Davis2,3, Sean Hewitt2, Nguyen Van Huong1, Tran Thi Uyen1, Doan Hanh Nhan1and Le Dinh Cong1 1 National Institute of Malariology, Parasitology and Entomology, Hanoi, Vietnam 2 Vietnam–Aus
Trang 1Comparison of three antigen detection methods for diagnosis and therapeutic monitoring of malaria: a field study from
southern Vietnam
Nguyen Mai Huong1, Timothy M E Davis2,3, Sean Hewitt2, Nguyen Van Huong1, Tran Thi Uyen1,
Doan Hanh Nhan1and Le Dinh Cong1
1 National Institute of Malariology, Parasitology and Entomology, Hanoi, Vietnam
2 Vietnam–Australia Malaria Control Project, Ministry of Health, Hanoi, Vietnam
3 Department of Medicine, University of Western Australia, Fremantle Hospital, Fremantle, Australia
Summary O B J E C T I V E S To compare the sensitivity, specificity and post-treatment persistence of three commonly
used rapid antigen detection methods
M E T H O D We studied 252 Vietnamese patients aged from 4 to 60 years, 157 with falciparum and 95 with vivax malaria and 160 healthy volunteers An initial blood sample was taken for microscopy, and OptiMALÒ, immunochromatographic test (ICT) malaria P.f./P.v.Òand Paracheck-PfÒtests Patients with falciparum malaria were treated with an artesunate-based combination regimen and those with vivax malaria received chloroquine Eighty-seven patients with falciparum malaria who were initially positive for one of the antigen tests and who remained blood smear-negative underwent follow-up testing over 28 days
R E S U L T S Paracheck-PfÒwas the most sensitive test for Plasmodium falciparum (95.8% vs 82.6% for ICT malaria P.f./P.v.Òand 49.7% for OptiMALÒ) Specificities were all 100% For vivax malaria, OptiMALÒperformed better than ICT malaria P.f./P.v.Ò(sensitivities 73.7% and 20.0%, respectively), with 100% specificity in both cases All tests had low sensitivities (£ 75.0%) at parasitaemias < 1000/ll regardless of malaria species During follow-up, Paracheck-PfÒremained positive in the greatest proportion of patients, especially at higher parasitaemias (> 10 000/ll) Residual OptiMALÒpositivity occurred only in a relatively small proportion of patients (< 10%) with parasitaemias > 10 000/ll during the first 2 weeks after treatment
C O N C L U S I O N S Although microscopy remains the gold standard for malaria diagnosis, Paracheck-PfÒ may prove a useful adjunctive test in uncomplicated falciparum malaria in southern Vietnam OptiMALÒ had the lowest sensitivity for P falciparum but it might have a use in the diagnosis of vivax malaria and perhaps to monitor efficacy of treatment for falciparum malaria where microscopy is unavailable
keywords falciparum malaria, vivax malaria, antigen detection, diagnosis, ICT malaria P.f./P.v.Ò, Paracheck-PfÒ, OptiMALÒ
correspondence Timothy M E Davis, Department of Medicine, University of Western Australia, Fremantle Hospital, PO Box 480, Fremantle, Western Australia 6959, Australia
Fax: +00 618 9431 2977; E-mail: tdavis@cyllene.uwa.edu.au
Introduction
Although examination of a blood smear by microscopy
remains the gold standard for malaria diagnosis, antigen
detection methods have been developed for situations in
which reliable microscopy may not be available In the case
of Plasmodium falciparum, these new rapid methods are based on detection of histidine-rich protein-2 (HRP-2; ICT malaria P.f.Ò, ParaSight-FÒ, Paracheck-PfÒ) or parasite lactate dehydrogenase (pLDH; OptiMALÒ) Species-specific pLDH isoforms have been used to develop a test for Plasmodium vivax (OptiMALÒ) Plasmodium vivax can
Trang 2also be detected through positive antibodies against
‘panmalarial’ antigen in the absence of those against
HRP-2 (ICT Malaria P.f./P.v.Ò)
The sensitivity and specificity of each of these tests have
been assessed in a range of clinical situations As might be
expected from their common antigenic target, the
perform-ance characteristics of ICT malaria P.f.Ò, ParaSight-FÒand
Paracheck-PfÒare similar Although the overall sensitivity
and specificity of each of these three tests is high (usually
>90%), their sensitivity falls off at parasite densities < 350/ll
(Lema et al 1999; Ricci et al 2000; Singh et al 2000; Proux
et al 2001) In the case of OptiMALÒfor falciparum
malaria, results of field studies appear more variable with an
overall sensitivity of between 60% (Fryauff et al 2000) and
96% (Moody et al 2000) Nevertheless, some of this
variability may also be explained by the relatively poor
performance of OptiMALÒat low parasitaemias (< 500/ll)
(Iqbal et al 1999; Fryauff et al 2000; Ricci et al 2000) The
ICT malaria P.f./P.v.Òhas a reported sensitivity of between
45% (Cho et al 2001) and 95% (Tjitra et al 1999) for vivax
malaria, but its ability to detect a parasitaemia at < 1500/ll
has been questioned (Singh et al 2000) The sensitivity of
OptiMALÒfor P vivax is between 70% (Fryauff et al 2000;
Cho et al 2001) and 96% (Moody et al 2000) but there
have been no reports of its relationship with parasitaemia
It has been suggested that the disappearance of pLDH
that accompanies parasite clearance might be used as a
measure of therapeutic response (Palmer et al 1999;
Moody et al 2000) By contrast, HRP-2 can persist for
several weeks after successful treatment (Shiff et al 1993)
Nevertheless, studies of OptiMALÒin this context have
not always taken into consideration the ability of
ga-metocytes to produce detectable levels of pLDH (Oduola
et al 1997), especially when regimens that do not include
artemisinin or primaquine have been used (Tjitra & Anstey
2001)
Because of the need for further studies (WHO 2000) and
as antigen detection methods have not been evaluated
formally in Vietnam, we compared the sensitivity,
speci-ficity and post-treatment persistence of OptiMALÒ, ICT
Malaria P.f./P.v.Òand Paracheck-PfÒin Vietnamese
patients and healthy volunteers To limit the effect of
gametocytaemia, the patients with falciparum malaria
received artesunate as initial therapy
Subjects and methods
Subjects
We studied 252 Vietnamese patients with slide-positive
malaria and 160 asymptomatic, aparasitaemic control
subjects The study was conducted between July and
September 2000 in Phuoc Long district, Binh Phuoc province (147 patients and 60 controls) and in Lac district, Dac Lac province (105 patients and 100 controls) The study districts in Binh Phuoc and Dac Lac provinces are in highly endemic areas of southern Vietnam containing dense jungle and rubber plantations Both areas are well known for multidrug resistant falciparum malaria
Patients aged 4–60 years with fever or a recent history of fever were eligible for recruitment Pregnant patients, and those with severe malaria or concomitant illness were excluded, as were those who had been treated for malaria
in the previous 4 weeks All controls were also aged between 4 and 60 years and were from the same areas as the patients Control subjects who tested positive by any of the three antigen detection methods were questioned regarding febrile episodes in the previous 4 weeks and excluded if they reported that they had received treatment for malaria during this period All patients and control subjects gave informed consent to study procedures that were approved by the Research Committee of the National Institute for Malariology, Parasitology and Entomology and by the Ethical Committee of the Ministry for Health, Vietnam
Methods
An initial fingerprick blood sample was taken from each patient and control for thick and thin smears and OptiMALÒ(DioMed AG, Switzerland), ICT malaria P.f./P.v.Ò(AMRAD, NSW, Australia) and Paracheck-PfÒ (Orchid Biomedical System, Goa, India) tests All tests used were within expiry date Three experienced technicians performed antigen testing according to the manufacturer’s recommendations, each taking responsibility for a single test type Test results were recorded without reference to one another and then results were cross-checked by each technician in turn Blood smears were stained with Giemsa and examined by a skilled microscopist, without reference
to the results of antigen testing Parasite densities expressed
as number per micro litre whole blood were determined by counting the number of asexual forms per 1000 white blood cells and assuming a total white cell count of
8000 · 106/l
Patients identified as positive by any test for vivax malaria were treated with chloroquine and discharged Patients diagnosed with falciparum malaria and asexual parasite density > 1000/ll whole blood were admitted to hospital and randomized to receive artesunate combined with either chloroquine or sulphadoxine/pyrimethamine in conventional doses Those with parasitaemias < 1000/ll were treated according to national guidelines with
Trang 3sequential artesunate and mefloquine All treatments were
supervised and patients were observed for at least 60 min
after dosing Any patients vomiting during this period were
retreated but excluded from the study All admitted
patients were kept in hospital for at least 7 days All
patients in Binh Phuoc, whether admitted to hospital or
not, and all admitted patients in Dac Lac were asked to
return for follow-up on days 7, 14, 21 and 28 or if they
became symptomatic On these occasions patients gave a
blood sample, underwent antigen testing and a clinical
assessment Those who failed to attend were contacted by
local health workers in an attempt to provide as complete a
follow-up as possible
Results
Overall, Paracheck-PfÒwas the best test for detecting
P falciparum [sensitivity (95% confidence intervals) 95.8
(92–99%)], followed by ICT malaria P.f./P.v.Ò[82.6
(77–89%)] and OptiMALÒ[49.7 (42–58%)] After
exclu-ding six control subjects (two from Binh Phuoc and 4 from
Dac Lac) who tested positive for any of the tests, the
specificities were all 100% For diagnosis of vivax malaria,
the OptiMALÒtest performed better than ICT malaria P.f./
P.v.Ò[sensitivities 73.7 (65–83%) and 20.0 (12–28%),
respectively], with 100% specificity in both cases
The results of sensitivity testing according to
parasita-emia and by study site are shown in the Table 1 It should
be noted that, because of the requirement for fever in
patients from a high transmission area with a degree of
semi-immunity, there were no cases in which the parasite
density was < 100/ll There were lower sensitivities
for each of the three tests at Dac Lac compared with
Binh Phuoc All three tests performed suboptimally for
P falciparum infections at parasitaemias < 1000/ll (overall
sensitivities £ 75.0%), but both Paracheck-PfÒand ICT
malaria P.f./P.v.Òhad good sensitivity above this cut-off
OptiMALÒdid not perform well in any situation except in
the highest parasitaemia subgroup at Binh Phuoc, although
it appeared to be a good test for P vivax at parasitaemias
> 1000/ll In the case of ICT malaria P.f./P.v.Ò, adequate sensitivity was seen only at the highest parasitaemias The persistence of positive antigen test results in 87 patients who did not represent with P falciparum asexual parasitaemia is shown in Fig 1 There was a stepwise increase in the proportion of patients with positive tests at
7 days post-treatment and beyond as the baseline para-sitaemia increased for all three tests Consistent with its high diagnostic sensitivity, Paracheck-PfÒwas the test with the greatest proportion of positive patients for any time-point and parasite density In the case of OptiMALÒ,
at parasitaemias > 10 000/ll the test remained positive in a relatively small proportion of patients during the first 2 weeks after treatment Serial results for the ICT malaria P.f./P.v.Òwere intermediate between those of the other two tests
Discussion Our data show that Paracheck-PfÒis the antigen testing device of choice for uncomplicated falciparum malaria in endemic areas of southern Vietnam This result is consis-tent with a Thai study (Proux et al 2001) in which the sensitivity of Paracheck-PfÒfor P falciparum was 92.3% (compared with 95.8% in the present study) and 84.6% for ICT malaria P.f./P.v.Ò(compared to our 82.6%) As is the case in Thailand (Proux et al 2001), Paracheck-PfÒis significantly cheaper than ICT malaria P.f./P.v.Òin Viet-nam The relatively high sensitivity of Paracheck-PfÒ appears to come at the cost of persistence of a positive result in successfully treated patients, a finding that may limit its usefulness in areas in which malaria transmission rates are very high Indeed, if a patient has a history of treated malaria within at least 4 weeks of presentation, the result should be interpreted with caution Six of our healthy control subjects tested positive for at least one of
Table 1 Sensitivity of the three antigen detection methods for patient subgroups classified by study site and baseline parasitaemia
Study site Parasite density (/ll) Number Paracheck-PfÒ
ICT malaria P.f./P.v.Ò OptiMALÒ Number
ICT malaria P.f./P.v.Ò OptiMALÒ
Trang 4the three tests used and, in each case, there was a history
consistent with recently treated malaria
The currently available OptiMALÒ test did not
perform well enough in our study to be recommended as
a diagnostic aid in Vietnam In fact, its overall sensitivity
(49.7%) was the lowest so far reported in the literature
(60%; Fryauff et al 2000), and a number of patients
whose parasitaemias were easily detectable by
micros-copy (>40 000/ll) tested negative The reason for this
result is unclear It may relate to the quality of the batch
used in the present study Alternatively, strain-specific
differences in the antigen–antibody interaction used in
the test may be to blame, as the reported sensitivity of
OptiMALÒ has varied significantly between different
geographical locations Nevertheless, OptiMALÒ is a
better test than ICT malaria P.f./P.v.Ò for diagnosing
vivax malaria and, as reported by others (Palmer et al
1999; Moody et al 2000), perhaps also in assessing the
response to treatment in falciparum malaria cases who
have an initially positive OptiMALÒ result None of our
subjects remained positive for OptiMALÒ beyond
14 days after treatment
The performance of all of the tests fell away at
relatively low parasitaemias, consistent with the findings
of others (Fryauff et al 2000; Iqbal et al 1999; Lema
et al 1999; Ricci et al 2000; Singh et al 2000; Proux
et al 2001) This can be potentially dangerous, as to miss
the diagnosis of malaria in a one-off assessment of an
ambulant, febrile patient may mean that complications
develop because appropriate treatment is not instituted
The assessment of a negative result in this situation will
be clearly influenced by the clinical features and, given the differences between our two study sites, perhaps also local test performance characteristics Depending on the avail-ability of microscopy, consideration should be given to bringing the patient back for review including repeat antigen testing Patients who present with established complications need to be managed in an inpatient facility, and repeated tests (whether blood smears or antigen detection) are usually more easily arranged if the diag-nosis is not confirmed initially In addition, antimalarial therapy is often given empirically to such patients on clinical grounds
Our results add to the evidence that antigen testing can prove a valuable adjunct to clinical assessment of the patient and blood film microscopy in certain
circumstanc-es, but their sensitivity indicates that they should not yet be regarded as a first-line diagnostic test As supported by the conclusions of a recent review (WHO 2000), careful examination of thick and thin blood smears by a trained microscopist should remain the goal of malaria control programmes in countries such as Vietnam
Acknowledgements
We would like to thank Ms Cath Barker and Dr Le Thi Nga of the Vietnam-Australia Malaria Control Project for their valuable assistance This study received financial support from AusAID (the Australian Agency for Interna-tional Development)
Parasitaemia
≤1000/µl 1000 to ≤10,000/µl 10,000 to ≤100,000/µl
(5)
100 90 80 70 60 50 40 30 20 10
(56)
(26)
(25)
Time (days)
0
0
(17) (21)
Parasitaemia Parasitaemia
Figure 1 Percentages of patients with
per-sistence of a positive test result for
Opti-MAL Ò (d——-d), ICT malaria P.f./P.v Ò
(s- - - - -s) and Paracheck-Pf Ò
(m— - —m) tests subdivided by presenting
parasitaemia Numbers in each subgroup
are shown in parentheses adjacent to each
line There were no patients with baseline
parasitaemia < 1000/ll who were both
positive for OptiMAL Ò and had full
follow-up data.
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