resistance of falciparum malaria to chloroquine and sulfadoxine-pyrimethamine in afghan refugee...

Tropical Medicine and International Health

volume 2 no 11 pp 1049–1056 november 1997

© 1997 Blackwell Science Ltd1049

Resistance of falciparum malaria to chloroquine andsulfadoxine-pyrimethamine in Afghan refugee settlementsin western Pakistan: surveys by the general health servicesusing a simplified in vivo test

Mark Rowland1, Naeem Durrani1, Sean Hewitt1 and Egbert Sondorp2

1 HealthNet International, Peshawar, Pakistan2 HealthNet International, Amsterdam, The Netherlands.

Summary Surveys of drug resistant falciparum malaria were conducted in several Afghan refugee

settlements, distributed over a 700 km range in western Pakistan, during the transmission seasons

of 1994 and 1995. Symptomatic malaria patients were recruited by a process of passive case

detection at the refugees’ basic health units. To facilitate follow-up by local health workers, a

modified version of the WHO extended in vivo test was adopted in which blood smears were taken

from each subject, and clinical symptoms recorded, at weekly intervals. Resistance to chloroquine

and sulfadoxine-pyrimethamine was identified in every settlement. The frequency of chloroquine

resistance ranged from 18% to 62%. Resistance occurred mostly as RI, with RII resistance never

exceeding 11%. Resistance to sulfadoxine-pyrimethamine occurred at much lower frequencies,

ranging from 4% to 25%. There was a resumption of clinical symptoms at the onset of parasite

recrudescence in over 90% of cases. The policy of using chloroquine as first-line treatment might

be changed in favour of sulfadoxine-pyrimethamine in most camps and areas of western Pakistan.

The modified in vivo test was almost as accurate as the normal WHO in vivo test in identifying the

grade of resistance, and should prove a useful tool for the monitoring of resistance to common

antimalarials by district health services.

keywords chloroquine, resistance, falciparum malaria, sulfadoxine-pyremethamine

correspondence Mark Rowland, HealthNet International, PO Box 889, University Town,

Peshawar, Pakistan

Introduction

Chloroquine-resistant falciparum malaria was first

identified in Pakistan in 1984 (Fox et al. 1985). Over the

last decade, resistance surveys conducted by the

National Institute for Malaria Research and Training

using the WHO extended in vivo test (WHO 1973) have

shown that chloroquine resistance is widespread in

many areas of the Punjab and North-west Frontier

Province (Shah et al. 1997). Resistance to sulfadoxine-

pyrimethamine has not been monitored or reported. The

Pakistan Ministry of Health currently recommends the

use of chloroquine as first-line treatment for the

treatment of falciparum malaria. This guideline may no

longer be appropriate.

Since the early 1980s up to 3 million Afghan refugees

have settled in camps – often in malarious areas – in

western Pakistan (Rowland et al. 1997). The United

Nations High Commissioner for Refugees presently

adopts the same treatment guidelines for falciparum

malaria as the Pakistan Ministry of Health. To develop

locally effective treatment guidelines for the refugee

communities, it is necessary to map the type, level, and

distribution of drug resistance to chloroquine and

Tropical Medicine and International Health volume 2 no 11 pp 1049–1056 november 1997

M. Rowland et al. Malaria drug resistance in Afghan refugees


sulfadoxine-pyrimethamine by in vivo survey. As

resources and technical manpower were limited,

HealthNet International (a non-government organization

responsible for malaria control in the refugee camps)

enlisted district general health staff to carry out the

survey work and designed a simplified extended in vivo

test in which smears were taken at weekly intervals. To

determine the operational significance of resistance on

disease, clinical symptoms were recorded concurrently.

This paper presents the results of these modified in vivo

and clinical surveys carried out at basic health units in

various refugee camps in North-west Frontier Province,

western Punjab, and Balochistan.

Methods

Study areas

Seven refugee villages, situated over a 700-km range,

were selected from North-west Frontier Province,

Balochistan, and western Punjab as sites for in vivo drug

resistance surveys (Figure 1). Each selected village had a

history of malaria and a good functioning basic health

unit (BHU) managed by either the United Nations High

Commissioner for Refugees (UNHCR), the Pakistan

government department responsible for refugee health

care, or an international non-government organization

(NGO). When selecting a range of representative

villages, we paid close attention to the wide diversity of

geographical situations in which refugees are settled.

Most villages were established over 15 years ago, when

the refugees first came to Pakistan. Houses were built

from mud or stone, depending on the nature of the

surrounding terrain. Some villages were built on dry

hillsides (e.g. Kahi in Kohat), some were waterlogged

(e.g. Adizai on the banks of the Kabul river in

Charsadda), others were situated on desert fringes (Pir

Alizai in Pishin). Settlement populations ranged from

120000 in Kot Chandna (Mianwali) to 5200 in Adizai

Chitral

Dir

Kohistan

Swat

MansehraMalakand

Abbott-abad

PeshawarKhyber

Kohat

KarakMiran Shah

Bannu

Znob

Pishin

Waziristan

Quetta

AFG

HA

NIS

TAN

Dera IsmailKhan

KASHMIR

PUNJAB

BALOCHISTAN

Parachinar

0 100 km

MardanCharsadda

Mianwali

Mohmand

Figure 1 Districts in which in vivo

resistance surveys were carried out (d).




(Charsadda). Altitude ranged from 380 m in Baghicha

(Mardan) to 1380 m in Azam Warsak (Waziristan).

Some settlements were peri-urban (e.g. Badaber in

Peshawar) while most were rural (e.g. Adizai in

Charsadda). Surrounding agriculture sometimes

included rice (e.g. in Mardan), orchards (in Waziristan),

maize (e.g. in Kohat), wheat or sugar (e.g. in

Charsadda). Local mosquito breeding sites were equally

diverse, and included water-filled borrow pits in Adizai

(Charsadda), irrigated rice fields in Baghicha (Mardan),

reservoir tanks in Azam Warzak (Waziristan), seasonal

rivers in Badaber (Peshawar), and springs in Pir Alizai

(Pishin). Most settlements remained densely populated

even though many Afghans had repatriated voluntarily.

Settlements were often situated within a few km of local

Pakistani villages, and malaria prevalence rates in

neighbouring local and refugee communities were often

homologous, suggesting a degree of ‘parasite exchange’

(Suleman 1988; Bouma & Rowland 1994). Because

hostilities have mostly ceased in eastern and southern

Afghanistan, cross-border movement of adult males was

frequent, leading to import and export of malaria.

Women and children were much less mobile, and hence

only a small proportion of malaria cases recorded in the

BHUs was acquired in Afghanistan (Rowland et al.

1996).

Survey procedures

The medical officer, malaria supervisor, and

microscopist from each BHU were trained by HealthNet

International (HNI) in survey methodology. Special

attention was paid to calculation of drug dose according

to body weight, to supervision of treatment, to

recording of clinical symptoms, and to patient follow-

up. The microscopists had been previously trained by

HNI and were highly proficient. Chloroquine resistance

was surveyed in 1994 and sulfadoxine-pyrimethamine

resistance in 1995. Subjects were recruited by passive

case detection during the transmission season from

August to December. Case data was recorded on

standard forms and printed registers provided by HNI.

A thick and thin blood smear was taken from any

patient presenting with fever or malaria-like symptoms.

Axillary temperature was taken with an electronic

thermometer and clinical symptoms were recorded

against a checklist. The patient was then given

presumptive treatment with chloroquine (10 mg per kg

body weight) or sulfadoxine-pyrimethamine (25 mg

sulfadoxine and 1.25 mg pyrimethamine per body

weight). The chloroquine was provided by WHO, the

sulfadoxine-pyrimethamine (Fansidar) was

manufactured by Roche. Patients were monitored for

30 min in case they vomited. Patients were requested to

return the next day by which time their smears had been

read by the microscopist. Confirmed falciparum cases,

enrolled into the chloroquine resistance survey, were

given an additional 15 mg chloroquine per kg body

weight during the next 24 h (total intake 25 mg/kg body

weight). Cases enrolled into the sulfadoxine-

pyrimethamine resistance survey were given no

additional treatment since the presumptive dose was

equivalent to a radical dose.

All subjects were requested to return a week after the

start of radical treatment so that staff could take a

follow-up smear and record clinical symptoms. This

procedure was repeated each week for 4 weeks for

subjects in the chloroquine resistance survey and each

week for 7 weeks for subjects in the sulfadoxine-

pyrimethamine resistance survey. If a subject failed to

report, the health worker visited him in his home,

usually that same day, to obtain the blood smear.

All slides were stained with Giemsa’s solution. A

smear was regarded as negative if 200 thick films failed

to show the presence of asexual parasites. Parasites were

initially scored using the plus system (Gilles & Warrell

1993). Unlike in the normal extended test, patients were

not excluded if their parasitaemias were less than

1000/ml of blood, and Dill-Glazko urine tests for

detection of prior intake of chloroquine were not

performed. If a chloroquine-treated patient became

falciparum-positive during the follow-up period, he was

retreated with sulfadoxine-pyrimethamine, and vice

versa. HNI monitors paid impromptu spot checks on

BHU survey staff, and questioned a number of subjects

in their homes to confirm that survey procedures had

been correctly followed. In order to verify diagnoses, all

slides were retained for re-examination by HNI’s

microscopists.

WHO extended in vivo tests

In 1993, one year before the modified test surveys, HNI

staff carried out 2 normal WHO extended in vivo tests

(WHO 1973) in refugee villages in the Charsadda and

Mardan districts. During these, blood smears were




taken at more frequent intervals, particularly during the

first 10 days. The results of these tests were used to

validate classifications based on modified test methods.

As in the modified test, the subjects’ axillary

temperature and clinical symptoms were recorded on a

checklist each time a slide was taken.

Analysis

Discrete variables, such as resistance frequencies, were

compared using x2-tests, sometimes after age structuring

using the method of direct standardization (Kirkwood

1988). Normally distributed variables, such as

temperature, were compared using analysis of variance.

Results

Verification of the modified test

The results of the WHO extended in vivo tests in

Charsadda and Mardan were used to assess the accuracy

of the modified test (Figure 2). According to the WHO

extended test, infections are classified as sensitive to

chloroquine or sulfadoxine-pyrimethamine if there is no

recrudescence during the respective 4 or 7 week study

periods after treatment; as resistant grade I (RI) if there

is trophozoite clearance for 2 or more days and

recrudescence after day 7; as resistant grade II (RII) if

parasitaemia reduces to less than 25% but fails to clear

completely; and as resistant grade III (RIII) if there is no

marked reduction in parasitaemia. In the modified

extended test, infections were classified as sensitive if

there was no trophozoite recrudescence after treatment,

as RI if trophozoites recrudesced 2–4 weeks after

chloroquine treatment or 2–7 weeks after sulfadoxine-

pyrimethamine treatment, and as RII if trophozoites

were present one week after treatment.

All infections classified as sensitive to chloroquine or

sulfadoxine-pyrimethamine in the WHO extended test

were negative by day 5 after the start of treatment

(Figure 2). All infections classified as chloroquine-

resistant RII in the WHO extended test were positive on

day 7. Thus for these two grades the modified test gave

classifications comparable to the WHO extended test.

No chloroquine-resistant RIII infections were recorded

during the two surveys, but had such cases arisen, the

modified test could not have distinguished them from

RII infections, since both grades of resistance are

trophozoite-positive on day 7. Of the infections

classified as chloroquine-resistant RI in the WHO

extended test 5% (3/60) cleared initially, but recrudesced

by day 7. These would be misclassified as RII under the

modified test. There were too few cases of RI resistance

to sulfadoxine-pyrimethamine in the WHO extended

test surveys to judge the accuracy of the modified test in

identifying this grade of resistance to this particular

drug combination.

Resistance frequencies

Seven chloroquine resistance surveys were undertaken in

1994 (471 subjects), but only 5 sulfadoxine-

pyrimethamine resistance surveys were possible in 1995

(258 subjects) owing to the low incidence of falciparum

malaria that year (Table 1). The default rate was less

than 2%.

Analysis revealed that the majority of chloroquine

resistant infections occurred in the youngest group

(Table 2). The age distribution differed slightly between

study sites, so in order to compare results, resistance

frequencies for individual villages were age-structured

by direct standardization (Kirkwood 1988), using all 729

subjects as the standard population.

The frequency of chloroquine resistance varied

significantly between study sites (x2 5 87, d.f. 5 12,

P 5 0.032). It was lowest in Kohat (18%) and highest in

Charsadda (62%) (Figure 3). Overall, 46% of infections

were resistant. Resistance existed mostly as RI; only

18% (36/213) of resistant infections were RII. The

10

100

00

Days

Freq

uen

cy (

%)

50

5

90

80

70

60

40

30

20

10

71 2 3 4

Figure 2 Frequency of trophozoite positive infections during

the first 10 days after the start of treament with either

chloroquine or sulfadoxine-pyrimethamine. ; ChlR2, j Chl

R1, m Chl S, d Fan S.




frequency of RII resistance relative to all infections

never exceeded 11%.

The frequency of sulfadoxine-pyrimethamine

resistance also varied significantly between study sites

(x2 5 15.6, d.f. 5 8, P 5 0.048). It was generally

present at a much lower frequency: range 4–25%.

Overall, only 12% of infections were resistant.

Compared with chloroquine resistance, a greater

proportion of sulfadoxine-pyrimethamine resistance –

32% (10/31) – existed as RII. There was no evidence of

an association between a high frequency of chloroquine

resistance and a high frequency of sulfadoxine-

pyrimethamine resistance (Figure 3).

Clinical symptoms

Our main purpose in recording clinical symptoms was

to assess whether parasite recrudescence had any

significance for disease management. The following

description refers only to patients in the normal WHO

extended test surveys whose infections were later shown

to be chloroquine resistant RI (N 5 76). This detailed

work was conducted by experienced HNI staff stationed

at BHUs in Mardan and Charsadda. The symptoms

recorded during the modified tests in other areas (by

general health workers) were not dissimilar. Since all

malaria patients were recruited by passive case detection

at the BHUs, all were symptomatic on admission to the

surveys. A recent history of shivering was the

commonest symptom on admission (occurring in 91%

of cases); this was followed by headache (79%), fever

(68%), vomiting (33%) and diarrhoea (21%) (Figure 4).

63% of patients exhibited 3 or more symptoms on

admission. The frequency of any particular combination

of symptoms was never significantly different from the

product of the individual symptoms. For example, the

frequency of patients with the fever and shivers

combination was, at 62% (47/76), identical to the

product of fever frequency and shivers frequency. That

for the headache and vomiting combination was, at 25%

(19/76), virtually identical to the product of headache

frequency and vomiting frequeny, 26%. This suggests

Table 1 Survey variables

District Village Persons completing survey Annual falciparum incidence

——————————————————— ————————————

Chloroquine (1994) Sulfadoxine- 1994 1995

pyrimethamine (1995)

Charsadda Adizai 25 34 94 95

Peshawar Badaber 162 61 17 9

Kohat Kahi 23 – 7 1

Mianwali Kot Chandna 81 34 8 2

Waziristan Azam Warsak 95 – 60 14

Pishin Pir Alizai 31 26 3 2

Quetta Mohammed Khel 54 103 12 14

Table 2 Frequency of resistance (%) within different age groups

Chloroquine resistance Sulfadoxine-pyrimethamine resistance

Age group ———————————————————— ———————————————————–

(years) Sensitive RI RII N Sensitive RI RII N

1–5 49 35 16 76 93 7 0 29

6–14 52 43 5 196 94 5 1 113

15–29 60 32 7 105 85 7 7 54

> 29 59 35 6 94 77 15 8 62

Total 55 38 8 471 88 8 4 258




that individual symptoms were expressed independently

of each other.

At the time of trophozoite recrudescence, 93% (71/76)

of subjects became clinically symptomatic again. No

true control group was available for comparison.

However, a satisfactory alternative was to match each

person at the time of his trophozoite recrudescence with

another member from the same group of 76 who on that

specific day after antimalarial treatment was negative

for trophozoites. When making the match, we always

selected a control of the same sex who was nearest in

age and weight to the case; the selection was done blind,

without knowledge of symptoms. Using this method, the

frequency of clinical symptomatics in the matched

District of western Pakistan

Freq

uen

cy o

f d

rug

res

ista

nce

(%

)

Quetta

100

0Charsadda

50

Peshawar

90

80

70

60

40

30

20

10

Mianwali Pishin

(b)

Quetta

100

0Charsadda

50

Peshawar

90

80

70

60

40

30

20

10

Mianwali PishinKohat Waziristan

(a)

Figure 3 Frequency of drug resistance in refugee settlements in various districts in western Pakistan. a, chloroquine resistance. b,

sulphadoxine-pyrimethamine resistance. h S, N R1, j R2.




control group was, at 32% (24/76), significantly less

than in the recrudescent group (x2 5 62, d.f. 5 1,

P , 0.0001).

The range of clinical symptoms reported during

recrudescence differed from that reported at admission

(Figure 4). The frequencies of headache (78%) and

diarrhoea (10%) were just as common as at admission,

but the frequencies of shivering (32%), fever (41%), and

vomiting (7%) were each significantly less (x2 . 15,

d.f. = 2, P , 0.002). As a consequence of this, only 21%

exhibited 3 or more different symptoms during

recrudescence, whereas 63% exhibited 3 or more at

admission. Nevertheless, the severity of symptoms

during recrudescence was serious: the frequencies of

fever, shivering, and headache were each significantly

greater than in the matched controls (x2 . 7, d.f. 5 2,

P , 0.005) (Figure 4).

The results for axillary temperature support the

above trends. Before treatment, 35% (27/76) had

temperatures > 37.5 8C. This was a significantly greater

proportion than the 17% (12/76) recorded at

recrudescence (x2 5 8, d.f. 5 1, P 5 0.005), which was

itself a greater proportion than the 5% (4/76) recorded

in the control group (2 5 3.8, d.f. 5 1, P 5 0.05). Mean

temperature was 37.3 8C before treatment, 36.6 8C at

recrudescence, and 36.3 8C in the control group

(F(3,225) 5 4.4, P 5 0.005).

Discussion

To develop or revise national guidelines for the

treatment of falciparum malaria, it is necessary to first

map the distribution and frequency of resistance

throughout the country. The WHO extended in vivo test

(WHO 1973) may not be convenient for this purpose

since it requires frequent parasitological follow-up

examinations and is labour-intensive. The WHO

standard 7-day test is also of limited value since it does

not distinguish between sensitive and RI infections. The

modified extended test described here, in which patients

were identified by passive case detection and follow-up

slides were taken at weekly intervals, proved accurate in

distinguishing sensitive from RI infections, 95%

accurate in distinguishing RI from RII infections, but

was unable to distinguish RII from RIII infections. The

modified test should prove useful for monitoring the

development or spread of resistance in countries such as

Pakistan where infections are mainly S, RI or RII. But

where infections are mostly RII or RIII, the standard 7-

day test, or the recently proposed 14-day test (WHO

1994) would be more appropriate – in Pakistan no case

of RIII has yet been identified (Shah et al. 1997). The

advantage of the modified test over the WHO extended

test was the ease with which it was accommodated into

the normal work schedules of mid-level BHU health

workers. Its use as a routine survey tool seems fully

justified.

Resistance to chloroquine and sulfadoxine-

pyrimethamine was identified in refugee villages in every

district surveyed in western Pakistan. The chloroquine

resistance results concur with those of surveys carried

out in local Pakistani villages (Shah et al. 1997). This

was expected, since refugee villages are often situated

near Pakistani villages and transmission has been shown

to occur locally (Suleman 1988; Bouma & Rowland

1994). Resistance to sulfadoxine-pyrimethamine has not

been reported in Pakistan before. Since male refugees

frequently travel into Afghanistan, malaria there is also

likely to be resistant (no in vivo surveys have yet been

undertaken in Afghanistan). Although the distribution

Freq

uen

cy o

f d

rug

sym

pto

m (

%)

diarrhoea

100

0shivering

50

headache

90807060

40302010

vomitingfever

Figure 4 Clinical symptoms associated

with patients exhibiting grade I

resistance to chloroquine. Controls

were nonparasitaemic when matched

with recrudescent cases.

h pretreatment, N recrudescence,

j control.




of resistance was significantly heterogeneous between

the 7 study sites, it showed no obvious geographical

gradation or pattern. In 5 of the 7 sites, chloroquine

resistance exceeded 45%. New infection may be

confused with recrudescence if transmission is intense,

and thereby exaggerate the true frequency of resistance.

However, in the refugee settlements the low incidence of

falciparum in 1994–95 would seem to rule out new

infections as a significant source of error. The estimates

of resistance frequency are therefore considered

reasonably accurate. Since parasite recrudescence is

associated with a resumption of clinical symptoms, it is

becoming difficult to justify the use of chloroquine as

first-line treatment in the majority of refugee

settlements. A recent WHO report considered it

unreasonable to maintain a drug as first-line treatment if

clinical failures exceed 25% (WHO 1994). To not

change the first-line drug may lead to a loss of faith in

the refugee health services, and to refugees seeking

private treatment with expensive or unnecessary drugs

(Guinness & Rowland, unpublished), as seems to be the

response of the Pakistani public to its own health

services (M. Donnelly, personal communication).

Sulfadoxine-pyrimethamine resistance, though present

in every locality, was comparatively rare and the

adoption of this drug as the first-line treatment would be

more efficacious. Indeed one NGO has already switched

from chloroquine to sulfadoxine-pyrimethamine to

reduce the burden of remissions on outpatient clinics

(Shah et al. 1997). A more rapid selection of

sulfadoxine-pyrimethamine resistance would, however,

be inevitable should this become official policy.

Acknowledgements

We thank the BHU health staff for carrying out the

surveys and the initial microscopy, and HealthNet field

staff for monitoring work. We are grateful to Professor

H. M. Gilles for his constructive criticisms. The

HealthNet malaria control programme is supported by

the United Nations High Commissioner for Refugees

and is funded by the European Commission.

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