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 follows: 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%. Compared 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 Giemsastained 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 transmitted 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,
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

Giemsa-stained peripheral blood smears. Serologic

testing in malaria is not recommended in the acute diagnosis of malaria but may be useful in other circumstances. These include the retrospective diagnosis of
malaria in a previously nonimmune individual, screening 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 parasitemia.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
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 performance to date. The Newmarket Malaria EIA
detects IgG, IgM, and IgA antibodies using recombinant merozoite-specic 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 characteristics 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 Giemsastained 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 microliters of conjugate (enzyme-labeled anti-human
immunoglobulin) was added to 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
J Travel Med 2007; 14: 105111

She et al.
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 (Spectramax 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 different from that obtained by the other test methods
used was repeated in duplicate.
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 repeated 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
P malariae separately by twofold dilutions between

Serologic Testing in Malaria

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 positive 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 Giemsa-stained smear. A result 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 divided 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

107
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 samples negative by both EIA kits, the left upper quadrant represents samples positive by the Cellabs EIA
and negative by the Newmarket EIA, the right upper quadrant represents samples positive by both
EIA kits, and the right lower quadrant represents
samples positive by the Newmarket EIA and negative 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 Giemsastained 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 Giemsa-Stained
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 sample drawn 4 days later that tested positive by the
Cellabs kit. Patients 34 and 49 were positive for malarial antibodies by all three serologic tests but negative by the Giemsa smear examination. Of the 36
J Travel Med 2007; 14: 105111

108
Table 1

She et al.
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 done
Not done
Not done
Not 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 81.994.4).
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 specicity. 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).
J Travel Med 2007; 14: 105111

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

109

Serologic Testing in Malaria

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 assays (Table 2). The Newmarket EIA did demonstrate higher specicity than the Cellabs EIA, but it
lacks sufcient sensitivity required for the purposes
of a serologic test for malaria, which include screening 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.

J Travel Med 2007; 14: 105111

110
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 negative by Giemsa-stained smear but positive by all
three serologic tests studied. While this may be due
to past exposure, these smears were evaluated under 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 prevalent in the general population, this may be benecial 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 antibodies, which occur within 7 days of onset of parasitemia.2,3
Therefore, one additional use for the malaria
EIA could be as an adjunctive diagnostic tool to the
J Travel Med 2007; 14: 105111

She et al.
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 consuming and more labor intensive than the EIA. It
has some ability to differentiate antibodies from different Plasmodium species, but cross-reactivity is a
well-documented occurrence.5,6,9 The fact that 13
of the 21 positive IFA samples in this study had positive titers for two or more Plasmodium species suggests a signicant degree of cross-reactivity of
antimalarial antibodies. Patient 72, interpreted as
P ovale by Giemsa-stained 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 addition to cross-reactivity, 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 negative 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 followed 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 determining positive cutoff OD value for optimal sensitivity, 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

111

Serologic Testing in Malaria

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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