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Journal of Clinical Microbiology, October 1999, p. 3260-3264, Vol. 37, No. 10
Unidad de Investigación en Medicina
Tropical y Salud Internacional,
Received 29 April 1999/Returned for modification 17 June
1999/Accepted 19 July 1999
The use of a new PCR-based method for the diagnosis of malaria in
the Spanish Malaria Reference Laboratory has promoted an increase in
confirmed cases of malaria. From August 1997 to July 1998, a total of
192 whole-blood samples and 71 serum samples from 168 patients were
received from the hospitals of the Spanish National Health System. Most
of the patients came from west-central African countries (85%). This
molecular method showed more sensitivity and specificity than
microscopy, detecting 12.4% more positive samples than microscopy and
13% of mixed infections undetectable by Giemsa stain. Plasmodium
falciparum was the main species detected, with 68% of the total
positive malaria cases, followed by Plasmodium malariae
(29%), Plasmodium vivax (14%), and Plasmodium
ovale (7%), including mixed infections in all cases. This report
consists of the first wide, centralized survey of malaria surveillance in Spain. The reference laboratory conducted the analysis of all imported cases in order to detect trends in acquisition. The use of a
seminested multiplex PCR permitted confirmation of the origins of the
infections and the Plasmodium species involved and
confirmation of the effectiveness of drug treatments. This PCR also
allowed the detection of the presence in Spain of primaquine-tolerant P. vivax strains from west-central Africa, as well as the
detection of a P. falciparum infection induced by transfusion.
Malaria has a major place among the
endemic tropical diseases. Malaria risk of varying degrees existed in
100 countries and territories in 1994 (26). The increase of
tourism and cooperation with developing countries and population
migrations forced by wars and socioeconomic factors have caused an
increase of malaria cases reported in immigrants and travellers
returning from areas where the disease is endemic, with rates of case
fatality up to 0.8% in countries like the United Kingdom
(4) and Germany (1), mainly due to late or
incorrect diagnosis. In Spain, malaria was eradicated in 1962, the last
year of reported autochthonous cases. From 1985 to 1992, the number of
imported malaria cases in Spain per year ranged between 100 to 150 and
has progressively increased to 230 cases. Malaria is a notifiable
disease in Spain, but supposedly only 30 to 40% of all cases are
reported (15). This situation also exists in other developed
countries, such as Switzerland or the United States, where only 25 to
50% of the cases are reported to health authorities (26).
Several molecular methods based on the amplification of DNA have been
developed for the detection of malarial infections in humans (8,
9, 18, 20, 21, 25), but only one of these, using five PCRs, can
differentiate between the four species of Plasmodium
(20, 21). The malaria laboratory of the National Center for
Microbiology in Spain is using a seminested multiplex malaria PCR
(SnM-PCR) with just two PCRs, capable of detecting and differentiating
between the four human malaria species in blood samples
(16). Moreover, this method includes a positive amplification control in each sample to prevent false negatives. The
incorporation of these molecular tools for the characterization of
parasite infections has allowed an increase of sensitivity in the
detection of human malarial parasites in blood, but these tests are
presently only used in field trials.
The World Health Organization (WHO) recommends the surveillance of all
imported malaria cases and the indirect surveillance of the spreading
of malaria resistance in developing countries by the reference
laboratories of developed countries. Therefore, these methodologies
could increase the specificity and sensitivity of diagnosis and, if
these methodologies are used in microbiology laboratories, could reduce
the rate of case fatalities due to incorrect diagnoses.
The main objective of this work was to provide the Spanish National
System of Health with a reference laboratory that surveilled imported
malaria cases. The laboratory criterion for diagnosis is the
demonstration of malarial parasites in blood films, and confirmation of
cases is by PCR (as a complementary tool to traditional microscopy).
Additionally, following the recommendations of the WHO, the reference
laboratory analyzed the confirmed cases, determining the origins of the
malaria infections and the Plasmodium species involved and
following patients after treatment in order to evaluate the
effectiveness of antimalarial drugs.
The study, approved by the Ethical Committee of the Carlos III
National Institute of Health, was carried out in the Parasitology Department of the National Center for Microbiology in Madrid, Spain.
The main duty of the department is to provide fast and accurate
diagnoses to the microbiology departments of state hospitals.
Patients, samples, and definitions of terms used.
From
August 1997 to July 1998, a total of 192 whole-blood samples and 71 serum samples from 168 patients with at least one symptom compatible
with clinical malaria were received from 25 hospitals of the Spanish
National Health System to be diagnosed for possible infection with
Plasmodium spp. In several cases, different samples were
sent from the same patients after confirmation of the malaria infection
for surveillance of treatment effectiveness.
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Usefulness of Seminested Multiplex PCR in
Surveillance of Imported Malaria in Spain
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
PCR test. The method of DNA extraction (from 3 µl of blood) was carried out by the Chelex method with minor modifications (16, 24). Detection and identification of malarial species were simultaneously performed with a sequence of two SnM-PCRs (16), and the sizes of the products were estimated after electrophoresis on a 2% agarose gel and staining with ethidium bromide.
The PCR mix for the first reaction consisted of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.001% (wt/vol) gelatin, 1% glycerol, a 200 µM concentration of each of the deoxynucleoside triphosphates, the PCR primers, 2.5 U of AmpliTaq DNA polymerase (Perkin-Elmer), and 41.1 µl of Chelex DNA as template in a final volume of 50 µl. The amounts of primers were 25 pmol for UNR (5'-GACGGTATCTGATCGTCTT-3'), 25 pmol for PLF (5'-AGTGTGTATCAATCGAGTTT-3'), and 1.25 pmol for HUF (5'-GAGCCGCCTGGATACCG-3'). This first reaction was expected to yield two products: the first being a band of 231 bp from UNR-HUF produced by the amplification of the small subunit of the human ribosomal gene (ssrDNA), the positive control for each individual sample, and the second being a band of 783 to 821 bp (depending on the Plasmodium species) from UNR-PLF that should detect the presence of any malaria species by amplification of the ssrDNA of Plasmodium spp. The UNR-HUF fragment is the individual positive control, and it must be present in every sample. The absence of this amplification product shows that the PCR was inhibited and the result of the second PCR must be ignored in order to avoid false negatives. The reaction mix for the second PCR (SnM-PCR) consisted of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl2, 0.001% (wt/vol) gelatin, 1% glycerol, a 200 µM concentration of each of the deoxynucleoside triphosphates, the PCR primers, 1 U of AmpliTaq DNA polymerase (Perkin-Elmer), and 4 µl of a 4/1,000 dilution of the PCR product of the first reaction as template in a final volume of 25 µl. The concentration of primers was 25 pmol for PLF, 3.12 pmol for MAR (5'-GCCCTCCAATTGCCTTCT-3'), 15 pmol for FAR (5'-AGTTCCCCTAGAATAGTTACA-3'), 6.25 pmol for OVR (5'-GCATAAGGAATGCAAAGAACAG-3'), and 2.5 pmol for VIR (5'-AGGACTTCCAAGCCGAAG-3'). Infections with different human Plasmodium species yield products of different sizes. A band of 269 bp indicates Plasmodium malariae infection, a band of 395 bp shows P. falciparum infection, a band of 436 bp suggests a Plasmodium ovale infection, and a band of 499 bp indicates a Plasmodium vivax infection. Mixed infections would show the appropriate bands. For both reactions, a 2400 GeneAmp PCR system (Perkin-Elmer) thermal cycler was used, beginning with a 5-min denaturation at 94°C, followed by (first reaction) 40 cycles of 45 s at 94°C, 45 s at 62°C, and 60 s at 72°C or (SnM-PCR) 35 cycles of 20 s at 94°C, 20 s at 62°C, and 30 s at 72°C. The final cycle was followed by an extension time of 10 min at 72°C. In order to avoid possible contamination, separate space was designated for the setup of different PCRs and the storage of filter tips, etc., and several negative controls (no DNA, uninfected blood, infected Rattus blood with Plasmodium bergei, and no extracted DNA) and positive controls for each Plasmodium infecting humans were used in order to localize any possible contamination.| |
RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Western or central African countries were the most common points of departure for malaria patients examined in this study (84% of patients) followed by Asia (8.5% of patients). Eastern African and Central American countries were the origin of 7% of the cases.
Sixty-one of 108 patients (56.5%) born in countries where malaria is endemic tested positive by SnM-PCR, while microscopy detected 51 infected individuals (47%). Twenty-seven of 59 (45.7%) nonimmune Spanish travellers tested positive by SnM-PCR, while microscopy detected 19 (32.2%) patients with malarial asexual parasites in their blood. The induced malaria (the Spanish woman who received the blood transfusion) corresponded to a P. falciparum infection by the SnM-PCR, and 10 days later she also tested positive by IFAT but continued to test negative by microscopy. In total, 89 of 168 patients (53%) tested positive by SnM-PCR, while 70 patients (41.7%) tested positive for malaria by microscopy. SnM-PCR proved capable of detecting malarial parasites in 11.3% of infected patients who had tested negative by microscopy of thin and thick films.
A total of 24 additional blood samples from 14 malaria patients were assayed to confirm the effectiveness of the treatment. SnM-PCR still showed the presence of malarial parasites in 11 (45.8%) of these cases, while microscopy detected malaria infection in six cases (25%). This result is 20.8% less effective than the detection of parasites by the amplification of DNA by PCR.
In global terms, the SnM-PCR detected 100 of 192 (52%) malaria
infections, while microscopy detected 76 (39.6%) positive samples, indicating that SnM-PCR detected 12.4% more malaria infections than
did microscopy (Table 1).
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The most prevalent Plasmodium species in the samples, considering the SnM-PCR results, was P. falciparum (68%), which was present in 59 samples as a single infection and in nine other cases as a mixed infection. P. malariae was detected by itself in 16 samples (20%) and in five mixed samples. This data confirms that quartan malaria was the second most prevalent infection detected, followed by P. vivax (14%) and P. ovale (7%), which were present in 10 and 6 samples, respectively, as single infections and were present in four and one samples, respectively, as mixed infections. The SnM-PCR was able to identify nine mixed infections, while microscopy detected two (detection of 13% more mixed infections by PCR than by microscopy). All the mixed infections detected were from people born in countries where the disease is endemic, except one triple infection which was from a Spanish missionary who had lived in western Africa, Asia, and South America over the last 20 years.
In 135 of 192 blood samples, the SnM-PCR and microscopy showed the same
results, including in the case of the triple infection. However, in 36 samples, SnM-PCR detected malarial species not identified by
microscopy. Out of 116 microscopically negative samples, 30 were
SnM-PCR positive, and SnM-PCR identified a second Plasmodium
species in six other samples (Table 2).
The only discrepancy resolved in favor of microscopy was a mixed
infection of P. falciparum and P. ovale in which
only the P. falciparum was detected by SnM-PCR. In this
case, the positive control of the first reaction was partially inhibited, probably due to the fact that the sample was of clotted blood.
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IFATs presented discrepancies with the data obtained by microscopy and SnM-PCR. In semi-immune people, six samples (8%) with serological titers higher than 1/10,240 and 8 (11%) with titers higher than 1/5,120 tested negative by both SnM-PCR and microscopy. In these individuals, antimalaria antibodies may still be detectable many months or years after detectable parasitemia. On the other hand, nonimmune people present antibodies in as little as several days after the appearance of malarial parasites in the blood. In accordance with this fact, only two (3%) of the total of IFAT-negative samples tested positive by both SnM-PCR and microscopy, corresponding to nonimmune individuals with a primary malarial infection.
Two siblings born in Equatorial Guinea, a boy and a girl (4 and 5 years old, respectively), tested positive by microscopy and SnM-PCR a few days after they returned to Spain. In the hospital, both children tested positive for infection by P. falciparum by microscopy and began the standard treatment of 7 days of quinine sulfate and clindamycin. In the reference laboratory, PCR determined that both children had a mixed infection with P. falciparum and P. vivax. Once the first treatment was finished, SnM-PCR detected a single P. falciparum infection in the boy, while the girl tested negative by SnM-PCR. This result demonstrates the failure of seven days of treatment with quinine sulfate and resulted in the start of a new course of treatment with quinine for 10 days and without treatment with primaquine diphosphate. Forty days later, the children tested positive for P. vivax infections by SnM-PCR. Afterwards, the children were treated with primaquine diphosphate (0.25 mg/kg of body weight) for 14 days. One month later, new samples showed that the boy was infected by P. vivax only. Higher dosages of primaquine diphosphate (0.35 mg/kg of body weight for 14 days) together with a new treatment of quinine sulfate for 7 days was used for treatment of this relapse. Three months later, after the appearance of new clinical malaria in the boy, a blood sample tested positive for P. falciparum infection by microscopy and a P. falciparum and P. vivax infection by SnM-PCR. The boy was then treated with mefloquine (15 mg per kg of body weight) plus a higher dosage of primaquine diphosphate (0.5 mg/kg of body weight for 14 days). After this treatment, clinical cure and absence of parasites in the blood were confirmed by microscopy and SnM-PCR.
The sample from the 63-year-old Spanish woman who presented symptoms of clinical malaria but never travelled outside Spain was confirmed as being a P. falciparum infection by the SnM-PCR. This case of induced malaria resulted from a blood transfusion from a patient with a history of malarial infection 7 months earlier. Retrospective analysis of the sera confirmed the acquisition of malarial infection in the following way: (i) a sample of serum dated prior to the transfusion tested negative for infection by IFAT (titer < 1/80), (ii) serum collected 20 days posttransfusion resulted in a positive titer of 1/2,560, and (iii) 1 month later the IFAT titer was 1/5,160.
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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This study of the sources of malarial infections detected in Spain indicates that west-central Africa is a major risk area for acquiring malarial infections. Among the samples with origins of infections in west-central Africa, the majority were from Equatorial Guinea, mainly due to the high rate of emigration from that country as well as the Spanish governmental collaboration with this former Spanish colony. The high number of malarial infections in Spain that originate from this country is explained by the high malaria morbidity (3).
P. falciparum is present in the 68% of the cases of imported malaria. P. malariae (20%) was the second most common species in these cases, followed by P. vivax (mainly from Asian and Central American regions) and P. ovale (which is restricted to west-central Africa).
SnM-PCR increased the detection of malarial parasites, including in cases of mixed infections. Data about mixed infections in which P. vivax and P. ovale are the second species involved makes clear the importance of correct diagnosis of the specific infection and the correct treatment of the liver parasite stages in order to prevent relapses.
IFAT is a good diagnostic method for people who have not been in previous contact with the infection. A few days after the blood is invaded, antibodies to erythrocytic asexual stages become detectable by the more sensitive tests. A few days later, high levels of antimalarial antibodies are reached, and these levels persist after the parasitological crisis has concluded. In this study, IFAT provided useful complementary information on several nonimmune patients and should be considered as a combined indicator of point prevalence and recent period prevalence of the disease. This data was confirmed for P. falciparum infections, because homologous P. falciparum antigen was used during IFATs. Although homologous P. falciparum antigens were used on IFATs, some cross-reactions were presented on samples when other species were involved in malaria infections.
The case of malaria induced by blood transfusion was detected by SnM-PCR and confirmed by IFAT and shows the necessity of using PCR or other molecular diagnostic methods to screen blood donors for possible malaria infection. IFAT could be adequate for persons who have had malaria in the past, although the test does not always indicate the actual presence of infection. In developed countries, serological testing is the current methodology to detect antimalarial antibodies during the screening of blood (5), but in countries where the disease was formerly endemic, where number of malaria cases increases every year due to immigration, tourism, etc., other methods of direct detection could be used. Regulations governing the acceptance of whole blood for transfusion vary considerably from one country to another, and it would be desirable for some internationally acceptable criteria to be established. PCR has been previously used for the screening of blood donors and demonstrates more sensitivity than microscopy (20, 23) and should be a useful tool to investigate potentially dangerous blood donors with histories of malaria or exposure to the infection.
Two mixed infections of P. falciparum plus P. vivax in two natives of Equatorial Guinea, a west-central African country where the majority of the population lacks the Duffy receptor necessary for the invasion of the erythrocyte by P. vivax (10, 11), confirmed the presence of a focus of P. vivax (16) in that country. Occurrences of P. vivax relapses in these patients after primaquine therapy would be assumed to be the most reliable indication of resistance. In this case, the failure of initial and secondary treatments was demonstrated by the detection of P. vivax by SnM-PCR, an infection accompanied by clinical features. The administration of higher dosages of primaquine (7, 19) after the third relapse gave good results and prevented any further malaria symptoms. The time of the appearance of the relapses (8 to 12 weeks) could be indicative of a short-term strain type, like some strains from southeast Asia (6). Until now, primaquine-tolerant strains have been described in southeast Asia (14, 22), the western Pacific, and Central America (12, 17), but only sporadic cases have been described in eastern Africa (13). It must be noted that the infection of this patient by P. falciparum was also resistant to standard treatment. In west-central Africa, an infection of two resistant malarial parasites (quinine-resistant P. falciparum and primaquine-resistant P. vivax) in the same individual had not been previously reported (22). This could suggest that the mixed infection originates in some Asiatic areas and was imported to west-central African countries where Asiatic populations have been increasing during recent decades.
Furthermore, three other cases of P. falciparum strains tolerant to quinine were found, two from Equatorial Guinea and one from Pakistan. The percentage of quinine-tolerant strains is still very low (7.3%) in comparison with chloroquine and mefloquine resistances, but the number of resistant strains is increasing (26), and in the future it could be necessary to develop new strategies for malaria therapies.
The appearance of a possible autochthonous case of P. vivax in Italy (2) and the increase of malaria cases in north Africa (27) have necessitated the establishment of a system of malaria surveillance in Spain. The risk of the reintroduction of malaria to countries where the disease has been eradicated is minimal (26) due to socioeconomic development and the high level of health care, but a system of surveillance of reemerging diseases, like malaria, needs to be established.
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ACKNOWLEDGMENTS |
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This work was supported by the Fondo de Investigaciones Sanitarias (FIS) (contract number 96/0216) and the Spanish Agency of International Cooperation (AECI). J. M. Rubio was granted a postdoctoral fellowship from the Comunidad Autónoma de Madrid, Madrid, Spain. J. Alvar was supported by a B.A.E. from the FIS (contract number 99/5038) and by the Christ's College, University of Cambridge, Cambridge, United Kingdom.
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FOOTNOTES |
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* Corresponding author. Mailing address: Servicio de Parasitología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Ctra. Majadahonda-Pozuelo km 2, Majadahonda, 28220 Madrid, Spain. Phone: 34-91-5097901. Fax: 34-91-5097966. E-mail: abenito{at}isciii.es.
Present address: Christ's College, Cambridge CB2 3BU, United Kingdom.
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Materials and Methods
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Discussion
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Journal of Clinical Microbiology, October 1999, p. 3260-3264, Vol. 37, No. 10
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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