PCR-based methods to the diagnosis of imported malaria.

Abstract

Rapid and precise diagnosis of malaria is needed to take care febrile patient returning from endemic areas. Since the first description of the diagnosis of Plasmodium infection by polymerase-chain-reaction (PCR), the role of this kind of molecular method in the laboratory diagnosis of imported malaria is still a topical question. PCR-based assays were found to be more sensitive and more specific than all conventional methods. The highest contribution of the molecular diagnosis is that a PCR negative result would ascertain the lack of any malaria infection, thus quickly orienting the investigations toward other aetiology. This technique should be now considered as the gold standard for the diagnosis of imported malaria.

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PCR-BASED METHODS TO THE DIAGNOSIS OF IMPORTED MALARIA
BERRY A.*, BENOIT-VICAL F.*,**, FABRE R.*, CASSAING S.* & MAGNAVAL J.F.*
Summary:
Rapid and precise diagnosis of malaria is needed to take care
febrile patient returning from endemic areas. Since the first
description of the diagnosis of Plasmodium infection by
polymerase-chain-reaction (PCR), the role of this kind of molecular
method in the laboratory diagnosis of imported malaria is still a
topical question. PCR-based assays were found to be more
sensitive and more specific than all conventional methods. The
highest contribution of the molecular diagnosis is that a PCR
negative result would ascertain the lack of any malaria infection,
thus quickly orienting the investigations toward other aetiology.
This technique should be now considered as the gold standard for
the diagnosis of imported malaria.
KEY WORDS : imported malaria, molecular diagnosis, PCR.
S
et al., 1990), the role of this kind of molecular method
in the laboratory diagnosis of imported malaria is still
a topical question. Albeit considered the gold standard
as regards sensitivity and specificity, the use of PCR-
based assays for routine detection of malaria infection
is debatable for many people, who consider the prac-
tical requirements (expensive apparatus, need for qua-
lified technicians, usage costs), together with the risk
of contamination and the problems of emergency dia-
gnosis, to be out of proportion given the known bene-
fits. However, permanent technological improvement
has made PCR faster and more and more user-friendly.
* Service de Parasitologie-Mycologie, Hôpital Rangueil, Centre Hos-
pitalier Universitaire de Toulouse, TSA 50032, 31059 Toulouse 9,
France.
** Laboratoire de Chimie de Coordination du CNRS, 205, route de
Narbonne, 31077 Toulouse 4, France.
Correspondence: Antoine Berry.
Tel.: 33 (0)5 61 32 28 92 – Fax: 33 (0)5 61 32 20 96.
E-mail: berry.a@chu-toulouse.fr
based test used (see above) or the nature of primers
that were used. At its best, the power of detection of
thick blood smear examination ranged from 10 to
50 parasites/µL (Guerin et al., 2002; Moody, 2002).
Quantitative Buffy Coat (QBC®) tests were not more
efficient (Wongsrichanalai et al., 1991) and dipstick
assays exhibited a poor sensitivity when the parasi-
temia value was under 100 parasites/µL (Moody, 2002).
Conversely, in 20 studies (Table I), PCR’s sensitivity for
the detection of P. falciparum ranged from 0.004 to
30 parasites/µL. However, some of these results should
be carefully considered, since they were experimental
and dilution ranges were done in very different man-
ners, using either blood from infected subjects, or
from P. falciparum cultivation (often with no indica-
tion concerning synchronization, so the presence of
multinucleated schizonts might be suspected), or from
extracted DNA (with different methods of extraction).
For species other than P. falciparum, comparing sen-
sitivity results were impossible due to the lack of any
means of cultivation and also to the variation of the
proportion of schizonts between different patients.
Concerning the diagnosis of imported malaria, only
papers that compared PCR and conventional micro-
scopy during a limited period of time and in all patients
suspected to be infected were analyzed. In 15 studies
including 2,416 subjects, 1,108 patients were found to
be malaria positive by conventional microscopy, and
1,204 (+ 10 %) by PCR (range: from 0 to + 13 %). All
the patients positive by microscopy were also positive
by PCR, whatever the Plasmodium species. When com-
pared to the set of the requests, the gain of sensitivity
was 4.4 % (range: from 0 to + 11 %) (Rubio et al., 1999;
Filisetti et al., 2002; Morassin et al., 2002; Richardson
et al., 2002; de Monbrison et al., 2003; Padley et al.,
2003; Patsoula et al., 2003; Perandin et al., 2003; Cal-
deraro et al., 2004; Fabre et al., 2004; Perandin et al.,
2004; Rougemont et al., 2004; Whiley et al., 2004;
Machouart et al., 2006; Vo et al., 2007). This improve-
ment in sensitivity affected mainly falciparum malaria.
P. falciparum is the most frequently detected agent of
imported malaria, especially in the European Union
where the closest endemic zone is sub-Saharan Africa
(Jelinek et al., 2002). Moreover, even chemoprophy-
ince the first description, in 1990, of the diagnosis
of Plasmodium falciparum infection by means of
polymerase-chain-reaction (PCR) (Jaureguiberry
A SENSITIVE ASSAY
F
or the laboratory diagnosis of malaria, PCR-based
assays were found to be more sensitive than all
conventional methods, whatever the type of PCR-

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laxis that is not correctly followed out remains rather
active on species other than P. falciparum, apart from
the chloroquine-resistant P. vivax strains, which are still
rarely encountered in Europe.
A SPECIFIC METHOD
T
Bank®, guarantees the specificity of PCR results. A fur-
ther precaution has often been the sequencing of PCR
products.
Concerning Plasmodium species identification, litera-
ture analysis reports frequent and sometimes substan-
tial discrepancies between the results from microscopy
and those from PCR. From the last 12 published stu-
dies that have compared the performance of thin blood
smear examination and PCR, 17.1 % (range: 1.5-39) of
923 species identifications by microscopy were cor-
rected after PCR checking (Rubio et al., 1999; Morassin
et al., 2002; de Monbrison et al., 2003; Perandin et al.,
2003; Calderaro et al., 2004; Di Santi et al., 2004; Ndao
et al., 2004; Perandin et al., 2004; Rougemont et al.,
2004; Whiley et al., 2004; Machouart et al., 2006; Vo
et al., 2007). The rate of misdiagnosis varied from 20 %
he nature of the target for primers or probes,
as determined by the studies of the Plasmodium
genome whose results are available in Gen-
to 50 % for P. malariae, P. ovale or P. vivax (Morassin
et al., 2002; de Monbrison et al., 2003; Perandin et al.,
2003; Calderaro et al., 2004; Di Santi et al., 2004; Ndao
et al., 2004; Perandin et al., 2004; Rougemont et al.,
2004). Concerning mixed infections, practically 100 %
of the microscopy results were false, either by excess
(diagnostic of a mixed infection when there was a sin-
gle species) or by omission (reverse situation). Plas-
modium falciparum identification appeared to be more
reliable, with no misdiagnoses found in eight studies
(Morassin et al., 2002; de Monbrison et al., 2003; Cal-
deraro et al., 2004; Ndao et al., 2004; Perandin et al.,
2004; Whiley et al., 2004; Machouart et al., 2006; Vo
et al., 2007), a 1.7 to 2.8 % misdiagnosis rate in three
studies (Rougemont et al., 2004; Rubio et al., 1999; Per-
andin et al., 2003) and 8.4 % of misdiagnoses in one
study (Di Santi et al., 2004).
However, the high specificity of PCR methods demons-
trated the other side of the coin, namely the intrinsic
risk of these tests being unable to detect new mole-
cular variants. This phenomenon was reported concer-
ning P. malariae in China (Liu et al., 1998; Kawamoto
et al., 2002), P. ovale in the Thai – Myanmar border
region (Kawamoto et al., 1996) or in Africa (Calderaro
et al., 2007) and also P. vivax in Papua-New Guinea,
where the so-called P. vivax-like species was described
(Qari et al., 1993). Moreover, newly emergent infec-
Sensitivity
for Pf
(parasite/µl)
PCR
type
Gene’s
targetAuthors/YearApparatusRevelationDetected species1
Arai et al., 1994
Ciceron et al., 1999
Tham et al., 1999
Fabre et al., 2002
Classical
Classical
Classical
Real-time
Nested
Simplex
Simplex
Multiplex
& simplex2
Simplex
Nested
Nested &
multiplex
Simplex
Multiplex
Multiplex
Nested
Simplex
Nested
Simplex
Simplex
Simplex
Simplex
Ethidium bromide (gel)
Probe (southern blot)
Probe (gel)
Syber green®
Pf
Genus Pf, Pv/
Genus Pf, Po, Pv/
Genus/Pm
DHFR
SSUrRNA
Cox 1/Plastid
Cox 1/Plastid/
SSUrRNA
STEVOR
SSUrRNA
SSUrRNA
1.3
3.0
0.01
0.035
Filisetti et al., 2002
Myjak et al., 2002
Rubio et al., 2002
Classical
Classical
Classical
Ethidium bromide (gel)
Ethidium bromide (gel)
Ethidium bromide (gel)
Pf
4
4
0.01
0.38
0.01
De Monbrison et al., 2003
Kho et al., 2003
Patsoula et al., 2003
Calderaro et al., 2004
McNamara et al., 2004
Montenegro et al., 2004
Perandin et al., 2004
Whiley et al., 2004
Mangold et al., 2005
Elsayed et al., 2006
Real-time
Classical
Classical
Classical
Classical
Classical
Real-time
Classical
Real -time
Real-time
Syber green®
Ethidium bromide (gel)
Ethidium bromide (gel)
Probe (enzyme-linked)
Probe (gel)
Ethidium bromide (gel)
Probe
Probe (enzyme-linked)
Syber green®
Probe (molecular braecon)
Genus & 4
Pf, Pv
Pf, Pv
4
4
Genus
Pf, Po, Pv
4
4
Pf, Genus
SSUrRNA
SSUrRNA
SSUrRNA
SSUrRNA
SSUrRNA
SSUrRNA
SSUrRNA
SSUrRNA
SSUrRNA
Cox 1/Plastid/
SSUrRNA
SSUrRNA
AQP/ECPR,
Pos25/CS
SSUrRNA
SSUrRNA
30
0.1
1.0
0.07
0.1
0.07
1.0
1.4
1.0
0.004
Machouart et al., 2006
Vo et al., 2007
Classical
Real Time
Simplex
Simplex
Probe (enzyme-linked)
Syber green‚
4
4
10
15
Gama et al., 2007 Real time
Classical
Simplex
Simplex
Probe (TaqMan®)
Ethidium bromide (gel)
Pf
Pf
0.5
0.5
1Pf, Pm, Po, Pv: Plasmodium falciparaum, P. malariae, P. ovale, P. vivax.
2For Pm, Po, Pv.
Table I. – PCR’s sensitivity for the detection of P. falciparum ranged from 0.004 to 30 parasites/µL in 20 studies.

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tions by zoonotic primate Plasmodium can go undia-
gnosed by species-PCR, which occurred with the
Borneo focus of P. knowlesi (Singh et al., 2004).
When primers or probes are chosen then designed,
these two points should be kept in mind and corre-
lated with future uses of the PCR test, e.g. routine dia-
gnosis in a westernized country or, conversely, epide-
miology surveys from specimens collected in the field.
The concomitant use of an assay detecting the Plas-
modium genus, whatever the origin – Man, subhuman
primate or even rodent – of the species involved,
appears to be an excellent safeguard against rare but
possible false negative results.
PCR AND THE DIAGNOSIS OF IMPORTED
MALARIA
E
is due to both high sensitivity and specificity, as detai-
led above. While PCR is used more and more in labo-
ratories of Parasitology, its exact place in the full range
of malaria diagnostic tests has yet to be explicitly esta-
blished.
Obviously, the emergency diagnosis of malaria will for
a long time remain the prerogative of microscopy
(thin smear or fast thick smear examination (Thellier
et al., 2002), QBC®) associated with immunochroma-
tographic methods. Such a test combination can detect
all infections displaying a moderate to high parasitemia,
which ensures within less than half an hour the diagno-
sis of severe malaria. PCR should therefore be consi-
dered a second-line method.
The first question about the routine use of PCR for the
diagnosis of imported malaria concerns the schedule
of use, daily, weekly or bimonthly, and also the type
of specimens concerned, all or only those posing a
problem. Based on a 8-year experience, we subse-
quently believe PCR should be done on a daily basis.
In this case, low parasitemia infections due to, e.g.
incorrect chemoprophylaxis or an immune status, would
be promptly identified, despite having escaped the first-
line methods. Anyone who deals with tropical medi-
cine has had a personal example of a patient displaying
chronic fever along with a recent history of travel in
a malaria-endemic area and who is finally diagnosed
as malaria-infected after many days of hospitalization
and multiple conventional investigations. A request for
PCR would quickly resolve this diagnostic quandary
(Puente et al., 2000; Antinori et al., 2001; Speers et al.,
2003). Moreover, it should be underlined that, accor-
ding to our experience, the predictive negative value
of a PCR-based result is 100 %. A negative result would
ascertain the lack of any malaria infection, thus quickly
ven now, PCR assay may be considered the gold
standard for the diagnosis of malaria, therefore
replacing thick smear examination. This situation
orienting the investigations toward other hypotheses.
With the expected reduction of hospitalization time,
substantial money saving could be achieved, as – in
France – daily costs are approximately 800 € in a Depart-
ment of Internal Medicine or Infectious/Tropical Diseases.
Parasitemia level can be quantified by real-time PCR.
However, this can be more easily achieved by the com-
bined use of blood count results and thin smear exa-
mination.
Since real-time PCR can detect and quantify very low
parasitic loads, it has been hypothesized that this assay
could be a valuable tool for the monitoring of falci-
parum malaria therapy. Two studies indicated that the
persistence of positive PCR results after a 5-day treat-
ment was consistent with treatment failure (Kain et al.,
1994; Ciceron et al., 1999). However, such a result can
also be due to the presence of gametocytes (Smalley
& Sinden, 1977), which removes any purpose from this
kind of molecular follow-up.
From a financial point of view, PCR is said to be a costly
method. It is clear that technical requirements, such as
room layout in a laboratory, together with the purchase
of a thermocycler and the required ancillaries, are not
inexpensive. However, one should consider that this
price is on a constantly decreasing slope and these
investments are also indispensable for other diagnoses
in Parasitology, such as that of congenital toxoplas-
mosis. Many hospitals have also chosen the solution of
a technical platform pooled with different laboratories.
In fine, it appears obvious that PCR use improves the
diagnosis of imported malaria, but also that this method
is restricted to health centers, such as University Hos-
pitals, for whom malaria identification is an important
and routine problem. For other structures, the combi-
nation of conventional microscopy with immunochro-
matographic methods is certainly the best choice.
CONCLUSION
C
robust predictive value of a negative result. PCR-based
methods should therefore be included in the panel of
diagnostic tools for imported malaria infection for any
center of reference. This is based on both scientific rea-
sons and also interesting from a “service” point of view,
whose importance is constantly increasing, to help
meet the duty of best efforts.
urrently, PCR assay may be considered the “gold
standard” for malaria diagnosis, given both its
high sensitivity and specificity, along with the
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