IMMXJNODIAGNOSIS OF MALARIA INFECTIONS BY THE NATIONAL
MALARIA ERADICATION SERVICE (SNEM) OF COSTA RICA1
Herman Weinstok,2 Jo& L. Gam% F.P Joaquin E, .sOt~,~ and Juan Rodrjguez R.5
Costa Rica’s National Malaria eradication Service has been using the indirect immunofluorescence test since 1974 for
fircr$oses of malaria survei&ance and diagnosis. The results obtained from determining the specific malaria antibodies in
a number of areas suggest the absence of malaria endemicity both in communities with high parasite rates in the fast and in vulnerable groufis where epidemic outbreaks were detected.
Introduction
Over the last two decades comparative data on the prevalence, geographic distri-
bution, and intensity of human malaria
infections have been based primarily on
direct microscopic examination of periph- eral blood samples treated with derivatives
of Romanowsky’s stain. However, depend-
ing upon the locale, population density, behavior of the etiologic agent, and the type and extent of host-parasite interac-
tions, this direct diagnostic method may
prove costly, difficult, insufficiently sensi- tive, or inconclusive. Considerable research has therefore been addressed to developing and evaluating indirect diagnostic methods for detecting malaria infections.
In Costa Rica, the natural history of
malaria, the response of the disease to
attack measures, and data obtained from
epidemiologic surveillance all indicated a
need to supplement the parasitoscopical
‘Paper presented at the IV Latin American Para- sitology Congress, III National Microbiology and Parasitology Congress, and XL11 National Medical Congress, held in San Jose. Costa Rica, 8-l 1 December 1976. Also appearing in Spanish in the Roletin de la Oficina Sanitaria Panamericana, 1979.
ZFormerly Minister of Health of Costa Rica. 3Director. National Malaria Eradication Service (SNEM) of Costa Rica.
4Director, SNEM Laboratory, San Jo&, Costa Rica. 5SNEM Laboratory, San Jose, Costa Rica.
diagnosis with seroepidemiologic fechniqves for better epidemiologic evaluation.
Past research had demonstrated the po- tential value of immunologic and serologic tests as diagnostic aids, and many subse-
quent investigations in this field had
sought ways of improving these tests’ sensi- tivity and specificity. Naturally, considera- ble thought was given to selection of the particular test to be used and its method of application -for although the fundamental principles of imniunology apply eqtially well to viral, bacterial, and protozoaa in- fections, significant quantitative and qual- itative differences exist between the anti- genie stimuli these different types of orga- nisms induce ili their hosts. Furthermore, despite the fact that the erythrocytic phase of malaria trrfection provides a powerful stimulus for production of specific anti- bodies against the parasite, serologic tests provide no indication of the degree of im-
munity acquired, because much of the
specific antibody has no protective func- tion.
Objectives
Principal reasons for seeking out a new serologic technique of this nature were as follows:
l To determine the prevalence of ma-
laria antibodies in those Costa Rican com-
258
PAHO BULLETIN lvol. 13, no. 3, I979
munities that had shown the highest ma-
laria parasite rates in previous years, in
order to confirm that transmission had
been interrupted.
l To determine the prevalence of ma-
laria antibodies among the population at
risk in areas with current parasitologically confirmed cases.
Materials and Methods
Of the many available immunodiagnostic tests for malaria (I) the indirect immuno- fluorescence test was selected because of its suitability for epidemiologic studies, its contribution to diagnosis, and its usefulness in investigating other matters relating to development of the infection. Additional factors considered in adopting this method were the ease of obtaining blood samples in the communities involved, the feasibility of obtaining suitable antigens, the malaria service’s ability to carry out the processing required, and the sensitivity and specificity of the test.
The populations included in the study
reported here were as follows:
l A total of 854 people were examined at
six localities in the district of Bejuco in 1974. This represented 73.6 per cent of the
district’s entire population, which was
1,160 at the time. The annual incidence of
parasitologically confirmed malaria cases
in these localities had ranged from 95.91 to 248.91 cases per 1,000 inhabitants in 1967 (Table 1).
l A survey was conducted in the Atlantic
seaboard district of Pococi in 1974 in the area of one imported case and another case introduced into the district. The survey sought to delimit the focus of transmission. In all, 512 inhabitants from four localities (representing 65 per cent of these localities’ total population of 787) were included in the survey.
l Another survey was carried out that
same year at two communities in the
district of Liberia, where routine surveil-
lance operations had detected two
P.
falciparum cases. Although the 1974 census recorded a population of 68 inhabitants for these communities, a total of 111 people were included in the survey. Many of these had recently arrived at local farms.
0 In 1975 and 1976 a total of 2,388
people were examined in response to 57
parasitologically confirmed cases in 24
localities of 16 districts in Costa Rica’s ori- ginally malarious area.
The indirect immunofluorescence test
was performed using the thick-smear anti- gen technique described by Sulzer et al. (2, 3). The antigens employed were as follows:
Table 1. Annual blood examination index (ABEIF’ and annual parasite incidence (ApI)b in six localities of Bejuco District, Costa Rica, in 1967.1973.
AU the infections indicated were caused by P. z&ax.
Locality 1967 1968 1969 1970 1971 1972 1973
ABE1 API ABE1 API ABE1 API ABE1 API ABEI API ABE1 API ABE1 API La Moravia - - 38 70.41 73 7.04 287 - 127 58 61 Quebrada de
Nando 199 126.91 96 42.32 62 5.29 141 5.29 132 5.29 49 - 33 La Javilla 136 95.91 44 5.05 98 50.51 245 10.10 196 ~ 147 39 Pueblo Nuevo
l P. viuax, achiote strain, adapted in the owl
monkey, Aotus tridgatus trimkgatus (4), com- posed of schizonts (80 per cent) at a density of approximately 20,000 parasites per cubic milli- meter of blood.
l P. falcipa7um from natural human infec-
tions, a proportion of t 80 per cent schizonts being obtained through maturation in vitro (5), at a density of approximately 20,000 parasites per cubic millimeter of blood.
Anti-human goat serum (IgM, IgA)
marked with commercial fluorescein was
used, and a microscope with ultraviolet il-
lumination was employed to detect fluo-
rescence.
Blood samples were obtained by pricking each subject’s finger and collecting the re- sulting blood within two circles 12 mm in diameter inscribed on a sheet of Watman No. 3 filter paper. To avoid contamina- tion, all the filter papers were separated by glassine sheets, and the resulting stacks of filter papers were placed in plastic bags containing silica gel. A dye was later used to cut out filter paper discs 10 mm in diam-
eter containing approximately 50 ~1 of
blood ( f 25 ~1 serum). Each disc was then placed in 0.4 ml of phosphate-buffered saline solution (PBS) (pH 7.2) to elute the serum, yielding a serum dilution of 1:16. The lowest accurate titer reading attain- able with this procedure is 1:16. Never-
theless, for purposes of calculating the
geometric mean of the reciprocal titers of the sera, the negative sera were arbitrarily distributed at the titers 1:4 and 1:s.
Results
Bejuco District
Seventeen sera from the six Bejuco Dis- trict localities yielded fluorescent reactions
at reciprocal titers of 264 when tested
against
P.
&ax antigen (Table 2). Although a large proportion of the localities’ total population was examined (at least 66 per cent of each age group beyond infancy and 36 per cent of all infants), the tests showed an extremely low proportion of seropositive subjects (i.e., subjects with serum recipro- cal titers of 2 16). This was true of all theage groups studied, especially of those
groups below age 15. Overall, only 7.49 per cent of the sera yielded positive results, the
highest percentage of positive responses
being found among subjects who had
reached 45 years of age. The geometric
mean reciprocal titer (GMRT) was 6. No
plasmodia were observed in the respective 854 thick blood films by microscopic exami- nation.
Pococi District
Of the sera obtained from 512 subjects in four Pococi localities, only six yielded titers of 1:16 with
P.
uivax antigen and only one yielded a titer of 1:64 (Table 3). This latter sample was from a subject 45 years of age. The percentage of seropositive subjects wasTable 2. Prevalence of P. v&x antibodies found among residents of six Bejuco District localities (see Table I), by serum titer and age group, in 1974.
<l 55 20 10 10 - - 6
l-4 174 165 80 80 4 1 - - 3.03 6
5-14 409 322 159 159 3 1 - - - 1.24 6
15.44 400 263 119 120 20 4 - - 9.12 6
245 122 84 27 26 20 7 4 - 36.91 11
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PAHO BULLETIN l vol.13,
no.3, I979
Table 3. Prevalence of P. z&x antibodies found among residents of four Pococi District localities,a by serum titer and age group, in 1974.
Age
group (Y-s)
No. of
SWa
tested
Serum titers obtained with Aotus/P. vivax antigen % positive
scra (titers GMRT’~ 1:4 1:8 1:16 1:64 1:256 1:1024 1:4096 I:16 or higher)
<l 3 2 1 - - - 6
1-4 85 50 35 - - - 5
5-14 216 115 100 1 - - - - 0.46 6
15-44 141 98 40 3 - - - 2.13 5
145 67 48 16 2 1 - - - 4.48 6
Total 512 313 192 6 1 - - 1.37 5
aBarra de Colorado Nortr, Barra de Colorado Sur, Agua Dulce, and Tortuguero. bCMRT = Geometric mean reciprocal titer.
Table 4. Prevalence of P. falc@arum antibodies found among residents of four Pococi District localities,a by serum titer and age gmup, in 1974.
Age
grow (Years)
No. of sera tested
Serum titers obtained with in vitro/P. falciparum antigen Y0 positive
sera (titers GMRTb 1:4 1:8 1:16 1:64 1:256 1: 1024 1:4096 1:16 or higher)
Cl 3 2 1- - - _ - - 6
1-4 85 50 35 - - - 5
5-14 216 116 100 - - - 6
15-44 141 99 40 1 1 - - 1.42 5
245 67 49 17 - 1 - - - 1.49 5
Total 512 316 193 1 2 - - 0.59 5
aBarra de Colorado Norte, Barra de Colorado Sur, Agua Dulce. and Tortuguero. bGMRT = Geometric mean reciprocal titer.
1:37 and the GMRT was 5. When tested
with P. fatciparum antigen (Table 4), these same sera yielded only three positive titers (2 16), the percentage of seropositive sub- jects was 0.59, and the GMRT was 5. All
thick blood smears yielded negative results.
Liberia District
Nine of the 111 sera collected from sub- jects in two Liberia localities were found to
have positive titers when tested with P.
vivax antigen. Most of these sera were ob-
tained from day laborers 15-44 years of
age, and all but two of the positive sera came from people in this age range. The other two positive sera were obtained from older subjects (Table 5). When tested with
P. falciparum, two of the Liberia sera yielded titers of 1:4,096. These were ob- tained from subjects with patent parasit- emia whose cases prompted the survey. In addition, sera from five nonresident work- ers yielded titers ~64 with P. falciparum
(Table 6). The GMRT was 7 with P. vivax
Table 5. Prevalence of P. V&X antibodies found among residents of two Liberia District localities,a by serum titer and age group, in 1974.
Age No. of
group Set-a
(years) tested
Serum titers obtained with Aotus/P. vivu.x antigen y0 positive
sera (titers GMRTb 1:4 1:8 I:16 1:64 1:256 I:1024 1:4096 1:16or higher)
<1 - - - -
1-4 4 2 2 - - - 6
5-14 8 4 4 - - - 6
15-44 85 38 38 2 4 3 - - 10.59 7
245 14 6 6 1 1 - - - 14.29 7
Total 111 50 50 3 5 3 - - 9.91 7
aBella Vista and Zopilota.
bGMRT = Geometric mean reciprocal titer.
Table 6. Prevalence of P. &?c$arum antibodies found among residents of two Liberia District localities,a by serum titer and age gmup, in 1974.
As
group (ye.4
No. of
Sera
tested
Sera titers obtained with in vitro/P.
falcifiawm
antigen To positivesera (titers GMRTb 1:4 1:8 I:16 1:64 1:256 1:1024 1:4096 1:16 or higher)
<l - - - -
l-4 4 2 2 - - - 6
5-14 8 4 4 - - - 6
15-44 85 38 38 4 3 - - 2c 10.59 8
245 14 6 6 - - 1 1 14.29 11
Total 111 50 50 4 3 1 1 2 9.91 8
aBella Vista and Zopilota.
bGMRT = Geometric mean reciprocal titer.
cThe two sera with titers of 1:4096 were obtained from subjects with patent P.
falcifimum
parasitemia, whose cases prompted the survey.24
Localities
A total of 2,388 sera were obtained from 57 subjects with patent
P. vivax
parasit- emia and from people living with or re- lated to these subjects. In all, 28 localities in 17 malaria-positive districts were in- volved. However, only 156 (6.53 per cent) of these sera yielded titers ~16 withP.
vivax antigen (Table 7). Forty-nine sera
yielded titers 264, but 46 of these came from the cases prompting the survey. The largest number of positive sera (71) was found in the 15-44 age group. The GMRT was
7.
Discussion
As these results demonstrate, determi- nation of malaria antibody levels can play a useful role in special epidemiologic studies-as well as in application of indi-
vidual or group chemotherapeutic mea-
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PAHO BULLETIN l YOZ.13,
no.3,
I979Table 7. Prevalence of
P.
&ux antibodies found among residents of 24 localities with confirmedP.
vhzx cases, by titer and age group, 1975-1976.Age
group (years)
No. of sera tested
Serum titers obtained with Aolus/P. vivax antigen y0 positive
sera (titers GMRI a 1:4 1:8 I:16 1:64 1:256 I:1024 I:4096 1:16 or higher)
<I 56 30 22 4 - - - - 7.14 8
1-4 348 170 158 14 3 3 - - 5.75 6
5-14 832 401 391 28 11 1 - - 4.81 11
15-44 893 418 404 44 19 a - 7.95 7
245 259 123 115 17 2 2 - - 8.11 7
Total 2,388 1,142 1,090 107 35 14 - 6.53 7
aGMRT = Geometric mean reciprocal titer.
roborating the absence of transmission, especially in areas where administration of antimalaria drugs has been considerably
reduced. The low prevalence of specific
malaria antibodies in the studied areas
suggests the absence of endemicity which could be suspected in most of the originally malarious areas of the country.
It would of course be very useful to carry out longitudinal studies in order to gain a chronological view of the study popula- tions’ serologic profiles, especially in areas with a history of high levels of patent
parasitemia.
Although the groups studied yielded
extremely low antibody levels suggesting absence of transmission, it will be necessary to carry out additional serologic surveys
employing stratified and random samples
in communities confronting a variety of
epidemiologic situations, and to implement parasitologic and serologic followup of all cases and malaria foci in the country, in order for interruption of transmission to be confirmed.
ACKNOWLEDGMENTS
We are especially grateful to all the peo- ple of the localities and scattered popula- tions studied who donated their blood to promote the work of these epidemiologic surveys. We are also indebted to all our friends and collaborators at the National Malaria Eradication Service. In addition, our thanks go to Dr. Francisco J. Lopez
Antuiiano (PAH O/WHO), whose great as-
sistance and effective guidance played a
decisive role in introduction of the indirect
immunofluorescent technique in Costa
Rica. Finally, we wish to hereby express appreciation of the support received from our colleagues at the Ministry of Health
and the Pan American Health Organi-
SUMMARY This article reports on use of the indirect im- munofluorescence test in Costa Rica to measure the prevalence of specific malaria antibodies among various vulnerable groups. The popula- tions surveyed included residents of one district with a history of numerous past cases; residents of two other districts with active cases; and residents of previously malarious areas who had activecases or were in close contact with malaria patients.
The test results indicate antibody levels suffi- ciently low to suggest absence of transmission among the populations surveyed. Nevertheless,
additional surveys of stratified and random sam- ples from communities facing a variety of epide- miologic situations will be needed in order for interruption of transmission to be confirmed.
In addition, the test results demonstrate the usefulness of serologic studies for delimiting transmission foci, for defining populations at risk, and for epidemiologic evaluation of areas with a history of high malaria incidence. In general, they also show how performance of such studies by specialized services can make it pos- sible to gain a clearer picture of the prevalence and distribution of the disease.
REFERENCES (I) World Health Organization. Develop- ments in Malaria immunology: Report of a WHO Scientific Group. WHO Technical Re- port Series No. 579. Geneva, 1975.
(2) Sulzer, A. J., M. Wilson, and V. C. Hall. Indirect fluorescent antibody test for parasitic diseases: V. An evaluation of a thick smear antigen in the IFA test for malaria antibodies. AmJ Trap Med Hyg 18:198, 1969.
(3) Sulzer, A. J ., and M. Wilson. The fluo- rescent antibody test for malaria: Critical re- views. Clinical Laboratory Sciences 2(4), 1971.
(4) Porter, J. A., Jr. Plasmodium uivax in- fections in Aotus ttivirgatus. Am J Trap Med Hyg 20(4):535-538, 1971.
(5) Lopez Antuiiano, F. J. Falciparum ma- laria antigen slides for indirect immuno-fluo- rescence test made from in vitro cultures. Trans R Sot Trap Med Hyg 68(3):257, 1974.
(6) Collins, W. E., M. W. Warren, J, C. Skinner, and H. S. Fredericks. Studies on the relationship between fluorescent antibody re- sponse and ecology of malaria in Malaysia. Bull
WHO 39:451, 1968.
SYMPOSIUM ON KETOCONAZOLX
The First International Symposium on Ketoconazole, a new anti- mycotic agent, will be held in Medellin, Colombia, 29 November-l December 1979.
Abstracts are invited on all aspects of Ketoconazole in -research and in clinical medicine. Studies making a significant contribution will be selected for possible presentation at the symposium.
