Am. J. Trop. Med. Hyg., 69(6), 2003, pp.

589592
Copyright 2003 by The American Society of Tropical Medicine and Hygiene

EVALUATION OF THE BINAX NOW ICT TEST VERSUS POLYMERASE CHAIN

REACTION AND MICROSCOPY FOR THE DETECTION OF MALARIA IN
RETURNED TRAVELERS
GABRIELLA A. FARCAS, KATHLEEN J. Y. ZHONG, FIONA E. LOVEGROVE, CHRISTOPHER M. GRAHAM, and
KEVIN C. KAIN
Institute of Medical Science, Faculty of Medicine University of Toronto, Toronto, Ontario, Canada; Global Health Program,
McLaughlin Center for Molecular Medicine, Toronto, Ontario, Canada; Tropical Disease Unit, Division of Infectious Diseases,
University Health Network-Toronto General Hospital, Toronto, Ontario, Canada

Abstract. Microscopic detection of Plasmodium species has been the reference standard for the diagnosis of malaria
for more than a century. However, maintaining a sufficient level of expertise in microscopic diagnosis can be challenging,
particularly in non-endemic countries. The objective of this study was to compare a new rapid malaria diagnostic device
(NOW ICT Malaria Test; Binax, Inc., Portland, ME) to polymerase chain reaction (PCR) and expert microscopy for
the diagnosis of malaria in 256 febrile returned travelers. Compared with PCR, the NOW ICT test showed a sensitivity
of 94% for the detection of P. falciparum malaria (96% for pure P. falciparum infection) and 84% for non-P. falciparum
infections (87% for pure P. vivax infections and 62% for pure P. ovale and P. malariae infections), with an overall
specificity of 99%. The Binax NOW ICT may represent a useful adjunct for the diagnosis of P. falciparum and P. vivax
malaria in febrile returned travelers.
non-falciparum infections and may possess technical advantages over its predecessors. The objective of this study was to
examine the performance of the NOW ICT test compared
with a blinded polymerase chain reaction (PCR) and expert
microscopic analysis for the diagnosis of all human malaria
species in febrile travelers returning from malaria-endemic
areas.

INTRODUCTION
In 2001 approximately 50 million people from western
countries visited malaria-endemic areas and at least 30,000
travelers contracted malaria.1 Despite treatment, between
1% and 4% of travelers who acquire Plasmodium falciparum
malaria will die as a result of infection.1 This fatality rate
increases to 20% or higher in patients who develop severe
malaria or those who are elderly.2 Since 90% of travelers who
contract malaria will not become ill until returning home,
accurate diagnosis and appropriate treatment depend on the
expertise of physicians and diagnostic laboratories in nonendemic areas.3,4
Although microscopic detection of parasites on Giemsastained blood smears has been the reference standard for
malaria diagnosis in laboratories for more than a century, it is
an imperfect standard highly dependent on the technical expertise of the microscopist.5 The ability to maintain the required level of expertise in malaria diagnostics may be problematic especially in peripheral medial centers in countries
where the disease is not endemic.5 The World Health Organization has recognized the urgent need for simple and costeffective diagnostic tests for malaria to overcome the deficiencies of [both] light microscopy and clinical diagnosis.6
Consequently, recent efforts have focused on developing sensitive and specific non-microscopic malaria diagnostic devices
including those based on the detection of malaria antigen in
whole blood. Many first-generation rapid diagnostic products
relied on the detection of the histidine-rich protein II (HRP
II) antigen of P. falciparum and therefore could not detect
other Plasmodium species. A newer generation of rapid diagnostic devices based on antigen capture with immunochromatographic (ICT) strip technology and use of monoclonal
antibodies to HRP II for the detection of P. falciparum as well
as aldolase, a pan-Plasmodium antigen, thus facilitating identification of non-falciparum infections. The performance of
one device, the ICT Malaria P.f/P.v (AMRAD-ICT Diagnostics, Sydney, Australia), has been previously evaluated.713
However, this device is no longer available.
A new rapid assay, the Binax, Inc. (Portland, ME) NOW
ICT malaria test, is designed to detect both falciparum and

MATERIALS AND METHODS

Patients. Patients presenting to the Tropical Disease Unit
of the Toronto General Hospital from July 1999 to January
2003 with fever ( 38C) or a history of fever (within 48
hours) and travel to a malaria-endemic area were eligible for
inclusion in the study. All patients with blood films containing
malaria parasites were enrolled. In addition, patients who had
repeatedly negative blood films during the first two months of
the study, (i.e., diagnosed with a febrile illness other than
malaria) were enrolled to provide a comparable control
group. The prevalence of malaria in returned travelers during
the study period was 15%.
Whole blood samples (pretreatment) were collected from
all patients for thick and thin blood film preparation, PCR,
rapid diagnostic tests, and complete blood counts. An expert
microscopist who was blinded to the results of additional diagnostic testing examined the blood films. Smears were considered negative if no parasites were seen in 500 oilimmersion fields (1,000) on a thick blood film. Parasite concentration was calculated by determining the number of
parasites per 200 or 500 white blood cells in a thick blood film.
Baseline white blood cell counts were used to calculate parasitemia (parasites per microliter). All PCR amplification,
species identification, and diagnostic assays were performed
in a blinded fashion. This study was reviewed and approved
by the Ethical Review Committee of the University Health
Network-Toronto General Hospital.
Polymerase chain reaction. Detection and malaria species
identification by PCR were performed as previously described.1416 Briefly, genomic DNA was extracted from
whole blood samples using Qiagen columns (Qiagen, Chatsworth, CA) following the manufacturers instructions. A

589

590

FARCAS AND OTHERS

5-L aliquot of the DNA extract was used in a nested PCR

assay to amplify a segment of the Plasmodium 18S ribosomal
RNA gene. The resulting PCR product was analyzed by electrophoresis on a 2% agarose gel stained with ethidium bromide as previously described.15
NOW ICT assay. The NOW ICT Malaria Test for
Whole Blood is a rapid, in vitro immunodiagnostic test for the
detection of circulating P. falciparum antigen and a panmalaria antigen in whole blood. The test card contains immobilized antibodies specific for the HRPII antigen of P. falciparum and antibodies specific for aldolase, a pan-malaria antigen. The assays were performed according to the
manufacturers instructions. The test results were independently examined and interpreted by three observers blinded
to the microscopic and PCR results. The final results of the
test were recorded as either negative or positive based on the
majority agreement. The readers also graded the assays results (as band intensity for the HRP II and pan-Plasmodium
antigen bands) ranging from 0 (negative: no visible reaction
for either HRP II or pan-malaria antigen) to 4+ (strongly
positive reaction for both or either antigen) (Figure 1).
Data analysis. The sensitivity and specificity of the Binax
NOWICT test were calculated with PCR results as the reference standard. Positive and negative predictive values were
calculated based on the prevalence of malaria infections in all
patients presenting to the Tropical Disease Unit of the Toronto General Hospital during the study period. The K statistic was used to measure agreement among the three independent observers.
RESULTS
During the study period, 256 individuals who presented
with fever after travel to a malaria- endemic area were enrolled. The ratio of male to female patients was 1.6 (63.5%
males and 36.5% females) with a mean SD age of 32 1.1
years (range 4 months to 71 years). Travel destinations
included Africa (58.1%), the Indian Subcontinent (17%),
Latin America (17%), Oceania (3.8%), Southeast Asia
(2.9%), and the Middle East (0.9%). Whole blood samples
from 101 of these individuals were confirmed by PCR to be
positive for P. falciparum malaria, 90 were PCR confirmed P.
vivax infections, 9 were P. ovale, 3 were P. malariae, 6 were
mixed infections, and 47 were PCR and blood smear negative.
Malaria-infected patients did not differ significantly from
other patients with respect to age, sex, or duration of illness.

The results of the NOW ICT test compared with PCR-based

diagnosis are shown in Table 1. The results of the NOW ICT
test compared with microscopic diagnosis are shown in Table 2.
When compared with the PCR, the sensitivity of the
NOW ICT assay was 95.5% for the detection of pure P.
falciparum infections, 94.3% for P. falciparum when present
either pure or as a mixed infection (i.e., P. falciparum mixed
with P. vivax, P. ovale, or P. malariae), 86.7% for pure P.
vivax infections, and 83.5% for all non-falciparum infections.
For pure P. ovale and P. malariae infections, the test had a
sensitivity of 61.5%. The overall specificity of the text was
98.7%. Based on a malaria prevalence of 15% during the
course of the study, the corresponding positive predictive
value for P. falciparum and non-P. falciparum infections were
89.8% and 88.4% and the negative predictive values were
97.7% and 93.8%, respectively. There was excellent agreement between the three independent observers, with a K
value of 0.99 for the P. falciparum line and 0.94 for the panmalaria line. When microscopy was used as a reference standard (Table 2) the corresponding sensitivity was 96.0% and
84.7% for falciparum and non-falciparum infections respectively, with a specificity of 98.7%.
When compared with the PCR, the NOW ICT test
yielded two false-positive and 23 false-negative results (Table
1), with nearly perfect agreement among all three independent observers. Compared with microscopy, the NOW ICT
test yielded 19 false-negative results, of which 15 occurred in
specimens with parasitemias < 1,000 parasites/L. The sensitivity of the rapid assay compared with parasitemia for P.
falciparum and P. vivax infections are shown in Tables 3 and
4. The sensitivity of the assay decreased to 75% for falciparum infections with parasitemias < 100 parasites /L (Table
3) and to 55% for vivax infections with parasitemias < 1000
parasites/L (Table 4).
DISCUSSION
In this study, we examined the performance characteristics
of the Binax NOW ICT rapid malaria diagnostic device using PCR as the primary reference standard based on its established advantages over microscopy, particularly in cases of
low parasitemia and in mixed infections.3,5,14,15,1720 Our results indicate that the NOW ICT test is sensitive (94.3%)
and specific (98.7%) for the detection of P. falciparum malaria in returned travelers. The assay was also specific but
somewhat less sensitive (83.5%) for the detection of nonfalciparum malaria (86.7% for pure P. vivax infections). The

TABLE 1
Results of the NOW ICT test compared with polymerase chain
reaction (PCR) as the reference standard for the diagnosis of malaria in febrile returned travelers
No. of samples with indicated PCR results

FIGURE 1. Binax ICT result showing A, low Plasmodium falciparum parasitemia (band intensity of 1+) and B, high P. falciparum
parasitemia (band intensity of 4+). C control band; P.f. Plasmodium falciparum; P.v. Plasmodium vivax.

NOW ICT
result

P. falciparum*

Non-P. falciparum

Negative

Total

Positive
Negative
Total

100
6
106

86
17
103

2
45
47

188
68
256

* Includes Plasmodium falciparum mixed infections with P. vivax, P. ovale, and P. malariae.
P. vivax, P. ovale, and P. malariae single and mixed infections.

591

EVALUATION OF THE BINAX NOW MALARIA TEST

TABLE 2
Results of the NOW ICT test compared with expert microscopy as
the reference standard for the diagnosis of malaria in febrile
returned travelers

TABLE 4
Binax Now ICT assay for the detection of Plasmodium vivax malaria according to the level of parasitemia
Parasitemia
(no. of parasites/L of whole blood)

No. of samples with indicated smear results*

NOW ICT
result

P. falciparum

Non-P. falciparum

Negative

Total*

Positive
Negative
Total

97
4
101

83
15
98

2
45
47

182
64
246

* There were 10 samples in which reliable identification to the species level by microscopy
was not possible, but which were identified as Plasmodium falciparum (6) and P. vivax (4)
by a polymerase chain reaction. These samples were excluded from analyses using microscopy as the reference standard.
Includes P. falciparum mixed infections with P. vivax, P. ovale, and P. malariae.
P. vivax, P. ovale, and P. malariae single and mixed infections.

sensitivity for the detection of pure P. ovale and P. malariae

infections was 61.5% with a specificity of 100%. The expression of the pan-Plasmodium antigen in P. ovale, and more so
in P. malariae, has not been fully characterized,13,21 and this
combined with low parasitemias in these infections likely accounts for relatively lower sensitivity for the detection of
these malaria species. It is important to note that this test does
not distinguish between the non-falciparum species (P. vivax,
P. ovale, and P. malariae), nor can it reliably distinguish pure
P. falciparum infections from mixed falciparum infections.
The test is simple to perform, rapid (< 15 minutes), and
easy to interpret, with excellent inter-reader agreement (K
value 0.99). A K value > 0.81 indicates almost perfect
agreement between observers. Discrepancies between readers occurred mainly when the test result was weakly positive,
most frequently when the sample had a low parasitemia. A
weak correlation (r 0.359) was observed between the intensity of the P. falciparum band and parasitemia, but a
slightly stronger correlation (r 0.637) was observed with
the intensity of the pan-Plasmodium band.
Rapid diagnostic assays may be most useful when expert
microscopy is not available. Although microscopy can be sensitive to a threshold of 550 parasites/L, depending on the
expertise of the microscopist and equipment limitations,12 the
average microscopist is likely to achieve a sensitivity closer to
100 parasites/L or higher. In this investigation, the sensitivity of the NOW ICT test for P. falciparum infections was >
95% for samples with > 100 parasites/L and > 94% for P.
vivax infections for parasitemias > 1,000 parasites/L; however, for both falciparum and vivax infections sensitivity decreased as parasitemia decreased.
False-negative results, particularly for P. falciparum, are of
concern. Five false-negative results did occur for P. falciparum infections (four with < 600 parasites/L and one with
> 10,000 parasites/L), 12 for P. vivax (nine with < 700 parasites/L and three with < 7,300 parasites/L), five for P. ovale

1100
1011,000
1,00110,000
>10,000

Microscopy
(no. positive)

NOW ICT
(no. positive)

Sensitivity
(%)

6
20
51
21

3
11
48
21

50.0
55.0
94.1
100

(all with < 600 parasites/L), and one for a P. falciparum, P.

ovale, P. malariae mixed infection in which only the P. falciparum infection was detected (520 parasites/L). The observation that false-negative results may occasionally occur even
at high parasitemia is of concern. Previous studies of HRP
II-based assays have reported the same limitation and potential explanations include a prozone effect (i.e., a high concentration of antibody may mask the antigen making it unavailable to be detected in these rapid assays) or the presence of a
mutation or deletion within the hrp ii gene.14 Thus, when the
clinical suspicion of malaria remains high despite a negative
rapid diagnostic test result, the assay should be repeated
within 1224 hours14 and these assays should be performed in
parallel with thick and thin blood smears.
There were two false-positive NOW ICT tests results in
this study. Occasional false-positive results due to the presence of rheumatoid factor have previously been reported with
diagnostic devices based on the detection of HRP II.22 Furthermore, detection of antigen may persist for up to 28 days
after cure of infection.18
Despite some inherent limitations, evidence suggests that
rapid malaria diagnostic devices might represent a useful diagnostic adjunct tool to microscopy in a clinical setting. Since
laboratories in areas where malaria is not endemic frequently
lack expertise in diagnostic microscopy, a rapid diagnostic
assay could provide a quick and accurate although still preliminary diagnosis, while definitive results are sought from a
reference laboratory. Importantly, due to the occurrence of
occasional false-negative results with rapid diagnostic assays,
malaria infection cannot be ruled out based on a negative
result. Microscopy remains essential for species identification,
parasitemia calculations, as well as a backup to exclude falsenegative results.
In conclusion, the Binax NOW ICT malaria test is a rapid
and easy to use diagnostic assay. The test achieves high specificity (> 95%) for all Plasmodium species and high sensitivity
for P. falciparum infections, but is less sensitive for the detection of non-falciparum malaria species, especially at parasitemias < 1000 parasites/L. Further studies are necessary to
establish the field performance of the NOW ICT assay.
Received July 16, 2003. Accepted for publication September 5, 2003.

TABLE 3
Binax Now ICT assay for the detection of Plasmodium falciparum
malaria according to the level of parasitemia
Parasitemia
(no. of parasites/L of whole blood)

1100
1011,000
1,00110,000
>10,000

Microscopy
(no. positive)

NOW ICT
(no. positive)

Sensitivity
(%)

4
26
37
34

3
25
36
33

75.0
96.2
97.3
97.1

Financial support: This study was supported in part by a grant from

the Canadian Institutes of Health Research (CIHR MT-13721 to
Kevin C. Kain). Kevin C. Kain is supported by a Career Scientist
Award from the Ontario Ministry of Health and a Canada Research
Chair (CIHR).
Authors addresses: Gabriella A. Farcas, Kathleen J. Y. Zhong, and
Fiona E. Lovegrove, Medical Sciences Building 7318, University of
Toronto, 1 Kings College Circle, Toronto, Ontario, Canada M5S
1A8. Christopher M. Graham and Kevin C. Kain, Tropical Disease
Unit, EN G-224, Toronto General Hospital, 200 Elizabeth Street,
Toronto, Ontario, Canada M5G 2C4, E-mail: kevin.kain@uhn.on.ca.

592

FARCAS AND OTHERS

REFERENCES
1. Ryan ET, Wilson ME, Kain KC, 2002. Illness after international
travel. N Engl J Med 347: 505516.
2. Muhlberger N, Jelinek T, Behrens RH, Gjorup I, Coulaud JP,
Clerinx J, Puente S, Burchard G, Gascon J, Grobusch MP,
Weitzel T, Zoller T, Kollaritsch H, Beran J, Iversen J, Hatz C,
Schmid ML, Bjorkman A, Fleischer K, Bisoffi Z, Guggemos
W, Knobloch J, Matteelli A, Schulze MA, Laferl H, Kapaun A,
McWhinney P, Lopez-Velez R, Fatkenheuer G, Kern P,
Zieger BW, Kotlowski A, Fry G, Cuadros J, Myrvang B, 2003.
Age as a risk factor for severe manifestations and fatal outcome of falciparum malaria in European patients: observations
from TropNetEurop and SIMPID surveillance data. Clin Infect
Dis 36: 990995.
3. Kain KC, Harrington MA, Tennyson S, Keystone JS, 1998. Imported malaria: prospective analysis of problems in diagnosis
and management. Clin Infect Dis 27: 142149.
4. Molyneux M, Fox R, 1993. Diagnosis and treatment of malaria in
Britain. BMJ 306: 11751180.
5. Ohrt C, Purnomo MA, Sutamihardja D, Tang, Kain KC, 2002.
Impact of microscopy error on estimates of protective efficacy
in malaria-prevention trials. J Infect Dis 186: 540546.
6. Anonymous, 1996. A rapid dipstick antigen capture assay for the
diagnosis of falciparum malaria. WHO Informal Consultation
on Recent Advances in Diagnostic Techniques and vaccines
for Malaria. Bull World Health Organ 74: 4754.
7. Cho Min N, Gatton ML, 2002. Performance appraisal of rapid
on-site malaria diagnosis (ICT malaria Pf/Pv test) in relation to
human resources at village level in Myanmar. Acta Trop 81:
1319.
8. Gatti S, Bernuzzi AM, Bisoffi Z, Raglio A, Gulletta M, Scaglia
M, 2002. Multicentre study, in patients with imported malaria,
on the sensitivity and specificity of a dipstick test (ICT Malaria
P.f./P.v.) compared with expert microscopy. Ann Trop Med
Parasitol 96: 1518.
9. Huong NM, Davis TM, Hewitt S, Huong NV, Uyen TT, Nhan
DH, Cong le D, 2002. Comparison of three antigen detection
methods for diagnosis and therapeutic monitoring of malaria:
a field study from southern Vietnam. Trop Med Int Health 7:
304308.
10. Iqbal J, Khalid N, Hira PR, 2002. Comparison of two commercial
assays with expert microscopy for confirmation of symptomatically diagnosed malaria. J Clin Microbiol 40: 46754678.
11. Mason DP, Kawamoto F, Lin K, Laoboonchai A, Wongsricha-

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

nalai C, 2002. A comparison of two rapid field immunochromatographic tests to expert microscopy in the diagnosis of malaria. Acta Trop 82: 5159.
Playford EG, Walker J, 2002. Evaluation of the ICT malaria
P.f/P.v and the OptiMal rapid diagnostic tests for malaria in
febrile returned travelers. J Clin Microbiol 40: 41664171.
Tjitra E, Suprianto S, Dyer M, Currie BJ, Anstey NM, 1999. Field
evaluation of the ICT malaria P.f/P.v immunochromatographic
test for detection of Plasmodium falciparum and Plasmodium
vivax in patients with a presumptive clinical diagnosis of malaria in eastern Indonesia. J Clin Microbiol 37: 24122417.
Pieroni P, Mills CD, Ohrt C, Harrington MA, Kain KC, 1998.
Comparison of the ParaSight-F test and the ICT Malaria Pf
test with the polymerase chain reaction for the diagnosis of
Plasmodium falciparum malaria in travellers. Trans R Soc
Trop Med Hyg 92: 166169.
Snounou G, Viriyakosol S, Zhu XP, Jarra W, Pinheiro L, do
Rosario VE, Thaithong S, Brown KN, 1993. High sensitivity of
detection of human malaria parasites by the use of nested
polymerase chain reaction. Mol Biochem Parasitol 61: 315320.
Zhong KJ, Kain KC, 1999. Evaluation of a colorimetric PCRbased assay to diagnose Plasmodium falciparum malaria in
travelers. J Clin Microbiol 37: 339341.
Brown AE, Kain KC, Pipithkul J, Webster HK, 1992. Demonstration by the polymerase chain reaction of mixed Plasmodium falciparum and P. vivax infections undetected by conventional microscopy. Trans R Soc Trop Med Hyg 86: 609612.
Humar A, Ohrt C, Harrington MA, Pillai DR, Kain KC, 1997.
Parasight F test compared with the polymerase chain reaction
and microscopy for the diagnosis of Plasmodium falciparum
malaria in travelers. Am J Trop Med Hyg 56: 4448.
Kain KC, Brown AE, Mirabelli L, Webster HK, 1993. Detection
of Plasmodium vivax by polymerase chain reaction in a field
study. J Infect Dis 168: 13231326.
Milne LM, Kyi MS, Chiodini PL, Warhurst DC, 1994. Accuracy
of routine laboratory diagnosis of malaria in the United Kingdom. J Clin Pathol 47: 740742.
Dyer ME, Tjitra E, Currie BJ, Anstey NM, 2000. Failure of the
pan-Plasmodium antibody of the ICT Malaria P.f/P.v immunochromatographic test to detect symptomatic Plasmodium
malariae infection. Trans R Soc Trop Med Hyg 94: 518.
Jelinek T, Grobusch MP, Nothdurft HD, 2000. Use of dipstick
tests for the rapid diagnosis of malaria in non-immune travelers. J Travel Med 7: 175179.