1.0; 3.0; 10.0; 30.0; 100.0 and 300.0 nmol per l BMM. The

concentrations are expressed in nmol / l BMM since the malaria parasite does not seem to show selective uptake of

halofantrine.

CTD/MAL/97.20

Page 8

3.5 Amodiaquine

Well

(1)

(2)

(3)

(4)

(5)

(6)

(7)

(8)

(9)

(10)

(11)

(12)

pmol/

well

0.25

0.50

1.00

2.00

4.00

8.00

16.00

Well A is the control.

Wells B - H represent amodiaquine concentrations of 0.05; 0.1; 0.2; 0.4; 0.8; 1.6 and 3.2 µmol per l blood. The

concentrations are expressed in

µmol / l blood as the malaria parasite shows selective uptake of amodiaquine.

3.6 Artemisinin

Well

(1)

(2)

(3)

(4)

(5)

(6)

(7)

(8)

(9)

(10)

(11)

(12)

pmol/

well

0.15

0.5

1.5

5.0

15.0

50.0

150.0

Well A is the control.

Wells B - H represent artemisinin concentrations of 3; 10; 30; 100; 300; 1000 and 3000 nmol per l BMM. The

concentrations are expressed in nmol / l BMM as the malaria parasite does not show selective uptake of artemisinin.

CTD/MAL/97.20

Page 9

3.7 Sulfadoxine / pyrimethamine

Ratio 80 : 1. Dose shown for sulfadoxine.

Well

(1)

(2)

(3)

(4)

(5)

(6)

(7)

(8)

(9)

(10)

(11)

(12)

pmol/

well

100

300

1 000

3 000

10 000

Well A is the control.

Wells B - H represent sulfadoxine concentrations of 0.2; 0.6; 2.0; 6.0; 20.0; 60.0 and 200.0 µmol per l BMM. The

corresponding concentrations of pyrimethamine in wells B - H are 0.0025; 0.0075; 0.025; 0.075; 0.25; 0.75 and 2.50

µmol per l BMM. The concentrations are expressed in

µmol / l BMM as malaria parasites show no selective uptake

of sulfadoxine and pyrimethamine

3.8 Pyrimethamine

(available on request)

Well

(1)

(2)

(3)

(4)

(5)

(6)

(7)

(8)

(9)

(10)

(11)

(12)

pmol/

well

0.125

0.375

1.25

3.75

12.5

37.5

125

Well A is the control.

Wells B - H represent pyrimethamine concentrations of 2.5; 7.5; 25; 75; 250; 750 and 2500 nmol per l BMM. The

concentrations are expressed in nmol / l BMM as the malaria parasite does not show selective uptake of

pyrimethamine.

CTD/MAL/97.20

Page 10

3.9 Pyronaridine

(available on request)

Well

(1)

(2)

(3)

(4)

(5)

(6)

(7)

(8)

(9)

(10)

(11)

(12)

Pmol/

well

0.1

0.2

0.4

0.8

1.6

3.2

6.4

Well A is the control.

Wells B - H represent pyronaridine concentrations of 2; 4; 8; 16; 32; 64 and 128 nmol per l BMM. Since it is not yet

known whether malaria parasites take up pyronaridine selectively, the drug concentrations are provisionally

expressed in nmol / l BMM.

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