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

4. Procedures 

 

4.1 Pre-evaluation of Test Subjects 

 

4.1.1  All subjects should be questioned with respect to their recent history of antimalarial drug 

use.  Persons who have received quinine, artemisinin or artemisinin derivatives within the last 7 

days, 4-aminoquinolines within the last 14 days, pyrimethamine and/or sulfonamides within the 

last 28 days, or mefloquine within the last 56 days should be excluded from the test.  Most urine 

tests give relatively reliable results only for a short period after drug intake, but rarely cover the 

indicated time-spans.  In cases of doubt, the micro-test may be performed, but it would be helpful 

to take a blood sample and have it analysed for residual drug(s). 

 

4.1.2  Written or oral consent, as appropriate, should be taken from all subjects whose blood is to 

be taken for the test. [NB: all patients with symptomatic malaria should be treated with the 

appropriate drug immediately after blood sampling for the micro-test.  In all cases, patient care 

must take priority over carrying out the test.] 

 

4.1.3  Thick and thin blood films should be taken from persons suspected of malaria, and stained 

with Giemsa or another reliable Romanowsky stain.  Patients who have mono-infections with 

Plasmodium falciparum and asexual parasitaemias in excess of 1 000 parasites, but less than 

80 000 parasites per µl blood, are considered suitable for testing.  The preselected patients should 

be subjected to a urine test for 4-aminoquinolines and sulfonamides (and tests for other drugs, if 

available), and those with positive readings should be excluded. 

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4.1.4  Biodata and pre-incubation parasite density should be entered in the appropriate test record 

form (see Annex II for example).  The parts of the form relating to schizont counts are filled in 

after the test has been read. 

 

4.2 Preparation of the growth medium 

 

(i) 

Take the following items from the test kit or cold storage, as appropriate: 

 

Χ

 

1 bottle of 20 ml RPMI 1640 LPLF liquid medium; 

Χ

 

Alcohol swab (for sterilizing the rubber seal of the medium bottle); 

Χ

 

1 sterile syringe, 5 ml; 

Χ

 

2 sterile injection needles for above; 

Χ

 

Number of sterile Falcon tubes, 6 ml, as required (1 per isolate to be 

tested);  mark the tubes with the patient number and the volume of 

medium required; 

Χ

 

1 plastic tube rack (to be used for the Falcon tubes). 

 

(ii) 

Calculate the volume of liquid LPLF medium which will be required for the 

number of planned tests.  Each 100 µl blood sample (isolate) requires 0.9 ml of 

RPMI 1640 LPLF medium to make a total of 1 ml blood-medium-mixture 

(BMM).  This volume is sufficient for a duplicate series (i.e. two columns of 

8 wells) of the same drug or two tests of different drugs (one column of each 

drug).  If more than two drugs are to be tested (or duplicate tests to be conducted 

with more than one drug), then it will be necessary to increase the amount of 

RPMI 1640 LPLF liquid medium (and that of the blood sample) accordingly.  For 

example, if 4 drugs (e.g. CHL, QNN, MEF and ART) are to be tested in single 

tests, then medium and blood samples will be required for 4 columns of 8 wells 

each: that is 2 x 100 µl blood and 2 x 0.9 ml liquid medium which is 200 µl blood 

and 1.8 ml of RPMI 1640 LPLF liquid medium.  If duplicated tests are required 

for the 4 drugs, then the corresponding amounts would be 4 x 100 µl blood and 4 

x 0.9 ml medium. 

 

(iii)  

Take the 5 ml syringe and the two needles, being careful to preserve sterility 

since there is no filtration phase in this test.  Remove the central cap shield of the 

RPMI 1640 LPLF liquid medium bottle, baring the central part of the rubber seal. 

 Wipe the rubber seal of the medium bottle clean with an alcohol swab, let it dry 

and then insert one needle.  Mount the other needle on the 5 ml syringe, pierce 

the rubber seal of the medium bottle and gently withdraw the required volume of 

RPMI 1640 LPLF liquid medium specific for the Falcon tube to be dosed. 

Remove the syringe, fill the medium into the scheduled Falcon tube, and recap 

Falcon tube.  Re-insert the syringe on the needle and withdraw the stipulated 

volume of medium for the next Falcon tube, fill the medium into the Falcon tube, 

and again, recap the tube.  This procedure is repeated until all scheduled Falcon 

tubes have been dosed, followed by the removal and safe disposal of the needles 

from the medium bottle.  If there is any medium left, the bottle should be stored 

in the refrigerator again (at 4

1C) and should be used up within one week. 

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NOTE:  The 6 ml Falcon tubes containing 0.9 ml (or multiples) of RPMI 1640 LPLF liquid 

medium can be stored in a refrigerator (4

1C) for up to 48 hours before use or can be 

transported in the field in a thermos container containing ice cubes or coolant blocks.  

Do ensure, however, that the tubes do not come into direct contact with the ice blocks 

and that they do not become inverted or immersed in water since this could result in the 

contamination of the BMM. 

 

4.3 Performance of the MARK III micro-test 

 

4.3.1  Disinfect the skin site of blood sampling (finger of children and adults, big toe of young 

infants) with an alcohol swab and let the site dry.  With the Autolet apparatus or a sterile, 

disposable haemolancet, prick the finger or big toe and withdraw 100 µl blood into a sterile 

heparinized capillary tube.  Transfer the blood quickly into the duly labelled 6 ml plastic Falcon 

tube containing an appropriate quantity of RPMI 1640 LPLF liquid medium (see section 4.2.b.).  

If more than two test lines are to be run, further capillary tubes need to be filled with blood and 

emptied into the medium. After the blood has been added to the medium, press the cap of the 

Falcon tube firmly into place and gently shake the tube to mix blood and medium.  The transfer 

of the blood from the heparinized capillary tube to the medium tube is facilitated by means of the 

small black rubber bulb supplied with the kit.  The bulb is slipped onto the distal end of the 

100 µl capillary tube before it is used to collect the blood.  By sealing the hole in the end of the 

rubber bulb with a fingertip and applying slight pressure on the bulb, the blood in the capillary 

tube will be expressed into the medium tube without difficulty. 

 

4.3.2  Prepare the pre-culture thick and thin films. 

 

4.3.3  The BMM is stable for several hours, and the tubes can be carried in a breast pocket to 

maintain the contents at approximately body temperature.  If transportation delays in excess of 4 

hours are foreseen, the wet ice technique described in the note at the end of section 4.2 should be 

followed. Ambient temperatures in excess of 40

1C will destroy the parasites. 

 

4.3.4  Before dosing, the test plates should be allowed to acquire ambient temperature while they 

are still in their wrappings in order to avoid the formation of moisture which could lead to 

contamination. When the test plates have acquired ambient temperature, they should be removed 

from the wrappings. The plastic sealing strips on the required number of test series of the 

appropriate plate(s) are removed by first cutting along the appropriate columns with the scalpel 

and then lifting off the required area of plastic with a forceps, taking care not to contaminate the 

wells. 

 

4.3.5  All the wells of the appropriate column are dosed with 50 µl of the BMM (1:9) using the 

50 µl fixed volume Eppendorf pipette and a disposable sterile tip as provided with the test kit.  

Dosing is always done starting with the control well (A) and following an increasing order of 

concentrations, ending with well H.  It is extremely important that the BMM in the Falcon tube 

be shaken from time to time to ensure that the blood is kept in suspension and thus evenly 

distributed to all the wells.  The sterile disposable tip is then removed and discarded. 

 

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4.3.6  A new sterile disposable tip is fitted to the Eppendorf pipette and the next column is set 

up in exactly the same way, and so on until all the scheduled wells have been dosed. 

 

4.3.7  Place the lid on the microplate and, with a glass pen, write the details of each test over 

the appropriate column of the plate. 

 

4.3.8  Shake the plate gently, without lifting it from the laboratory bench, so that the drug 

deposits in the wells are completely dissolved. 

 

4.3.9  Take the candle jar from the incubator (set to give an internal temperature in the candle jar 

of 37.5

1C; a prewarming of at least one hour is important) and load it with the plates to be 

incubated. Light two candles (only the pure paraffin candles as supplied with the kit should be 

used) and place one on each side of the stacked plates.  Do not put the candles on top of the 

plates.  Replace the candle jar lid, being careful to sealit well, leaving the exhaust cock in the 

open position.  When the second candle is about to go out, close the exhaust cock. 

 

4.3.10  Place the candle jar in the incubator set at 37.5

1C, noting the time. 

 

4.3.11  Incubate at 37.5

1C (∀ 0.51C) for 24-30 hours, depending on the development stage of the 

trophozoites in the preculture slide.  Experience to date indicates that isolates which have not 

produced schizonts within 30 hours are influenced by factors which invalidate the test procedure. 

 By far the most common reason for delayed maturation is the previous intake of antimalarial 

drugs by the test subject. 

 

4.3.12  After incubation, the contents of the test wells are 

>harvested= by removing the 

supernatant with an Eppendorf pipette, and the red blood cells deposited on the flat bottom of the 

wells are transferred to a clean microscope slide to form a series of thick films as detailed in the 

format below: 

 

WELL A 

WELL B 

WELL C 

WELL D 

Γ

 

 

Γ

   

Γ

 

 

Γ

 

 

WELL E 

WELL F 

WELL G 

WELL H 

Γ

 

 

Γ

   

Γ

 

 

Γ

 

 

 

4.3.13  The same Eppendorf tip can be used if the 

>harvest= starts with well H and proceeds, 

well by well, up to well A (which is the last to be harvested).  The Eppendorf tip is then 

discarded before starting the harvest of a new series, proceeding again from well H to A.  

 

NOTE:  If a different harvesting sequence is chosen, the Eppendorf tip must be changed after 

every well. 

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4.3.14  The resultant thick films must be carefully dried before staining, as they will otherwise 

spontaneously detach from the slide.  If air-dried, 24-48 hours are normally required, but this 

period can be reduced (and the growth of contaminants avoided) by drying the films in an 

incubator set at 37.5

1C for 30 minutes or with a hair dryer.  If the latter method is used, care 

should be taken not to overheat the films, as this would result in autofixation. 

 

4.6.15  Some workers report highly satisfactory results from acetone-treated slides.  The thick 

films are air-dried until visibly dry, which usually takes 30 minutes.  The slide is then dipped 

into pure acetone, air-dried and stained as described below. 

 

4.3.16  The thick films are stained for 30 minutes in a Giemsa stain (reliable brand) at a dilution 

of 1% (v/v) in buffered water of pH 6.8.  Great care must be exercised in handling the stained 

films until they are completely dry. 

 

4.3.17  Drying can be performed by air, in an incubator (37.5

1) or with a hair dryer, but again, in 

this latter case, care must be taken to avoid overheating and degradation of the stain. 

 

5. Examination of the Post-culture Blood Slide 

 

5.1 Background 

 

It is most important to realize that the schizont counting procedure for PYR and SDX/PYR is 

different from that for the other drugs (CHL, MEF, QNN, AMO, ART, HAL, PND) in the 

Mark III micro-test system. 

 

5.2 Counting procedure for the CHL, MEF, QNN, AMO, ART, HAL and PND tests 

 

The basis for the count is: 

 

--------------------------------------------------------------------------------------------------------------------

- 

   NUMBER OF SCHIZONTS WITH THREE OR MORE NUCLEI OUT OF A TOTAL 

     OF 200 ASEXUAL PARASITES (i.e. SCHIZONTS AND TROPHOZOITES) 

------------------------------------------------------------------------------------------------------------------ 

For an acceptable test, schizont maturation in the control (well A) must be 10% or more (i.e. 20 

schizonts with three or more nuclei per 200 asexual parasites).  The counts read in the drug wells 

can then be expressed as a percentage of the control as in the following example: 

 

 

CONTROL 

 

DRUG UNDER TEST  

To be repeated for each test well

 

 

Number of schizonts, i.e. parasites with 3 or 

more nuclei, per 200 parasites after 

incubation 

 

No. of schizonts per 

200 parasites  after 

incubation 

 

% of schizonts relative to control 

samples  

(control = 100%)

 

98 

 

49 

98

49

x 100 = 50% 

 

 

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5.3 Counting procedure for the SDX/PYR post-culture thick films 

 

In the WHO standard micro-test the action of PYR or SDX/PYR on the developing schizont is 

not as clear cut as that of the drugs mentioned in section 5.2, since these drugs may affect nuclear 

division.  Schizonts with three nuclei or more develop even when the drug is effective, although 

these nuclei are often not well defined and the schizont may appear abnormally developed.  

Thus, it is more difficult to establish the 

>breakpoint= or >end point= of sensitivity/resistance, 

and the presence of schizonts with three nuclei or more cannot be used to define schizont 

formation as is the case for CHL, MEF, QNN, AMO, ART, HAL and PND.  

 

To overcome this difficulty and to avoid confusion in distinguishing normal from abnormal 

nuclei, the threshold of schizont growth has been changed to one of eight or more normal nuclei. 

The reason for this is that schizonts which develop to the stage of eight nuclei or more in the 

presence of PYR or SDX/PYR are usually not abnormal, and thus indicate normal growth 

(maturation).  The criterion for PYR and SDX/PYR is schizonts of 8 nuclei or more.   

 

NOTE:  parasites with a large number of minute chromatin corpuscles scattered within a swollen 

mass of cytoplasm are not to be counted as schizonts.  Such bodies may typically occur 

under the impact of PYR or SDX/PYR.  (These do not produce merozoites.) 

 

PYR and SDX/PYR comparative asexual parasite counts are therefore slightly more complex 

than those of the standard WHO micro-test for CHL, MEF, QNN, AMO, ART, HAL and PND, 

but with some practice can be mastered relatively easily.  The procedure for PYR and SDX/PYR 

is almost identical to that for the seven other drugs, except that it is necessary to enumerate 

schizonts with eight or more normal nuclei against the total asexual parasite count of 200. 

 

 

6. Interpretation and Reporting of Test Results 

 

For data analysis, the investigator may opt for a simple evaluation from a table showing the 

original data and those derived after standardizing for control growth, or for a more elaborate. 

computer-adapted procedure [Wernsdorfer & Wernsdorfer (1995).  The evaluation of in vitro 

tests for the assessment of drug response in Plasmodium falciparum. 

Mitt.Österr.Ges.Trop.Med.Paras. 17: 221-228], which provides a wide array of parameters, 

including those required for a graphic display of results, and for parallel or longitudinal 

comparison of drug response data. Diskettes of this programme are available on request from the 

Malaria Unit, Division of Control of Tropical Diseases, World Health Organization, CH-1211 

Geneva, Switzerland. 

 

6.1 CHL, MEF, QNN, AMO, ART, HAL and PND Tests 

 

The definition of resistance in vitro has been derived from the in vivo drug response in non-

immune patients.  In such patients, there is generally a good relationship between in vivo and 

in vitro response with well-absorbed drugs, e.g. CHL, AMO, MEF, QNN, in the absence of 

vomiting or diarrhoea.  With poorly or variably absorbed drugs (e.g. HAL), pharmacokinetic 

factors may produce drug failure in vivo in the presence of sensitive malaria parasites.  The  

 

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indication of in vitro resistance in the table below is correlated to in vivo response in non-

immune patients. 

 

Provided that: 

 

Χ

 

there is satisfactory growth in the control (i.e. 20 or more schizonts with three or more 

nuclei in 200 parasites); 

Χ

 

the original isolate did not contain  > 80 000  parasites / µl blood;  and 

Χ

 

the infection was indeed P.falciparum alone. 

 

then: 

 

 

Satisfactory response 

 

Indication of resistance 

 

Test Drug 

 

 

Complete schizont inhibition at 

 

Schizont formation at

 

 

Chloroquine 

 

4 pmol or less 

 

8 pmol or more 

 

Mefloquine 

 

16 pmol or less 

 

32 pmol or more 

 

Quinine 

 

128 pmol or less 

 

256 pmol or more 

 

Amodiaquine 

 

2 pmol or less 

 

4 pmol or more 

 

Halofantrine 

 

1.5 pmol or less* 

 

5 pmol or more* 

 

Artemisinin 

 

** 

 

** 

 

Pyronaridine 

 

** 

 

** 

* Provisional figures 

** Determination of critical concentration pending (on the basis of comparative in vivo and in vitro tests).

 

 

More meaningful data are obtained when a series of tests (ideally 30 or more, but a minimum of 

10) are carried out at the same time and place and are grouped.  These grouped data are achieved 

by listing, as a first step, the test results as they were read.  Subsequently, the readings obtained 

in the drug wells of each series are transformed into percentages of the control (control = 100%) 

and listed to the first decimal.  The standardized data are added up, column by column, and the 

sums divided by 

>n= (the number of tests) in order to calculate the mean percentage of schizont 

maturation at each drug concentration.  Finally, the degree of inhibition of schizont maturation 

(in %) is calculated by subtracting the percentage of schizont maturation from 100. In the table 

opposite, the procedure is shown using a series of 10 acceptable chloroquine tests. 

 

The procedure is simplified when using the computer programme mentioned in Section 6. After 

entering study area, year, drug and its concentrations in nmol or µmol / l (blood or BMM as 

appropriate), the schizont counts are entered as they were read for each isolate between wells A 

and H.  Standardization for schizont maturation in the control wells is part of the programme 

which yields practically every important regression parameter.  The Probit Caculus Sheet for the 

above series is given as an example in Annex I.  The series shows a good fit of the observed data 

points to the regression line (chi-square for heterogeneity = 0.1992 at maximum permissible 

11.1). 

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Schizont counts (per 200 parasites) in well

 

 

A 

 

B 

 

C 

 

D 

 

E 

 

F 

 

G