105

Comparison of Immunouorescence Antibody Testing and Two

Enzyme Immunoassays in the Serologic Diagnosis of Malaria
Rosemary C. She, MD,* Mindy L. Rawlins, BS, Ramsey Mohl, MT (ASCP), *
Sherrie L. Perkins, MD, PhD,* Harry R. Hill, MD,* and Christine M. Litwin,
MD*
*Departments of Pathology, Pediatrics, and Medicine, University of Utah School of Medicine, Salt Lake City, UT,
USA; Associated Regional and University Pathologists (ARUP) Institute for Clinical and Experimental Pathology,
Salt Lake City, UT, USA
DOI: 10.1111/j.1708-8305.2006.00087.x

Background. Serologic testing in malaria has traditionally been done by immunouorescence antibody testing (IFA),
but the use of commercially available enzyme immunoassays (EIAs) has become more widespread.
Methods. We compared IFA with two commercial EIA kits, the Cellabs Pan Malaria CELISA and the Newmarket
Malaria EIA. Seventy-ve samples from 74 patients with clinically suspected malaria were examined by both EIA kits.
The samples were also examined by IFA (n = 48) and/or Giemsa-stained blood smear (n = 48).
Results. Using a consensus result as a gold standard, the agreement, sensitivity, and specicity were, respectively, as
fol- lows: Cellabs EIA 93.2, 95.5, and 92.2%; Newmarket EIA 87.7, 68.2, and 96.1%; and IFA 89.1, 86.4, and 91.7%.
Com- pared to positive Giemsa-stained smears, the sensitivities were as follows: Cellabs EIA 90.9% (10/11),
Newmarket EIA
54.5% (6/11), and IFA 100% (11/11). Antinuclear antibody (ANA)positive sera (n = 11) and rheumatoid factor (RF)
positive sera (n = 11) showed no cross-reactivity with the Newmarket EIA, while the Cellabs EIA yielded positive
results in one ANA-positive and two RF-positive sera. Among healthy blood donors (n = 50), the Newmarket EIA
showed
100% specicity (50/50) and the Cellabs EIA showed a specicity of 92%
(46/50).
Conclusions. While the Newmarket EIA was a generally more specic assay, it was insufciently sensitive relative to
the IFA and the Cellabs EIA for screening purposes for malaria antibodies. The Cellabs EIA demonstrated the best
overall sensitivity and is a reasonable choice as a serodiagnostic tool for malaria. It may also be useful as an adjunct to
Giemsa- stained smear examination, to aid in cases of low parasitemia in previously nonimmune individuals.

ore than 300 million cases of malaria occur

per year worldwide, of which more than
1 million result in death. Malaria is not endemic in
the United States but in recent years, approximately
1,200 to 1,400 new cases have been reported each
year. The vast majority of these are individuals who
acquired malaria in endemic areas, such as travelers
and immigrants. Malaria is caused by the protozoan
parasites Plasmodium falciparum, P vivax, P
ovale, and P malariae, and is transmitted by species
of the female Anopheles mosquito. Rarely, malaria is
trans- mitted by transfusion of blood products.1
The diagnosis of malaria in the acute clinical setting has traditionally relied upon examination of

Corresponding Author: Christine M. Litwin, MD,

Giemsa-stained peripheral blood smears. Serologic

testing in malaria is not recommended in the acute
di- agnosis of malaria but may be useful in other
circum- stances. These include the retrospective
diagnosis of malaria in a previously nonimmune
individual, screen- ing for chronic malaria, and blood
bank screening of potential donors.1,2 Serologic IgG
response is rapid and usually occurs within 1 week
of onset of parasit- emia.3,4 Antibodies begin to wane
after approximately
1 month and persist for several months to
years.2,3
Immunouorescence antibody testing (IFA) has
been a reliable serologic test for malaria in past
decades. It is highly sensitive and specic,5 but
on the other hand, time consuming and subjective.
Enzyme immunoassays (EIAs) for malaria have
Department of Pathology, University of Utah School

of Medicine, 50 N, Medical Drive, Salt Lake City, UT

84132, USA. E-mail: christine.litwin@path.utah.edu

become commercially available. The capability to

handle large sample pools, the objectivity, and
the potential for automation of the EIA have
prompted studies focused on screening blood

2007 International Society of Travel Medicine, 1195-1982

Journal of Travel Medicine, Volume 14, Issue 2, 2007, 105111

106
donations with EIA rather than with IFA. Several
studies have validated the use of EIA in blood bank
screening in Europe and Australia.69
The Cellabs EIA detects IgG antibody captured
by recombinant antigens from P falciparum, P
vivax, P ovale, and P malariae. Further
specications on the nature of the antigens used
are not available. There are no published studies
evaluating its per- formance
to date. The
Newmarket Malaria EIA detects IgG, IgM, and
IgA antibodies using recom- binant merozoitespecic proteins of P falciparum and P vivax. It
has been evaluated with satisfactory results for the
purpose of blood bank screening.6,8 In this study,
we compared the performance charac- teristics of
the IFA and two commercial EIA kits, the Cellabs
Pan Malaria Antibody CELISA and the
Newmarket Malaria EIA.
Materials and Methods
Human Plasma and
Sera
The study was approved by the Institutional
Review Board of the University of Utah, IRB 7275.
Between January 2002 and June 2005, serum or
plasma from clinical samples tested for malaria by
either Giemsa- stained blood smear or malaria IFA
were collected at our reference laboratory in Salt
Lake City, Utah. Seventy-four samples from
individual patients were evaluated. Of these, 46
were evaluated by Giemsa stain, 27 by IFA, and
two by both Giemsa stain and IFA. A second
plasma sample from patient 72 drawn
4 days after the original sample was included in this
study, for a total of 75 specimens. To test for specicity of the malaria EIA in healthy adults, 50 serum
samples were collected from random healthy blood
donors in the Salt Lake City, Utah area (all
collected in 2003 and stored at 20C).
Cellabs Pan
Malaria
Antibody
CELISA
All specimens were tested according to the recommended protocol of the manufacturer (Cellabs,
Sydney, New South Wales, Australia). Briey, samples and controls were diluted 1 to 100 in phosphate-buffered saline (PBS)/Tween. One hundred
microliters of each sample was added to the coated
wells and incubated for 1 hour at room
temperature. The wells were washed four times
with PBS/Tween using an automatic washer
(Columbus Pro, Tecan Trading AG, Switzerland).
One hundred micro- liters of conjugate (enzymelabeled anti-human immunoglobulin) was added to
J Travel Med 2007; 14: 105111

She et
al.
each well and allowed to incubate for 1 hour at
room temperature. The wells were washed four
times with PBS/Tween. One hundred microliters
of chromogen substrate

Serologic Testing in Malaria

was added to each well and incubated for 15
minutes at room temperature in the dark. Fifty
microliters of stop solution (6.5% hydrochloric
acid) was then added to each well. Optical
densities (ODs) were immediately read by a
microplate reader (Spectra- max Plus; Molecular
Devices Corp., Sunnyvale, CA, USA) at 450 nm
with a reference of 620 nm. The cutoff value for
each run was determined to be the mean OD of
two negative controls plus 0.100. Values were then
expressed as an index value of OD sample/OD
cutoff. Any specimen with a result dif- ferent from
that obtained by the other test methods used was
repeated in duplicate.

107
P malariae separately by twofold dilutions between

Newmarket
Malaria
EIA
Tests were carried out on all samples according to
specications of the manufacturer (Newmarket
Laboratories Ltd, Newmarket, UK). Briey, 50 L
of undiluted sample or control was added to the
wells. The wells were placed on a plate shaker for
30 seconds and incubated for 30 minutes at 37C.
The wells were washed ve times with 30-second
soaks between each wash using the automated
plate washer. Fifty microliters of recombinant
antigen conjugated to horseradish peroxidase was
added to each well and allowed to incubate for 30
minutes at
37C. The wells were again washed ve times with
30-second soaks between each wash. Fifty microliters of substrate solution (urea peroxide and tetramethylbenzidine) was added to each well and
incubated for 30 minutes in the dark at room temperature. Fifty microliters of stop solution (0.5 M
sulfuric acid) was added to each well. ODs were immediately read at 450 nm using a reference of 650
nm. The cutoff value for each run was determined
to be the mean OD of three negative controls plus
0.100. Index values were calculated by dividing
results by the cutoff OD value. Any specimen with
discrepant results versus the other test methods
used was re- peated in duplicate.
Immunouorescence Antibody Testing
Of the patient samples collected, 29 had IFA testing performed prior to the study. An additional 19
IFA tests were performed on the specimens with
discrepant results between EIA testing and/or the
Giemsa-stained blood smear. All IFA testing was
performed
by Parasitic Disease Consultants
(Tucker, GA, USA) based on a method previously
described using thick smears of washed, heparanized blood containing Plasmodium sp. schizonts
from infected primates as antigen.5 Serum samples
were tested for P falciparum, P vivax, P ovale, and
J Travel Med 2007; 14: 105111

1:4 and 1:64. Those samples having titers of at least

1:64 were considered positive.
Cross-Reactivity
Studies
The cross-reactivity of the Cellabs and the Newmarket assays was determined by testing sera from
11 antinuclear antibody (ANA)positive samples
and 11 rheumatoid factor (RF)positive samples.
Any specimen that tested positive was repeated
in duplicate by the respective EIA kit. The crossreactivity of the two EIA kits with Babesia microti
was tested using a serum sample that had tested
posi- tive for IgM (1:80, reference <1:16) and
negative for IgG antibodies by B microti IFA.
Consensus
Result
A consensus result was obtained for each sample to
be used as the gold standard, taking into account results obtained from the two EIA kits and, if performed, the Giemsa-stained smear and/or the IFA.
A result was considered consensus positive if at
least two of three or if at least three of four test
methods were positive, or if two of four test
methods were positive including the Giemsastained smear. A re- sult was considered consensus
negative if at least two of three or if at least three
of four test methods were negative. A result was
considered consensus equivocal if two of four test
methods were positive and the Giemsa-stained
smear was negative.
Statistical Analysis
Specicity for the Newmarket and Cellabs EIA assays among healthy subjects (n = 50) was
determined by the percentage of negative results
among this group. The 95% condence intervals
(CIs) for the sensitivity and specicity of the IFA
and two EIA kits were determined according to a
general method.10 Agreement between test methods
was determined as number of concordant results
di- vided by total number of results.
Results
Giemsa-Stained
Blood
Smears
Eleven of the 48 samples evaluated by Giemsastained thick and thin smear preparations were
interpreted as positive by an experienced hematopathologist. Five cases were interpreted as P falciparum, four as P vivax, one as P ovale, and one as
P vivax versus P ovale (Table 1).

Immunouorescence Antibody Testing

A total of 48 samples were tested by IFA. Of the 29
original samples evaluated by IFA, 7 were
interpreted

as positive, which included various combinations

of all four malarial species of Plasmodium. There
were an additional 14 positive samples by IFA
among the other 19 samples tested because of discrepant EIA and/or Giemsa stain results. Results of
all IFA-positive specimens are included in Table 1.
Of the 21 total positive IFA results, 13 were positive for two or more species of Plasmodium.
Enzyme
Immunoassays
All 75 specimens were tested by the two EIA kits.
By the Cellabs EIA, 27 tested positive and 48
tested negative. By the Newmarket Malaria EIA,
19 tested positive and 56 negative. Comparison of
the two EIA kits is shown (Figures 1 and 2). Figure
1B uses the logarithm of the index values to
compare the two kits. The left lower quadrant
represents sam- ples negative by both EIA kits, the
left upper quad- rant represents samples positive
by the Cellabs EIA and negative by the
Newmarket EIA, the right up- per quadrant
represents samples positive by both EIA kits, and
the right lower quadrant
represents samples
positive by the Newmarket EIA and nega- tive by
the Cellabs EIA. Agreement between the two kits
was 84.0%.
Comparison of Serologic Tests to Consensus
Results
By consensus results, the 75 total samples consisted
of 22 positive, 2 equivocal, and 51 negative results.
Agreement, sensitivity, and specicity for each test
method are shown in Table 2. As demonstrated by
95% CIs, the Cellabs EIA was signicantly more
sensitive than the Newmarket EIA. The 95% CIs
otherwise showed no other statistically signicant
differences between the serologic tests. Samples
positive by one or more test method are summarized in Table 1. Among these samples, the most
frequently observed pattern, seen in ve cases, was
one in which the IFA, the two EIA kits, and
Giemsa- stained smear were positive for a given
sample. In four cases, the Newmarket EIA
tested negative when the Cellabs EIA, IFA, and
Giemsa-stained smear were interpreted as positive.
Comparison of EIA and IFA to GiemsaStained
Blood
Smear
The IFA detected 11 of 11 positive Giemsa-stained
smears, followed by the Cellabs EIA (10/11) and the
Newmarket EIA (6/11). The one case missed by
the Cellabs EIA was patient 72, who had a second

sam- ple drawn 4 days later that tested positive

by the Cellabs kit. Patients 34 and 49 were positive
for ma- larial antibodies by all three serologic tests
but neg- ative by the Giemsa smear examination.
Of the 36

Table 1

Results of cases positive by at least one test method

Case no.

Newmarket EIA

Cellabs EIA

IFA*

Giemsa stain

Consensus result

3
5
6
12

Pos
Pos
Pos
Pos

Pos
Neg
Pos
Pos

Not
Not
Not
Not

done
done
done
done

Pos
Neg
Pos
Pos

13
14
16
19
20
22
24
26
30
31
33

Pos
Neg
Pos
Neg
Pos
Neg
Neg
Pos
Pos
Neg
Pos

Pos
Neg
Pos
Pos
Neg
Pos
Neg
Pos
Pos
Pos
Pos

Pos
Neg
Pos
Pos
Neg
Pos

Pos
Pos
Pos
Pos
Pos
Pos

Not done
Not done
Not done
Not done
Not done
Not done
Not done
Not done
P falciparum
P vivax
P vivax versus
P ovale
Neg
Neg
Neg
Neg
P vivax
P vivax

Pos
Neg
Pos
Neg
Neg
Neg
Neg
Pos
Pos
Pos
Pos

34
47
49
50
51
53
55
62
64
68

Neg
Pos
Pos
Pos

Pos
Pos
Pos
Pos

Neg
Neg
P falciparum
P falciparum

Neg
Equ
Pos
Pos

69

Neg

Pos

P falciparum

Pos

70
71

Neg
Pos

Pos
Pos

P falciparum
P vivax

Pos
Pos

72
72-2
73

Neg
Neg
Pos

Neg
Pos
Pos

P ovale
Not done
Not done

Pos
Pos
Pos

74

Neg

Pos

Neg
Neg
Neg
Plasmodium malariae,
P vivax, P ovale
P falciparum, P vivax
P falciparum
P falciparum, P malariae
Neg
Neg
Neg
P malariae
Neg
P vivax
P vivax, P malariae
P falciparum, P vivax,
P ovale, P malariae
P falciparum
Neg
P falciparum
Neg
P ovale
P falciparum, P ovale,
P malariae
Neg
Neg
P falciparum
P falciparum, P vivax,
P ovale, P malariae
P falciparum, P vivax,
P ovale
P falciparum
P falciparum, P vivax,
P ovale
P ovale, P vivax
P ovale, P malariae
P falciparum, P vivax,
P malariae
P vivax, P ovale

Not done

Pos

Pos
Neg
Pos
Equ
Pos
Pos

EIA = enzyme immunoassay; IFA = immunouorescence antibody testing; pos = positive; neg = negative; equ = equivocal.
*Positive titers for IFA tests are greater than or equal to 1:64.

Consensus results: positive = 2/3, 3/3, 3/4, or 4/4 test methods are positive for a given sample, or 2/4 positive including positive Giemsa stain; negative =
0/3, 1/3, 0/4, or 1/4 positive; equivocal = 2/4 positive and Giemsa stain is negative.

negative smears, 30 were negative by the Cellabs

EIA (specicity 83.3%, 95% CI 76.185.6) and 32
by the Newmarket EIA (specicity 88.9%, CI 81.9
94.4).
Healthy Subjects
With the Newmarket EIA, all 50 healthy blood donor samples tested negative, yielding a specicity of
100% (95% CI 94.9100). With the Cellabs EIA, 4
of the 50 healthy blood donor samples tested positive
for a specicity of 92% (95% CI 86.493.6), which
is signicantly lower than the Newmarket EIA
speci- city.
All four
positive
samples
demonstrated low positive OD readings, ranging

between 0.150 and

0.230, with a positive cutoff of 0.145 (Figure
2).

Cross-Reactivity Studies
Using the Newmarket EIA, 0 of 11 ANA-positive
serum samples and 11 RF-positive serum samples
gave positive results. Using the Cellabs EIA, 1 of 11
ANA-positive serum samples and 2 of 11 RF-positive samples gave positive results. Both Newmarket
and Cellabs EIA kits gave negative results with the
single Babesia antibodypositive sample.
Discussion
The serologic tests examined yielded disparate results in many cases. This may be explained by the
fact that each method uses different antigens to
capture

Figure 1 (A) Two-by-two table comparing the Cellabs

and Newmarket EIA results from patients with suspected
malaria (n = 75). (B) Graphical illustration of A,
comparing the Cellabs and Newmarket EIA using the
logarithm of the index value (OD sample/OD cutoff)
for each test result. The consensus result corresponding
to each specimen is also represented (refer to legend).
The left upper quadrant contains 10 samples that were
negative by the Newmarket EIA and positive by the
Cellabs EIA. Consensus results of these samples
consisted of six positive and four negative results. The
two points in the right lower quadrant represent samples
that were negative by consensus, negative by the Cellabs
EIA, and positive by the Newmarket EIA. In the right
upper quadrant, the 17 specimens positive by both EIA
kits are plotted. These 17 points include 2 consensus
equivocal cases and 15 consensus positive cases. In the
left lower quadrant are the 46 specimens negative by
both EIA assays. Of these, 45 were consensus negative
and 1 was consensus positive. EIA = enzyme immunoassay; OD = optical density.

Figure 2 Distribution of OD index values for the

Cellabs and Newmarket EIA, separated into the
following groups: healthy controls (n = 50), RF- or
ANA-positive samples (n = 22), samples negative by
consensus result (n = 51), samples equivocal by
consensus results (n = 2), and samples positive by
consensus results (n = 22). The horizontal dashed line
indicates the cutoff (OD signal/OD cutoff = 1), above
which are positive results and below which are negative
results. EIA = enzyme immunoassay; OD = optical
density; ANA = antinuclear antibody; RF = rheumatoid
factor.

anti-Plasmodium antibodies. Different thresholds

for detection of antibodies may also be an issue for
the Newmarket EIA kit, which used undiluted sera
or plasma but demonstrated the lowest sensitivity of
the methods tested. The antigens used to capture
antibodies in the Newmarket kit are directed
against P falciparum and P vivax, but the kit failed
to detect two P falciparum and two P vivax cases.
These four cases were, however, detected by
Giemsa stain, IFA, and the Cellabs EIA. Despite the
limited number of positive specimens in this study,
there is no overlap of 95% CIs for the sensitivities
of the two EIA as- says (Table 2). The Newmarket
EIA did demon- strate higher specicity than the
Cellabs EIA, but it lacks sufcient sensitivity
required for the purposes of a serologic test for
malaria, which include screen- ing for chronic
malaria and potential blood donors for antimalarial
antibodies. It is acknowledged that

Table 2 Agreement, sensitivity, and specicity of the Cellabs EIA, Newmarket EIA, and IFA test using
consensus results as the gold standard
Cellabs EIA (n = 75)
Newmarket EIA (n = 75)
IFA (n = 48)

% Agreement

% Sensitivity*

% Specicity*

93.2
87.7
89.1

95.5 (82.799.2)
68.2 (54.174.6)
86.4 (73.792.4)

92.2 (86.793.8)
96.1 (90.098.8)
91.7 (80.197.2)

EIA = enzyme immunoassay; IFA = immunouorescence antibody testing.

*95% condence intervals are given in parentheses.

clinical history of the patients in this study was not

known; therefore, demographic information was
not available to aid the interpretation of results that
were discrepant between the various methods
tested.
Recent larger studies have yielded results different from those of this study in regard to the performance of the Newmarket EIA. Kitchen and
colleagues7 demonstrated that it had comparable if
not superior sensitivity to a traditional IFA method
in acute, Giemsa stainproven malaria. In this study,
the Newmarket EIA detected 82.6% (114/138) of
acute P falciparum cases, 84.6% (11/13) of acute
P vivax cases, and 70% (7/10) of acute P ovale
cases. In comparison, the IFA, employing P
falciparum schizont as antigen, detected 74.3%
(103/138) of acute P falciparum cases, 61.5%
(8/13) of acute P vivax cases, and 80% (8/10) of
acute P ovale cases. Seed and colleagues8
demonstrated the Newmarket EIA to have a
sensitivity of 98% (106/108) in acute P falciparum
cases and 100% (12/12) in acute P vivax cases. Both
studies reported specicities of near
100% for this assay among healthy blood donors,
similar to ndings in the present study. There have
been few published studies on the performance of
the Cellabs Pan Malaria IgG CELISA to date.
Ozbilge and colleagues11 found that when compared
to light microscopy, the kit had a sensitivity and
specicity of 83 and 85%, respectively, similar to
the results found here.
There were two cases in this study that were
neg- ative by Giemsa-stained smear but positive
by all three serologic tests studied. While this may
be due to past exposure, these smears were
evaluated un- der the clinical suspicion of malaria
and thus there is a concerning possibility of low
parasitemia not detectable by smear examination.
Reexamination of a negative smear may be done
in future cases should a concomitant serologic test
be positive in a previously unexposed individual.
Although this would not be a useful approach in
malaria-endemic areas, in which antimalarial
antibodies
are preva- lent in the general
population, this may be bene- cial at institutions
in nonendemic countries that often have limited
experience examining blood smears for malaria.
The Cellabs EIA kit has in this study shown to be
reasonably sensitive in the acute setting, detecting
10 of 11 cases of malaria. The single case that
was missed was detected in plasma collected from
the same patient 4 days later. This corresponds to
the development of antimalarial an- tibodies, which
occur within 7 days of onset of para- sitemia.2,3

Therefore, one additional use for the malaria

EIA could be as an adjunctive diagnostic tool to the

Giemsa-stained peripheral blood smear examination in the acute setting of suspected malaria. The
methods for EIA used in this study yielded results in
less than 3 hours. IFA, while correlating well in
this study with Giemsa smear evaluation, is time
con- suming and more labor intensive than the
EIA. It has some ability to differentiate antibodies
from dif- ferent Plasmodium species, but crossreactivity is a well-documented occurrence.5,6,9
The fact that 13 of the 21 positive IFA samples in
this study had posi- tive titers for two or more
Plasmodium species sug- gests a signicant degree
of cross-reactivity of antimalarial
antibodies.
Patient 72, interpreted as P ovale by Giemsastained smear evaluation, was positive by IFA for
P ovale and P vivax on the rst sample submitted.
The sample submitted 4 days later was positive for
P ovale and P malariae. In addi- tion to crossreactivity,
subjectivity
likely
affects
the
reproducibility of the IFA.
There are diagnostic methods available that are
more rapid, such as antigen detection (eg, immunochromatography) and nucleic acid detection. One
disadvantage of immunochromatography is its decreased sensitivity as parasitemia falls below a certain threshold, such as 100 parasites/ L, and these
are the instances in which an adjunctive test to the
Giemsa smear would be most useful. Polymerase
chain reaction has shown to be an excellent diagnostic tool in the acute setting and may eventually
become more widely available.12
The specicity of the Cellabs EIA kit among
healthy blood donors was only 92% using the manufacturers recommended method for determining
the cutoff OD value. On the other hand, the manufacturer has previously published a study using an
OD of 3 standard deviations above the mean of
neg- ative controls as a method of determining the
cutoff value.13 If this method were used, the
Cellabs EIA would have yielded a higher
specicity without compromise of the sensitivity
(data not shown). Studies of the Newmarket
Malaria EIA have fol- lowed the manufacturers
protocol for determining cutoff OD values for each
run.6,8 Standardization of these assays by testing
additional random healthy donors will yield a
more accurate method of deter- mining positive
cutoff OD value for optimal sensi- tivity,
specicity, and normalization of between-run
variations.
Acknowledgments
This work was supported by the ARUP Institute for
Clinical and Experimental Pathology. A special

thanks to Cellabs Pty Limited and Newmarket

Laboratories Ltd for supplying the EIA reagents

used in this study.
Declaration of Interests

7.

The authors state that they have no conicts of

interest.
8.

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