Am. J. Trop. Med. Hyg.. 53(5), 1995. pp. 526—531 Copyright 0 1995 by The American Society of Tropical
IN VITRO
Medicine
and Hygiene
SUSCEPTIBILITY
OF PLASMODIUM
CHLOROQUINE, AMODIAQUINE, SULFADOXINE/PYRIMETHAMINE
FALCIPARUM
TO
QUININE, MEFLOQUINE, AND IN EQUATORIAL GUINEA
A. BENITO, J. ROCHE, R. MOLINA, C. AMELA, ANDJ. ALVAR Department of Parasitology, National Center of Microbiology, and National Center of Epidemiology. Instituto de Salud Carlos ill, Ministry of Health, Madrid, Spain; Hispano Guinean Center of Tropical Diseases, Spanish Agency of International Cooperation, Malabo, Equatorial Guinea Abstract.
Between
March
1990
and
June
1992,
a study
was
carried
out
in Equatorial
Guinea
on the
in vitro
response of Plasmodium falciparum to different antimalarial drugs. Field work for the study was conducted both in the country's island region as well as on the mainland, and resistant isolates were found to exhibit interregional differences. On the island of Bioko, 204 tests were performed with 16% (1 1 of 69) resistant to chloroquine, 9% (4 of 46) resistant to quinine, 14% (6 of 43) resistant to a combination of sulfadoxine/pyrimethamine, and 6.5% (3 of 46) resistant to amodiaquine. In the mainland area of Bata, the same antimalarial drugs and mefloquine were tested with the following results: 9% (5 of 58) resistant to chloroquine; 2% (1 of 58) resistant to amodiaquine, and 3% (2 of 58) resistant to a combination of sulfadoxine/pyrimethamine. No isolates resistant to quinine or mefloquine were
found. Effective concentrations (EC@,O, EC@, and EC@) and regression lines (log dose/response) for each antimalarial drug were calculated to establish a surveillance system for antimalarial drug chemosensitivity in Equatorial Guinea. Finally, 12 isolates from 12 patients previously treated with chloroquine were studied to compare both tests (in vivo in vitro) and obtain a correlation between the RI! and RI!! types of in vivo and in vitro resistances. No correlation for the RI type was found between the two methods. One way of controlling the morbidity and mortality in duced by Plasmodium falciparum infections is to effectively treat all malaria cases. However, not only has P. falcipa rum ‘sgrowing resistance to chloroquine and other antima larial drugs had considerable impact on the epidemiology of malaria, increasing morbidity and mortality alike, it has also produced serious obstacles to control programs. Since resis tance to chloroquine in Africa was first detected in Tanzania and Kenya,' it has spread to the point that it extends to practically all parts of Africa.2 3 This has led to the use of other antimalarials such as quinine or a combination of sul fadoxine/pyrimethamine, as well as amodiaquine, which is widely used in central Africa. Equatorial Guinea is a small country situated on the west coast of central Africa, consisting of two islands (Bioko and Annobon) and a mainland region (Rio Muni). There are sev en provinces with 2 1 administrative areas. The islands are situated in the Gulf of Guinea: Bioko (3°43'N, 8°43'E), 45 km from the Cameroon coast, has 60,000 inhabitants, and Annobon, situated in the southern hemisphere (next to Sao Tome island), 670 km from Bioko, has 2,174 inhabitants. The mainland region, with 200,000 inhabitants, is situated between Cameroon and Gabon. Bata (1051 ‘N,9°4l‘E)is its capital. The population of Equatorial Guinea belongs pre dominantly to three ethnic groups of Bantu origin: the Fang (mainly in the mainland region), the Bubi (Bioko island), and the Annobones, originating from slaves that colonized the island of Annobon. There are seven health regions with a net of district hos pitals. The primary health care programme is run by the Agencia Espanola de Cooperacion Internacional and by the Ministry of Health. There arc a small number of health cen ters, hospitals, and dispensaries run by nongovernment or ganizations. The National Malaria Control Programme is supported by the Ministry of Health, the United Nations Dc velopment Program-World Bank, and the Centro Hispano
The main causes of mortality in this country are malaria and diarrheal and respiratory diseases. Vaccination coverage (diphtheria-pertussis-tetanus [DPT], bacille Calmette-Guerin [BCG], oral polio vaccine [OPV], and measles) is 90%, and there are a small number of cases of tuberculosis and ac quired immunodeficiency syndrome when compared with other countries in Africa. Malaria in Equatorial Guinea is hyperendemic to holoen demic and exhibits a stable year-round pattern of transmis sion with small seasonal fluctuations.@ To create a surveil
Guineano de Enfermedades Tropicales.
age in accordance
lance
system
to monitor
P. falciparum
sensitivity
to anti
malarial drugs, a reference center has been set up for Equa torial Guinea, with the first in vitro and in vivo tests being carried out in the country's island and mainland areas. The aims
of this
study
were
1) to ascertain
resistance
levels
to
different antimalarial drugs in the two regions using the of ficial World Health Organization (WHO) in vitro and in vivo tests (Payne D, unpublished data), 2) on the basis of results of various effective concentration (ECSO, EC%, and EC@J) tests,
to set up a surveillance
system
to monitor
the
che
mosensitivity of P. falciparum within the overall framework of Equatorial Guinea's National Control Program, and 3) to compare these results with those from studies conducted in neighboring
territories.
MATERIALS
AND
METHODS
Area and population studied. The study was carried out in two regions of Equatorial Guinea: the island of Bioko, located
45 km from
the Cameroon
coast
in the Gulf
of Gum
ea and Bata, which is at sea level and is the capital of the mainland region (Rio Muni), situated between the Cameroon and Gabon. On Bioko, a total of 410 tests were conducted with selec tion being made from 1,587 children less than 10 years of
526
with WHO-standardized
incorporation
cri
527
IN VITRO SUSCEPTIBILITY OF P. FALCIPARUM TABLE 1 to chloroquine of 69 Plasmodium
TABLE 3 iso
In vitro response to amodiaquine of 46 Plasmodium falciparum iso
lates from Bioko Island, Equatorial Guinea, and 58 P. falciparum isolates from Bata (mainland region), 1990—1991
lates from Bioko Island, Equatorial Guinea, and 58 P. falcipa rum isolates from Bata (mainland region), 1990—1991
In vitro response
Drug concen uation (pmol/ well)No. Bata119(28)
(%) of isolates with complete inhibition
falciparum
inhibition
maturationBioko of schizont maturation%
Drug concen Uation (pmoL/ well)No. Bata0.2515(33)
of
schizont
BatsBioko
845.64234
48 (83)96.53 51 (88)99.15 53(91)99.63 55 (95)99.64
57 (98)99.67 58 (100)99.98
in vitro response
to quinine
TABLE 2 of 46 Plasmodium
Dnig concen tration (pmoll well)No. Bata42
57.9183
region),
falciparum
isolates
1990—1991
(%) of isolates with complete inhibition of maturationBioko schizont maturation%
inhibition schizont
of
BatsBioko
(4.3)
92.45168(17) (7) 99.863218(39)
1006435(76) 10012839(85) 10025639 (85)
5 (9)22.41
criteria
used
a protocol were
those
100
of 14 days. The RI—RI!! of the standard
WHO
in
vivo test (unpublished data). Twelve patients were tested with the aim of comparing the degree of the in vivo chlo roquine resistance (RI—lUll) and the results of in vitro par asite sensitivity. In vitro procedure. The test consisted of measuring the development of parasites in culture in the presence of dif ferent concentrations of antimalarials. In all cases, use was made of the microtest (MARK II; supplied by WHO, Ma nila, The Philippines). This test uses 96-well microtiter cul ture plates, in which each column corresponds to a patient and each row corresponds to a given concentration of anti malarial, with the exception of the topmost well in all col umns, which contains no drug and is used as control. Using the control as the starting point and moving down the re spective columns, wells contain increasing concentrations of each antimalarial drug. A total volume of 50 p.1 of a 1:9 mixture of peripheral blood and RPM! 1640 culture medium was added to each well in the cultivation plates (each column corresponding to an isolate). Each plate was cultured at 37°Cfor 28 hr in a candle jar in which oxygen was reduced by means of con sumption of a paraffin sail. In Bata, cultures were prepared with the aid of a battery-powered field stove supplied by the WHO Manila Regional Office, while in Bioko, electric-pow ered insulators were used. On conclusion of the culture pe riod, medium in each well was extracted and eight thick blood films of each isolate were taken. Schizonts containing
In vitro response to sulfadoxine/pyrimethamine of 43 Plasmodium falciparum isolates from Bioko Island, Equatorial Guinea, and 58
P. falciparum isolates from Bata (mainland region), 1990—1991 No. (%) of isolates with Drug complete inhibition of concentration schizont maturation (sulfadoxine/ maturationBiokoBataBioko pyremethamine). pmollwell% Bats10/0.1253(7)4(7)62.71
inhibition schizont
of
47.7430/0.37515
23 (39)53.26 36(62)56.91
43(74)92.11 53(91)99.79 57(98)99.90 58 (100)99.79
7, 10, and 14 following resistance
of
TABLE 4
from Bioko Island, Equatorial Guinea, and 58 P. falciparum iso lates from Bata (mainland
29(50)68.81 49(84)94.39 55 (95)99.65 56 (96)99.90 57 (98)99.97 58(100)100 58(100)100
100
teria for microtest performance (Payne D, unpublished data). The selection of patients for the clinical tests was carried out in the consulting rooms of the pediatric service. Exclusion criteria included severe malaria requiring immediate therapy, malaria complicated by other infectious diseases, and infec tions with P. malariae or P. ovale in addition to P. falcip arum. The technique of Dill and Glazko7 was used to detect the presence of 4-aminoquinolines in urine. Of the total test ed, 204 (50%) isolates exhibited sufficient transformation in cultivation for interpretation to be possible. In Bata, 885 schoolchildren less than nine years of age were given blood tests: 96 met selection requirements. Of these 96, 58 isolates (60%) presented sufficient transforma tion in cultivation for each of the antimalarial drugs. Four antimalarials, chloroquine, quinine, amodiaquine, and sulfadoxine/pyrimethamine, were studied on the island and five (the previous four and mefloquine) were studied on the mainland. With the aim of correlating the results of both in vivo and in vitro tests, 14-day in vivo protocols standardized by WHO (unpublished data) were carried out in 12 patients. The ac tual drug base of chloroquine sulfate (lot 0020; Ludeco, Brussels, Belgium), was confirmed in the Centro Nacional de Farmacobiologia, Instituto de Salud Carlos Ill (Madrid, Spain). A total dose of 25 mg/kg of chloroquine sulfate was given over three consecutive days (10 mg on days one and two and 5 mg on day 3). Patients were chosen and super vised at the Pediatric Service of the General Hospital of Malabo. Deithametrin-impregnated bed nets were installed on all beds; thick blood smears were taken daily on days 0—
inhibition schizont
BathBioko
83.310.5028(61) 98.79136 99.79237 (79) 99.90439 (80) 99.96846(100) (85) 1001646(100)
38(65)84.10
99.15448(70)(50) 99.66855(80) 1001656 1003256 (82) 1006465 (82) (94)
(%) of isolates with complete inhibition of schizont maturationPercentage maturationBioko
78.57100/1.2520(47)31(53)92.15 (35)17
97.06300/3.7526 99.241,000/12.533 99.723,000/37.534 99.9010,000/12537 100
(60)50 (77)56 (80)56 (86)57
(30)83.39
(86)90.62 (96)99.94 (96)99.00 (98)99.48
99.92
528
BENITOAND OTHERS TABLE 5
-
Cross-resistances (maximum inhibitory concentrations) of various antimalarial drugs with schizonts of eight Plasmodiumfalciparum isolates from Bioko Island, Equatorial Guinea, l990@l99l*
Probit
5 4
CQ-QNCQ-AMDCQ-SDXQN-SDXCQ-QN-SDX16-25616-1616-3
xl0@32-256-10@32-25664-4256 xl0@256-3
3
x HP 2 @
a
chloroquine;
the
maximum
QN = quinine;
inhibitory
concentration
AMD = amodiaquine;
with
schizonts
(pmol/well).
CQ
=
SDX = sulfadoxine-pyrimethamine.
three or more nuclei for chloroquine, amodiaquine, meflo quine, and quinine and eight or more for sulfadoxine/pyri methamine per 200 parasites were enumerated. The per centage of schizonts for each drug concentration was cal culated by dividing the schizont count per 200 parasites by the schizont count per 200 parasites in the control and mul tiplied by 100. Resistance was indicated if schizonts appeared in the pres ence of 8 pmol/well of chloroquine, 4 pmol/well of amodia quine, 256 pmol/well of quinine, and 64 pmol/well of amo diaquine. In the case of sulfadoxine/pyrimethamine, a break point was established in any well registering inhibition of 90% of schizonts with eight or more nuclei. For each drug, the respective EC50, EC%, and EC@ were computed by conducting a logarithmic dose-response test where transformation from trophozoites to schizonts took place in 50%, 90% and 99% of the parasites (Grab B, Werns dorfer WH, unpublished data). Regression lines for the re spective antimalarial drugs were plotted using the SAS/ STAT®Release 6.03 program (SAS, Inc., Cary, NC). RESULTS
On Bioko, 16% (1 1 of 69) of all isolates tested were re sistant to chloroquine, 9% (4 of 46) to quinine, 14% (6 of 43) to sulfadoxine/pyrimethamine, and 6.5% (3 of 46) to amodiaquine. The response to these four antimalarials is de picted in Tables 1—4.The breakpoint for sulfadoxine-pyri methamine was between 300/3.75 and 1,000/12.5 pmol. Fur thermore, cross-resistance was detected among nearly all an timalarial drugs tested (Table 5). The logarithms for the EC@, EC@, and EC@ of the four antimalarial drugs are given in Table 6, and represent the concentrations in which 50%, 90%, and 99% of the parasites failed to undergo transfor mation. The regression lines for the four same antimalarials are shown in Figure 1. In Bata, 9% (5 of 58) of the isolates were found to be resistant to chloroquine, 2% (1 of 58), to amodiaquine, and
TABLE
EC5O
EC99
EC9O
—,-. Chloroquin.
FIGURE
1.
—I—Quinine
Probit
amodiaquine, and modium falciparum
regression
—— Amodiaqulne
lines
of
204
-o-
8DX/PYR
(chloroquine,
quinine,
sulfadoxine/pyrimethamine [SDXIPYRJ) Plas isolates from Bioko island, Equatorial Guinea.
EC = effective concentration (50%, 90%, and 99% inhibition of schizont
maturation).
3% (2 of 58) to sulfadoxin&pyrimethamine. study
revealed
no isolates
resistant
The present
to mefloquine
or quinine.
The breakpoint for sulfadoxine/pyrimethamine was between 1,000/l 2.5 and 3,000/37.5 pmol. Tables 1—4and 7 depict the responses of 58 isolates to each of the drugs used. Figure 2 shows the regression lines for chioroquine, quinine, amodia quine, and mefloquine. Figure 3 shows the regression line for sulfadoxine-pyrimethamine. Table 6 shows the EC50, EC@, and EC@ values. Island EC@ readings (pmol) were 1.183 for chioroquine, 2.394
for quinine,
0.476
for amodiaquine,
and 3.095
for sul
fadoxine/pyrimethamine. In contrast, equivalent EC50 values for the mainland region were 0.489 for chloroquine, 3.607 for quinine, 0.125 for amodiaquine, 10.340 for sulfadoxine/ pyrimethamine, and 0.870 for mefloquine. Both in vivo and in vitro tests were carried out for 12 individuals. Table 8 shows the correlation between the in vivo (14 days) and in vitro tests. In the 14-day test, four isolates were susceptible at minimum effective concentra tions of 0.4 x l0-6M and 0.8 X 10M, and in vivo type RI! resistance was detected in two isolates. One isolate presented schizonts in vitro at a chioroquine concentration of 1. 16 X l06 M, but was sensitive in the 14-day test. Table 8 shows the agreements between the in vitro and the in vivo tests. The two isolates resistant in vitro gave RI! responses in vivo. DISCUSSION
This is the first report of in vitro studies on parasite sus ceptibility to different antimalarial drugs in Equatorial Gum
6
Effective concentrations (EC in pmol) (log10of dose) of chioroquine, amodiaquine, quinine, and sulfadoxine/pyrimethamine against Plasmodium falciparum
from Bioko
Island, Equatorial
Guinea,
and Bata and for mefloquine
against P. falciparum
from Bata
SulfadoxineChloroquineQuinineAmodiaquinepyrimethamineMefloquineBiokoBatsBiokoBatsBiokoBatsBioko
[email protected]
[email protected] [email protected] 251.4292.919Slope1.2740.6502.0481.9801.4781.4001.043
1.8401.200SD0.7840.6900.4880.7020.6760.6450.957 0.5250.815
IN VITRO
in vitro response
TABLE 7 to mefloquine of 58 Plasmodium
SUSCEPTIBILITY
529
OF P. FALCIPARUM
14 @roblt falciparum
iso
lates from Bata (mainland region), Equatorial Guinea
12 i;@
Drug (%) of isolates with concentration complete inhibition of inhibition of (pmol/well)No. maturation240(69)94.43454(93)99.81858(100)1001658(100)1003258(100)1006458(100)10012858(100)100 schizont maturation% schizont
ECSO
EC9O
EC99
CHLOROQUINE.+AMODIAOUINE-a--QUININE-$--MEFLOQUINE
ea. Two reports have been published citing cases of chlo roquine resistance demonstrated by in vitro techniques in parasites from patients returning home after a period of stay in the
9 No resistance
had previously
been
detected
to the other drugs used in this study. With respect to chloroquine, the in vitro resistance level is moderate (16%) and lower than that recorded by other investigators in other countries in the same geographic area. In Cameroon for example, there is evidence of in vivo re sistance of 64% of the patients,'° and a range of 6—30% of in vitro isolates tested were resistant to 4-aminoquinolines.9 Higher in vitro resistance percentages have been reported from Nigeria,― where there has been a 10—25% increase in resistance from 1987 to 1989. In other parts of Nigeria, such as Oyo State in the southern part of the country, in vivo and in vitro sensitivity has been almost 100%, with no in vitro resistance being detected.'2 In Liberia, 34% of the isolates have proven to be chloroquine-resistant in vitro.'3 In The Gambia, an increase in resistance has been documented, with I 1% of the isolates tested being resistant.'4 In Cameroon, the percentage of isolates that are chloro quine resistant ranged from 2% in the region of Bertoua (in the east), to 10% in Garoua (in the north), 86% in Limbe (40 km northeast of Malabo), and 56% in Kribi (near the border with mainland Equatorial Guinea).'5 In Yaounde, in vitro tests have revealed resistance percentages to chloro quine of 60% in 1987 and 1988, 55% in 1989, and 42% in 1990 and 199l.16 In Benin, resistance percentages are similar to those in Cameroon.'7 In Zaire, the percentages of resistant isolates vary between 80% and 90%.18 19 In Brazzaville, Congo, chloroquine-resistance levels were 59% in 1985, 60% in 1985, 50% in 1987, and 50% in 1990.20 The moderate levels of amodiaquine-resistant isolates en countered in this study could well lead to this drug being considered as a second line of attack or as a possible front line substitute in resistance
in the event to chloroquine.
of there There
FIGURE
2.
Probit
regression
lines
of
230
(chloroquine,
quinine,
amodiaquine, and mefloquine) of 230 Plasmodium falciparum iso lates from Bata (Rio Muni), Equatorial
Guinea.
EC = effective
con
centration (50%, 90%, and 99% inhibition of schizont maturation).
Resistance to quinine has also been encountered in central Africa, with percentages of 0% in 1985, 1% in 1987, 2% in 1988, 9% in 1989, 3% in 1990, and 4% in 1991 •20. 25On the island of Bioko, 9% of the isolates were found to be resistant to quinine, mainly in the southern area of Luba where most of the isolates resistant to this antimalarial were detected (25%).
Rates
of in vitro
resistance
to quinine
did
not
cor
relate well with levels of in vivo resistance. The clinical assays showed quinine to be a very good antimalarial for second-line treatment for those cases sent to distric hospitals or health centers because of treatment failures with first-line therapy (chloroquine) at the village level. Quinine is also the usual drug of choice for the treatment of severe P. falcipa rum malaria. Finally, the percentage of isolates resistant to sulfadoxine/ pyrimethamine seem to constitute valid grounds for it being regarded as a second-line antimalarial on the island of Bio ko. On the mainland, EC data indicated greater sensitivity for all antimalarials, with the exception of sulfadoxine/pyr imethamine. Nine percent (5 of 58) of all mainland isolates tested
were
resistant
to chloroquine,
a lower
percentage
than
being a further increase has been an increase in
the use of amodiaquine, which has proved extremely effec tive in zones where there is a high degree of resistance to chioroquine. This efficacy has already been demonstrated in different countries, e.g., Kenya.2124 In Brazzaville, amodia quine remains effective provided the dose is increased from 25 to 35 mg/kg/3
days;25 in Rwanda,
it has also proved
most
effective, offsetting the high degree of local resistance to chloroquine.26 The percentage of resistant isolates found on Bioko would argue in favor of amodiaquine being used should resistance to chloroquine dramatically increase.
-e—SOX/PYR
F@ouar 3. Probit regression line of 58 sulfadoxine/pyrimetham inc Plasmodium falciparum isolates from Bata, Equatorial Guinea. EC = effective concentration (50%, 90%, and 99% inhibition of schizont maturation).
BENITOAND OTHERS
530
Sensitivity
of Plasmodium
falciparum
(14-day
TABLE 8 in vivo tests and in vitro tests) on the island of Bioko, Equatorial
M)Results
Results of in vitro test (MIC,
2.8TotalS of in vivo test0.2Sensitivity—40.40.81
RI RH 12a Total4 MIC
= minimum
1990—1991*
l06
. 16 4—Resistance3.26.41
4 2
3 41 inhibitory
concennation
41 of chloroquine
11 that caused a total inhibition
that found on Bioko. The most important difference with respect to the Bioko study was in the choice of individuals; on the mainland, this group were made up of schoolage chil dren more than five years of age, whereas on Bioko, most of the samples were taken from children less than five years of age. With respect to those drugs usually used as second-line antimalarials, 2% (1 of 58) of the isolates were found to be resistant to amodiaquine and 3% (2 of 58) to sulfadoxine/ pyrimethamine. This confirms the potential efficacy of these drugs in this region. The present study detected no isolate resistant to mefloquine or quinine, which are considered third-line antimalarials to be used in the treatment of com plex or serious cases. The data for Equatorial Guinea are consistent with comparable data for most of the surrounding countries, in which the majority of isolates exhibit good sen sitivity to these antimalarials. Perhaps of greatest importance is the fact that a firm foun dation, in the form of an in-place surveillance system, has now been implemented for keeping track of chemoresistance in Equatorial Guinea. Moreover, calculation of the EC@,
11
11
of schizonts
lution of isolates. These are the first studies in Equatorial Guinea to evaluate the sensitivity pattern of P. falciparum to chloroquine using in vitro-in vivo tests, and to quinine, amodiaquine, and sul fadoxine/pyrimethamine using the in vitro tests. In vivo results obtained from patients a with an RI level of resistance cannot be correlated directly with the in vitro results with parasites from the same persons (Table 8). There are at least two explanations for this finding. The host's im mune system activity impacts the in vivo response but not the in vitro results with the same parasites. An infected, im mune host can be treated and the disappearance of parasites from the peripheral blood would be interpreted as indicating that the P falciparum strain was sensitive to the drug used. Cultured parasites from this patient may well show resis tance to the same drug. Disturbances in the uptake of the drug can result in the erroneous interpretation that the par asites are resistant to the treatment whereas the in vitro re sults clearly indicated that they are susceptible. Therefore, the evaluation of the immune status and drug concentration in the blood of the subject are absolutely necessary to clarify levels of in vivo drug sensitivity. The application of tech niques that are more precise than the detection of 4-amino quinolines in the urine (Dill and Glasko test) such as the immunobsorbent
assay
or high-performance
liquid chromatography will be valuable. According to strat ification in central Africa, for the in vivo test results, the
16
grown.
island of Bioko should be classified as Stratum III (chioro quine resistance present at the RI!! level),27 a region with frequent and high resistance, similar to the foci in Gabon, Congo, Zaire, and southwestern Cameroon. Acknowledgment: We thank Dr. Manuel Nguema, National Director of the Equatorial Guinea Health Ministry's Malaria Control Pro gram, for kind support. Financial
support:
This study was funded
by the Agencia
Espanola
de Cooperacion Internacional (Spanish Agency for International Co operation), and by Spain's Fondo de Investigaciones Sanitarias (Health Research Foundation) (Expte.91/0002). Authors'
addresses:
A. Benito, R. Molina,
and J. Alvar, Servicio
de
Parasitologia, Centro Nacional de Microbiologia, Instituto de Salud Carlos III, 28220-Majadahonda, Madrid, Spain. J. Roche, Hispano Guinean Center of Tropical Diseases, Spanish Agency of Interna tional Cooperation, Malabo, Equatorial Guinea. C. Amela, National Center of Epidemiology, Instituto de Salud Carlos II!, Ministry of Health, Madrid, Spain. Reprint requests: A. Benito, Servicio de Parasitologia, Centro Na cional de Microbiologia, Instituto de Salud Carlos III, 28220-Ma jadahonda, Madrid, Spain.
EC@,and EC@will allow for future observation of the evo
enzyme-linked
Guinea,
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