THE DETERMINATION OF CHLOROQUINE-RESISTANT FALCIPARUM MALARIA IN NIMBORAN, IRIAN JAYA
Bulletin Penelirian Kesehatan
Vol. 1% No. 2
Health Studies in Indonesia
1981
TBE DETERMINATION OF CHLOROQUINE-RESISTANT
FALCIPARUM MALARIA IN NIMBORAN, IRIAN JAYA +
A.J. Dimpudus *, S. Gandahusada*", S. Gunawan*""
ABSTRACT
Di Kecamatan Nimboran, Iiian Jaya dalam bulan Agustus I979 dilaksanakan test resistensi Plasmod&alciparum
terhadap chloroquind
Metode yang digunakan adalah in-vivo dun macro in-vitro test menurut prosedur standard dari WHO.
Tujuan penelitian ini adalah untuk mencari fokus dun P. falcipmum yang resisten terhadap chloroquine
dan membandingkan hasil dari kedua cara tersebut.
Endemisitas malaria di Nimboran adalah sangat tinggi dengan spleen rate 72,7 % pada usia 5 -14
tahun dan parasite rate 15,8 % pada senzua umur, meskipun telah diadakan penyemprotan rumah
dengan DDT. Species malaria yang terbanyakditemukan ialah P, fakipantm.
Telah dikerjakan 21 in-vivo test dan 28 macro in-vitro test dan diternukan 2 kasus yang resisten invivo pada derajat R f dun KIlIserta 5 kasus yang resisten in-vitro: 1 kasus menunju kkun inhibisi pembentukan schizont pada 3,O nanomol, sedangkan 4 kasus sama sekali tidak menunjukkurz inhibisi pemberztukan schizont pada 3,O nanomol.
Dengan macro in-vitro test dialami banyak kegagalan (57%)dan kemungkinan-kemung-kinan sebab
kegagalan tersebu t dibicarakan.
Hasil dari penelitian membuktikaiz bahwa Kecamatan Nimboran, suahf daerah yilny terpilih untuk
menjadi daerah transmigrasi, merupakan fokus resistensi P. falciparum terhadap chloroqlr ,c.
INTRODUCTION
Of the four species of human malaria parasites, Plasmodium falciparum and P. vivax
account for more than 95 % of the malaria
cases in Irian Jaya (unpublished provincial
malaria report). P. rnalariae is found unevenly
distributed throughout this area in small
numbers, while P. ovule has recently been
reported in a small island, Owi, in the Teluk
Cenderawasih (Biak) regency.
Resistance of falciparlim malaria to a
standard dose of chloroquine in Irian Jaya in
the town of Jayapura was reported by
Verdrager et a1 in 1976 (1) and by Hutapea in
1979 (3), while in 1961 resistance cases of
P. falciparum to pyrimethamine and proguanil
were reported by Meuwissen (2). Those chloro-
+ This investigation is part of the project "Regional
*
**
Collaborative Studies on Drug Resistant Malaria
with Special Emphasis on P. falciparurn jesistance
to 4-aminoquinoline" with financial support from
WHO/SEARO.
Provincial Health Office, Irian Jaya.
National Institute of Health Research and Development.
quine resistant cases of falciparum malaria were
in-vivo tested.
The objective of this study was to determine
the susceptibility of P. falcipanin: to chloroquine in Nimboran, lrian Jaya, using both tlie
in-vivo and the macro in-vitro tests and to
compare the results of both tests. T i e study
was conducted in August 1979 in Ger~srem
town and surroundings, subdistrict Ninibaran.
The Nimboran area is situated about 100 lun
west of Jayapura at about 2'36's and 140'12'~.
Its elevatioi is 25 m and the at-ea is intersected
by numerous small and medium sized rivers
and surrounded by primary forest, cocoa
plantations, and swamps.
The main vectors in this area were Anopheles
koliensis and At?. purlchrlatus and they were
still suscceptible to DDT.
The subdistrict of Nimboran has been
scheduled to be a resettlement area and since
1974 groups of transmigrants from Java have
been resettled there.
Indoor resldual house spraylng has been
performed in this area since 1974 using DDT
L) g / q
~ . ~ i l . , ; i j ~ p~lW~I~C ~C al year. Parasite surveys
conducted in tlie past several years have
revealed parasite rates between 8.6 %and 41.3 70
Thc Determination of Chloroquine Resisten l~alciparumMalaria
The drug used was not coated and at the
time of each drug administration precautions
were taken to ensure that the drug was
swallowed and retained. Severely ill patients,
subjects who vomited or had mixed infections
with other malaria species or had complications
wlth otherdiseases such as heart, lung, liver,
kidney, intestional or metabolic diseases, were
excluded from the test. Duplicate thick and
thin blood smears were taken starting on day
0 before the administration of the first test
dose of chloroquine and repeated daily up to
day 7 and stained with Giemsa. Asexual
parasites were counted per 300 white blood
cells or more in some cases. Urine tests using
the Dill Glazko reagent were performed on day
0 prior to the drug administration and on day
1.
The WHO grading of falciparum resistance
to chloroquine is as follows:
Sensitive-S : Clearance of asexual parasitaemia within 7 days of initiation
of treatment, without subsequent recrudescence.
R I
: Clearance of asexual parasitaemia as in S but followed by
recrudescence.
R I1
: Marked reduction (
75 %)
of asexual parasitaemia, but no
clearance.
R 111
: No marked (
75 %) reduction of asexual parasitaemia.
for all ages. Proportional falciparum infection
rates varies between 53.7 % and 79.0 %. Slide
positivity rates from passive case detection
ranged between 30 % - 100 % (unpublished
malaria report).
The total population of this area amounted
to 9,482 people living in 55 villages. Chloroquine was the primary drug available and it
was easy to obtain this drug from the health
centre or to purchase it from the small shops
locally.
MATERIAL AND METHODS
To study the response of P. falciparum to
chloroquine, the standard procedure of WHO
for the in-vivo (4) and the macro in-vitro tests
based on the work of Rieckman et a1 (5) was
used.
Drug resistance in malaria is defined as the
ability of a parasite strain to survive and/or
to multiply despite the administration and
absorption of a drug given in doses equal to or
higher than those usually recommended but
within the limits of tolerance of the subject
(WHO, TRS No. 296,1965).
To find suitable cases for the in-vivo and the
macro in-vitro tests a preliminary survey was
performed. The thick blood films collected and
dried up were immediately stained with 10 %
Giemsa solution for 5 minutes and examined
in the field. If examination of 10 - 20 microscopic fields did not detect at least 10 trophozoites, the case was disregarded for the in-vivo
and in-vitro test. If at least 10 trophozoites
were found, but all were young rings, then the
case was accepted for the in-vivo but excluded
for the macro in-vitro test.
>
<
The macro in-vitro test.
Ten cu nun venous blood were extracted
from falciparum cases which met the following
criteria:
1 . Not severely ill, with trophozoite count of
at least 500, preferably more than 1000 per
cu mm blood. Most of the trophozoites had
to be of average (half grown rings) or large
size (large rings), which ment they were at
lest 12 hours old.
2. The trophozoite count should not exceed
80,00O/cu mm blood.
3. Dill-Glazko urine test should be negative
and the person had not taken within the last
month pyriinethamine and/or sulfonamides.
The in-vivo test.
Basically the 7 days in-vivo test was used,
but if time permitted, the observation was
extended to more than 7 days. The test consisted of the administration of 25 mg chloroquine
base per kilograin body weight over three days,
the first day (day 0) 10 rng, the second day
(day 1) also 10 mg and tile third day (day 2)
5 mg chloroquine base per kg body weight.
Tlie drug used was Resochin tablets containing
150 mg chloroquine base per tablet. Cases
eligible for the test had trophozoite count of
at least 500 per cu mm blood, preferably more
than 1000 per cu mm blood. Young children
were preferred to older children and adults.
The venous blood was then transferred into
a sterile 25 ml Erlenmeyer flask with stopper
and containing glass beads. The blood was defribinated bv rotating the flask gently for 5 minutes. Witlz a sterile 1 cc pipette, 1 cc blood
19
A.J.
Dirnpudus, d k k
smears was used as a standard (100 5%). The
count in 'each of thc test smears was then calculated as a percentage of this average count in
the control. If after examining 100 microscopic fields or per 1,500 white blood cells no
schizonts were found, the smear was
considered negative or clear of schizonts for
that concentration of chloroquine. For the
macro in-vitro test n o clearance of schizonts
at 1.0 nanomol chloroquine hut comnlete
inhibition at 1.5 nanomol was considered as a
possihility of parasite resistance at a low grade
level. If the schizont formation was not inhibited between 1.5 and 3.0 nanoniol, then this
was regarded as comnarable with the R1 to RII
level of resistance with the in-vivo test, depending on the immunity level of the case.
was then placed into the test vials of various
chloroquine concentrations, the lowest belng
0.25 nanomol and the highest 3.0 nanomol.
The vials were swirled gently t o mix the
contents well and were then placed in a rack
with caps loosely screwed in ascending order
of cliloroquine concentration in a waterbatll
at 38.5O - 4 0 ° c hours. This must be done less
than three hours after extracting the venous
blood. Two of each set of test vials did not
contain clllorocluine b u t only growth media and
these two vials served as controls.
After incubation, the content in the vials
was stirred with a stick to resuspend cells in the
plasma. From every vial 1 set of thick and thin
smears was made and thick smears from all of
the test vials of one series were made into one
slide. The smears were dried for a t least 4 0
hours at room temperature and then stained
with Giernsa 2 % solution with a pH of
6.6 - 6.8 for 3 0 minutes.
Examination o f the smears consisted of
counting schizonts with three o r more pieces of
chromatin per 3 0 0 white blood cells. The
average schizont count in the two control
in years
15
9
12
26
23
24
586
5
7
15
9
14
100
55.6
58.3
57.7
39.1
58.3
11.7
2
4
13
5
11
70
3
2
1
1
1
26
1
1
1
2
3
2
Total
950
150
15.8
105
34
8
3
-
1
The preliminary survey in the Nimboran
study area covered 9 5 0 people, living in 12
villages. The overall parasite rate was 15.8 %
and falciparum infections comprised 70 % of
all (1 0 5 cases) malaria cases (Table 1 ).
Parasite rates in sub-district of Nlmboran
Tabel 1
Age group
RESULTS
1
*) F = P. falciparum; V = P. vivax and M = P. malariae
The in-vivo test was done in three cases of
children less than 5 years old. and 18 cases
aged 1 0 years and over. In this last group two
cases of resistance at RI and RIIl level were
found (table 2 a r d 4). 'Uie other 17 cases were
susceptible and 2 cases were considered
failures because of low counts on day 0.
Of the 28 macro in-vitro tests, 16 tests
(57 %) were failures, in which there was almost
n o growth of trophozoites into schizonts. Tlle
outcome of the remaining 1 2 successful tests
A total o f 121 people, most of them
between 5-14 years old, were examined for
spleen enlargement and 8 8 ~ e o n l e(72.7 %)
were found with a ~ a l n a b l esnleen. The everage
enlarged spleen was 2.8.
Of the 105 falciparum cases, only 17 were
eligible for both tests, 4 qualified fpr the
in-vivo test and 11 for the macro in-vitro test.
Thus the total in-vivo tests were 2 1 cases and
the total in-vitro tests were 2 8 cases.
20
I
T h e D e t e r m i n a t i o n of Chloroquine Resisten Falciparum Malaria
was 7 susceutihle cases (58 %) and 5 resistant
falciparum cases (42 %), of which 3 cases had
no schiyont clearance at 3.0 nanornol and 1
case had schizont clearance at 3.0 nanomol
(Table 3 and 4).
In-vivo test of falciparum cases by age group
Tabel 2
Age group
Number
(years)
tested
Failure
Resistant
Susceptible
Less than 5 vrs
3
0
0
3
5- 9
10-1 4
-
-
-
-
0
2
0
2
1
13
2
2
17
1 5 years and over
1
17
Total
21
Table 3
.
In-vitro test of falciparum cases by age group
I
Number
Age group
5years
>
9
10 - 14
15
5-
Total
Susceptible
1
2
3
22
1
0
3
12
0
1
0
4
0
1
0
6
28
16
5
7
Falciparum cases resistant t o chloroquine by in-vivo
and macro in-vitro tests.
Table 4
Case
Res~stant
tested
(years)
<
Failure
N o.
Age
(years)
1
3
5
18
25
28
20
32
16
17
17
5
In-vivo
In-vitro test
test
R I
Clearance at 3.0 nanornol
R Ill
Susceptible
n o clearance
n o clearance
n o clearance
no clearance
-
-
at
at
at
at
3.0 nanornol
3.0 nanornol
3.0 nanornol
3.0 nanornol
* not tested
DISCUSSION
High levels of immunity mask the effect of the
in-vivo test especially among cases aged 10
years and over. The two in-vivo resistant cases
were military personnel from Ujung Pandang,
South Sulawesi, who came to Jayapura in
March 1979. Their first malaria attack was one
P. falcipamnz resistance to chloroquine
exists in that area. In a hyper or holoendeniic
area the macro in-vitro test reveals more
resistant cases than the 7 days in-vitro test.
21
A.J. Dimpudus, dkk
week after arrival in a village on the border with
Papua New Guinea. Most probably the infection was contracted in Waena, a village near
Jayapura which was already known to be a
focus of chloroquine resistant falciparum malaria. These two cases might have such a low
level of immunity that they could not be cured
with a standard dose of chloroquine.
In case no. 3 the in-vivo test showed
resistance, while the macro in-vitro test gave
a susceptible reaction to chloroquine. The cause
is unknown. Drug failure was not considered as
the cause because the urine test was positive
on day 1. The macro in-vitro test showed a high
percentage of failure (57 %). This might be
attributed to various factors e.g. a low pretest
trophozoite counts, too young trophozoites,
fluctuation of the incubation temperature, the
time lapse between blood extraction and
incubation of more than 3 hours, faulty
technique in performing the test, deterioration
of the growth media in the control vials since
many screw caps were loosely fitted and
moulds may grow in the control vials, too much
bumping of the flasks containing the venous
blood during transportation from the field to
the laboratory, or the patient has already been
treated with an antimalaria drug. The team was
aware of these factors that might jeopardise the
test and care had been taken to avoid them.
Table 5 shows the average percentage of small,
medium and large size trophozoites for the
group of failed (n=16) and successful (n=12)
tests.
Average preculture counts i n percentage of the different
sizes of trophozoites
Table 5
Size of trouhozoits
Small
Medium
Large
16 failed tests
28.9
62.4
8.7
12 successfcl tests
22.2
52.8
24.9
There is difference in percentage of young
trophozoites (small rings), 28.9 % and 22.2 %
for the group of failed and successful tests
respectively. However this difference is statistically not significant, because of the small
number of tests. Most likely the causes of
failures were in selecting patients with too
young trophozoites or faulty diagnosis of trophozoite age. Another likely cause might be
the content of control vials, which had
deteriorated due to long storage, and vial caps,
which were not well screwed during the storage.
This high percentage of failures (about 50 %
or more) was also experienced in Kalimantan
Timur and other provinces of Indonesia. In
Africa the failure was almost 100 % (Rooney,
personal comn~unication)and it was thought
that the P. falciparum strain may be a' factor,
since the same batch of test vials gave good
results in other places.
RECOMMENDATION
The Malaria Control Programme applied in
that area was not successful. Transmission continued outdoors. Therefore that area is considered not suitable for resettlement project
until new methods for interruption of malaria
transmission are found. Meanwhile efforts
should be taken to prevent the further spread
of drug resistant falciparum cases in Irian Jaya
to other areas of Indonesia.
SUMMARY
Twenty one in-vivo and 28 niacro in-vitro
tests were performed and the result of the
investigation was the detection of 2 resistant
cases of the RI and RI[I type with the in-vivo
test and 5 resistant cases with the in-vitro test.
A high percentage (57 %) of failure in the
macro in-vitro test was encountered.
22
The Determination of Chloroquine Resisten Falciparum Malaria
ACKNOWLEDGEMENTS
The authors are grateful to dr Damri Laksamana for his guidance in the field, to Mr M.
Sitanggang for preparing the Dill Glazko
reagent and to all the provincial health
personnel for their kind assistance and cooperation. Specifically we would like to thank the
Mandosir, S. Nari, and H
microsco~ists,
Finthay of the provincial malaria laboratory
for their participation in this study.
MT
REFERENCES
1. Verdrager, J. Frwati, Sirnandjuntak, C. and
Saroso, J.S. (1976). Response of falciparum malaria to a standard regimen of
chloroquine in Jayapura, Irian Jaya.
Bull. Hlth. Studies in Indonesia, IV :
19.
2. Meuwissen, J.H.E.T., (1961). Resistance of
Plasmodium falciparum t o pyrimethamine and proguanil in Netherland New
Guinea, Amer. J. Trop. Med Hyg, 10 :
135
3. Hutapea, A.M., (1979). In vivo strain sensitivity test to a standard dose of chloroquine in falciparum malaria in Jayapura,
Indonesia. Maj. Kedok Indonesia,29 :49.
4. WHO Technical Rep. Series, (1973). No. 529.
5. Rieckmann, K.H., Namara, J.V., Frisher,
H., Stockart, T.A. Carson. P.E. and Powell, R.D., (1968). Effects of chloroquine, quinine and cycloguanil upon the
maturation of asexual erythrocytic forms
of two starins of Plasmodium falcipamm
in vitro. Amer. J. Trop. Med. ~Hyg.,
17: 661.
Vol. 1% No. 2
Health Studies in Indonesia
1981
TBE DETERMINATION OF CHLOROQUINE-RESISTANT
FALCIPARUM MALARIA IN NIMBORAN, IRIAN JAYA +
A.J. Dimpudus *, S. Gandahusada*", S. Gunawan*""
ABSTRACT
Di Kecamatan Nimboran, Iiian Jaya dalam bulan Agustus I979 dilaksanakan test resistensi Plasmod&alciparum
terhadap chloroquind
Metode yang digunakan adalah in-vivo dun macro in-vitro test menurut prosedur standard dari WHO.
Tujuan penelitian ini adalah untuk mencari fokus dun P. falcipmum yang resisten terhadap chloroquine
dan membandingkan hasil dari kedua cara tersebut.
Endemisitas malaria di Nimboran adalah sangat tinggi dengan spleen rate 72,7 % pada usia 5 -14
tahun dan parasite rate 15,8 % pada senzua umur, meskipun telah diadakan penyemprotan rumah
dengan DDT. Species malaria yang terbanyakditemukan ialah P, fakipantm.
Telah dikerjakan 21 in-vivo test dan 28 macro in-vitro test dan diternukan 2 kasus yang resisten invivo pada derajat R f dun KIlIserta 5 kasus yang resisten in-vitro: 1 kasus menunju kkun inhibisi pembentukan schizont pada 3,O nanomol, sedangkan 4 kasus sama sekali tidak menunjukkurz inhibisi pemberztukan schizont pada 3,O nanomol.
Dengan macro in-vitro test dialami banyak kegagalan (57%)dan kemungkinan-kemung-kinan sebab
kegagalan tersebu t dibicarakan.
Hasil dari penelitian membuktikaiz bahwa Kecamatan Nimboran, suahf daerah yilny terpilih untuk
menjadi daerah transmigrasi, merupakan fokus resistensi P. falciparum terhadap chloroqlr ,c.
INTRODUCTION
Of the four species of human malaria parasites, Plasmodium falciparum and P. vivax
account for more than 95 % of the malaria
cases in Irian Jaya (unpublished provincial
malaria report). P. rnalariae is found unevenly
distributed throughout this area in small
numbers, while P. ovule has recently been
reported in a small island, Owi, in the Teluk
Cenderawasih (Biak) regency.
Resistance of falciparlim malaria to a
standard dose of chloroquine in Irian Jaya in
the town of Jayapura was reported by
Verdrager et a1 in 1976 (1) and by Hutapea in
1979 (3), while in 1961 resistance cases of
P. falciparum to pyrimethamine and proguanil
were reported by Meuwissen (2). Those chloro-
+ This investigation is part of the project "Regional
*
**
Collaborative Studies on Drug Resistant Malaria
with Special Emphasis on P. falciparurn jesistance
to 4-aminoquinoline" with financial support from
WHO/SEARO.
Provincial Health Office, Irian Jaya.
National Institute of Health Research and Development.
quine resistant cases of falciparum malaria were
in-vivo tested.
The objective of this study was to determine
the susceptibility of P. falcipanin: to chloroquine in Nimboran, lrian Jaya, using both tlie
in-vivo and the macro in-vitro tests and to
compare the results of both tests. T i e study
was conducted in August 1979 in Ger~srem
town and surroundings, subdistrict Ninibaran.
The Nimboran area is situated about 100 lun
west of Jayapura at about 2'36's and 140'12'~.
Its elevatioi is 25 m and the at-ea is intersected
by numerous small and medium sized rivers
and surrounded by primary forest, cocoa
plantations, and swamps.
The main vectors in this area were Anopheles
koliensis and At?. purlchrlatus and they were
still suscceptible to DDT.
The subdistrict of Nimboran has been
scheduled to be a resettlement area and since
1974 groups of transmigrants from Java have
been resettled there.
Indoor resldual house spraylng has been
performed in this area since 1974 using DDT
L) g / q
~ . ~ i l . , ; i j ~ p~lW~I~C ~C al year. Parasite surveys
conducted in tlie past several years have
revealed parasite rates between 8.6 %and 41.3 70
Thc Determination of Chloroquine Resisten l~alciparumMalaria
The drug used was not coated and at the
time of each drug administration precautions
were taken to ensure that the drug was
swallowed and retained. Severely ill patients,
subjects who vomited or had mixed infections
with other malaria species or had complications
wlth otherdiseases such as heart, lung, liver,
kidney, intestional or metabolic diseases, were
excluded from the test. Duplicate thick and
thin blood smears were taken starting on day
0 before the administration of the first test
dose of chloroquine and repeated daily up to
day 7 and stained with Giemsa. Asexual
parasites were counted per 300 white blood
cells or more in some cases. Urine tests using
the Dill Glazko reagent were performed on day
0 prior to the drug administration and on day
1.
The WHO grading of falciparum resistance
to chloroquine is as follows:
Sensitive-S : Clearance of asexual parasitaemia within 7 days of initiation
of treatment, without subsequent recrudescence.
R I
: Clearance of asexual parasitaemia as in S but followed by
recrudescence.
R I1
: Marked reduction (
75 %)
of asexual parasitaemia, but no
clearance.
R 111
: No marked (
75 %) reduction of asexual parasitaemia.
for all ages. Proportional falciparum infection
rates varies between 53.7 % and 79.0 %. Slide
positivity rates from passive case detection
ranged between 30 % - 100 % (unpublished
malaria report).
The total population of this area amounted
to 9,482 people living in 55 villages. Chloroquine was the primary drug available and it
was easy to obtain this drug from the health
centre or to purchase it from the small shops
locally.
MATERIAL AND METHODS
To study the response of P. falciparum to
chloroquine, the standard procedure of WHO
for the in-vivo (4) and the macro in-vitro tests
based on the work of Rieckman et a1 (5) was
used.
Drug resistance in malaria is defined as the
ability of a parasite strain to survive and/or
to multiply despite the administration and
absorption of a drug given in doses equal to or
higher than those usually recommended but
within the limits of tolerance of the subject
(WHO, TRS No. 296,1965).
To find suitable cases for the in-vivo and the
macro in-vitro tests a preliminary survey was
performed. The thick blood films collected and
dried up were immediately stained with 10 %
Giemsa solution for 5 minutes and examined
in the field. If examination of 10 - 20 microscopic fields did not detect at least 10 trophozoites, the case was disregarded for the in-vivo
and in-vitro test. If at least 10 trophozoites
were found, but all were young rings, then the
case was accepted for the in-vivo but excluded
for the macro in-vitro test.
>
<
The macro in-vitro test.
Ten cu nun venous blood were extracted
from falciparum cases which met the following
criteria:
1 . Not severely ill, with trophozoite count of
at least 500, preferably more than 1000 per
cu mm blood. Most of the trophozoites had
to be of average (half grown rings) or large
size (large rings), which ment they were at
lest 12 hours old.
2. The trophozoite count should not exceed
80,00O/cu mm blood.
3. Dill-Glazko urine test should be negative
and the person had not taken within the last
month pyriinethamine and/or sulfonamides.
The in-vivo test.
Basically the 7 days in-vivo test was used,
but if time permitted, the observation was
extended to more than 7 days. The test consisted of the administration of 25 mg chloroquine
base per kilograin body weight over three days,
the first day (day 0) 10 rng, the second day
(day 1) also 10 mg and tile third day (day 2)
5 mg chloroquine base per kg body weight.
Tlie drug used was Resochin tablets containing
150 mg chloroquine base per tablet. Cases
eligible for the test had trophozoite count of
at least 500 per cu mm blood, preferably more
than 1000 per cu mm blood. Young children
were preferred to older children and adults.
The venous blood was then transferred into
a sterile 25 ml Erlenmeyer flask with stopper
and containing glass beads. The blood was defribinated bv rotating the flask gently for 5 minutes. Witlz a sterile 1 cc pipette, 1 cc blood
19
A.J.
Dirnpudus, d k k
smears was used as a standard (100 5%). The
count in 'each of thc test smears was then calculated as a percentage of this average count in
the control. If after examining 100 microscopic fields or per 1,500 white blood cells no
schizonts were found, the smear was
considered negative or clear of schizonts for
that concentration of chloroquine. For the
macro in-vitro test n o clearance of schizonts
at 1.0 nanomol chloroquine hut comnlete
inhibition at 1.5 nanomol was considered as a
possihility of parasite resistance at a low grade
level. If the schizont formation was not inhibited between 1.5 and 3.0 nanoniol, then this
was regarded as comnarable with the R1 to RII
level of resistance with the in-vivo test, depending on the immunity level of the case.
was then placed into the test vials of various
chloroquine concentrations, the lowest belng
0.25 nanomol and the highest 3.0 nanomol.
The vials were swirled gently t o mix the
contents well and were then placed in a rack
with caps loosely screwed in ascending order
of cliloroquine concentration in a waterbatll
at 38.5O - 4 0 ° c hours. This must be done less
than three hours after extracting the venous
blood. Two of each set of test vials did not
contain clllorocluine b u t only growth media and
these two vials served as controls.
After incubation, the content in the vials
was stirred with a stick to resuspend cells in the
plasma. From every vial 1 set of thick and thin
smears was made and thick smears from all of
the test vials of one series were made into one
slide. The smears were dried for a t least 4 0
hours at room temperature and then stained
with Giernsa 2 % solution with a pH of
6.6 - 6.8 for 3 0 minutes.
Examination o f the smears consisted of
counting schizonts with three o r more pieces of
chromatin per 3 0 0 white blood cells. The
average schizont count in the two control
in years
15
9
12
26
23
24
586
5
7
15
9
14
100
55.6
58.3
57.7
39.1
58.3
11.7
2
4
13
5
11
70
3
2
1
1
1
26
1
1
1
2
3
2
Total
950
150
15.8
105
34
8
3
-
1
The preliminary survey in the Nimboran
study area covered 9 5 0 people, living in 12
villages. The overall parasite rate was 15.8 %
and falciparum infections comprised 70 % of
all (1 0 5 cases) malaria cases (Table 1 ).
Parasite rates in sub-district of Nlmboran
Tabel 1
Age group
RESULTS
1
*) F = P. falciparum; V = P. vivax and M = P. malariae
The in-vivo test was done in three cases of
children less than 5 years old. and 18 cases
aged 1 0 years and over. In this last group two
cases of resistance at RI and RIIl level were
found (table 2 a r d 4). 'Uie other 17 cases were
susceptible and 2 cases were considered
failures because of low counts on day 0.
Of the 28 macro in-vitro tests, 16 tests
(57 %) were failures, in which there was almost
n o growth of trophozoites into schizonts. Tlle
outcome of the remaining 1 2 successful tests
A total o f 121 people, most of them
between 5-14 years old, were examined for
spleen enlargement and 8 8 ~ e o n l e(72.7 %)
were found with a ~ a l n a b l esnleen. The everage
enlarged spleen was 2.8.
Of the 105 falciparum cases, only 17 were
eligible for both tests, 4 qualified fpr the
in-vivo test and 11 for the macro in-vitro test.
Thus the total in-vivo tests were 2 1 cases and
the total in-vitro tests were 2 8 cases.
20
I
T h e D e t e r m i n a t i o n of Chloroquine Resisten Falciparum Malaria
was 7 susceutihle cases (58 %) and 5 resistant
falciparum cases (42 %), of which 3 cases had
no schiyont clearance at 3.0 nanornol and 1
case had schizont clearance at 3.0 nanomol
(Table 3 and 4).
In-vivo test of falciparum cases by age group
Tabel 2
Age group
Number
(years)
tested
Failure
Resistant
Susceptible
Less than 5 vrs
3
0
0
3
5- 9
10-1 4
-
-
-
-
0
2
0
2
1
13
2
2
17
1 5 years and over
1
17
Total
21
Table 3
.
In-vitro test of falciparum cases by age group
I
Number
Age group
5years
>
9
10 - 14
15
5-
Total
Susceptible
1
2
3
22
1
0
3
12
0
1
0
4
0
1
0
6
28
16
5
7
Falciparum cases resistant t o chloroquine by in-vivo
and macro in-vitro tests.
Table 4
Case
Res~stant
tested
(years)
<
Failure
N o.
Age
(years)
1
3
5
18
25
28
20
32
16
17
17
5
In-vivo
In-vitro test
test
R I
Clearance at 3.0 nanornol
R Ill
Susceptible
n o clearance
n o clearance
n o clearance
no clearance
-
-
at
at
at
at
3.0 nanornol
3.0 nanornol
3.0 nanornol
3.0 nanornol
* not tested
DISCUSSION
High levels of immunity mask the effect of the
in-vivo test especially among cases aged 10
years and over. The two in-vivo resistant cases
were military personnel from Ujung Pandang,
South Sulawesi, who came to Jayapura in
March 1979. Their first malaria attack was one
P. falcipamnz resistance to chloroquine
exists in that area. In a hyper or holoendeniic
area the macro in-vitro test reveals more
resistant cases than the 7 days in-vitro test.
21
A.J. Dimpudus, dkk
week after arrival in a village on the border with
Papua New Guinea. Most probably the infection was contracted in Waena, a village near
Jayapura which was already known to be a
focus of chloroquine resistant falciparum malaria. These two cases might have such a low
level of immunity that they could not be cured
with a standard dose of chloroquine.
In case no. 3 the in-vivo test showed
resistance, while the macro in-vitro test gave
a susceptible reaction to chloroquine. The cause
is unknown. Drug failure was not considered as
the cause because the urine test was positive
on day 1. The macro in-vitro test showed a high
percentage of failure (57 %). This might be
attributed to various factors e.g. a low pretest
trophozoite counts, too young trophozoites,
fluctuation of the incubation temperature, the
time lapse between blood extraction and
incubation of more than 3 hours, faulty
technique in performing the test, deterioration
of the growth media in the control vials since
many screw caps were loosely fitted and
moulds may grow in the control vials, too much
bumping of the flasks containing the venous
blood during transportation from the field to
the laboratory, or the patient has already been
treated with an antimalaria drug. The team was
aware of these factors that might jeopardise the
test and care had been taken to avoid them.
Table 5 shows the average percentage of small,
medium and large size trophozoites for the
group of failed (n=16) and successful (n=12)
tests.
Average preculture counts i n percentage of the different
sizes of trophozoites
Table 5
Size of trouhozoits
Small
Medium
Large
16 failed tests
28.9
62.4
8.7
12 successfcl tests
22.2
52.8
24.9
There is difference in percentage of young
trophozoites (small rings), 28.9 % and 22.2 %
for the group of failed and successful tests
respectively. However this difference is statistically not significant, because of the small
number of tests. Most likely the causes of
failures were in selecting patients with too
young trophozoites or faulty diagnosis of trophozoite age. Another likely cause might be
the content of control vials, which had
deteriorated due to long storage, and vial caps,
which were not well screwed during the storage.
This high percentage of failures (about 50 %
or more) was also experienced in Kalimantan
Timur and other provinces of Indonesia. In
Africa the failure was almost 100 % (Rooney,
personal comn~unication)and it was thought
that the P. falciparum strain may be a' factor,
since the same batch of test vials gave good
results in other places.
RECOMMENDATION
The Malaria Control Programme applied in
that area was not successful. Transmission continued outdoors. Therefore that area is considered not suitable for resettlement project
until new methods for interruption of malaria
transmission are found. Meanwhile efforts
should be taken to prevent the further spread
of drug resistant falciparum cases in Irian Jaya
to other areas of Indonesia.
SUMMARY
Twenty one in-vivo and 28 niacro in-vitro
tests were performed and the result of the
investigation was the detection of 2 resistant
cases of the RI and RI[I type with the in-vivo
test and 5 resistant cases with the in-vitro test.
A high percentage (57 %) of failure in the
macro in-vitro test was encountered.
22
The Determination of Chloroquine Resisten Falciparum Malaria
ACKNOWLEDGEMENTS
The authors are grateful to dr Damri Laksamana for his guidance in the field, to Mr M.
Sitanggang for preparing the Dill Glazko
reagent and to all the provincial health
personnel for their kind assistance and cooperation. Specifically we would like to thank the
Mandosir, S. Nari, and H
microsco~ists,
Finthay of the provincial malaria laboratory
for their participation in this study.
MT
REFERENCES
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Saroso, J.S. (1976). Response of falciparum malaria to a standard regimen of
chloroquine in Jayapura, Irian Jaya.
Bull. Hlth. Studies in Indonesia, IV :
19.
2. Meuwissen, J.H.E.T., (1961). Resistance of
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Guinea, Amer. J. Trop. Med Hyg, 10 :
135
3. Hutapea, A.M., (1979). In vivo strain sensitivity test to a standard dose of chloroquine in falciparum malaria in Jayapura,
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4. WHO Technical Rep. Series, (1973). No. 529.
5. Rieckmann, K.H., Namara, J.V., Frisher,
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