T R A N S F U S I O N C O M P L I C AT I O N S Plasmodium genome in blood donors at risk for malaria after several years of residence in Italy Sonny Michael Assennato,1 Alessandra Berzuini,2 Barbara Foglieni,2 Marta Spreafico,2 Jean-Pierre Allain,1 and Daniele Prati2
BACKGROUND: At present, the main risk of transfusion-transmitted malaria (TTM) in nonendemic countries is chronic, asymptomatic immigrants from malaria-endemic areas. Semi-immune donors may carry undetected parasitemia. This study examines Plasmodium infection in at-risk blood donors in Northern Italy. STUDY DESIGN AND METHODS: Plasma samples from 97 candidate donors and 80 controls were tested for malarial antibodies using a commercial enzyme immunoassay. The conserved 18S rRNA and the mitochondrial genes of Plasmodium were amplified to detect and quantify parasite genomes (copies/mL). Plasmodium species were identified with a species-specific nested polymerase chain reaction. Parasitemic samples were further tested by amplification of polymorphic repetitive regions in MSP-1 Block 2, MSP-2 Block 3, and glutamate-rich protein (GLURP) confirmed by sequencing. RESULTS: Three of 83 seropositive (3.6%) and one of 14 seronegative at-risk candidate donors carried Plasmodium genome (4 × 103-8.5 × 104 copies/mL): two P. falciparum, one P. malariae (seronegative sample), and one coinfection with P. malariae and P. ovale. Alleles of MSP-1 (MAD20 and K1), MSP-2 (3D7 and FC27), and GLURP were amplified from Sample 261. In Sample 282 only one allele in MSP-2 (FC27) and GLURP was amplified. No alleles were detected in Samples 283 and 331. CONCLUSIONS: Immigrants from endemic countries might carry infectious Plasmodium after 2 to 5 years of continuous residence in Italy. Serologic screening may miss donors carrying P. malariae. Permanent exclusion or screening for both antibodies and genome are needed to prevent TTM.
G
lobally, malaria presents a significant disease burden with 150 million infections reported every year, mainly in sub-Saharan Africa,1 especially among children and pregnant women,2 and it remains a serious adverse effect of transfusion because of the asymptomatic persistence of parasites in some blood donors.3 Malaria is usually transmitted by the bite of an infected Anopheles mosquito, but cases of transfusion-transmitted malaria (TTM) have been reported,4 even in countries in which malaria is not endemic, such as Italy, where the incidence of TTM ranges from 0 to 2 cases per million blood donations.5 TTM is an important clinical and public health problem especially because, in recipients with no immunity to malaria, it can be rapidly fatal if it is not recognized and treated quickly.6 This can be particularly important in the large proportion of transfused patients in Europe who are immunocompromised. Over the past few years in Western Europe, increasing numbers of potential donors have been immigrants from malaria-endemic areas and, unwittingly, this situation has an impact on the resources of Italian blood centers.7 Italian laws guiding blood donation comply with current European regulations, which require a deferral
ABBREVIATIONS: GLURP = glutamate-rich protein; qPCR = quantitative, real-time polymerase chain reaction; S/CO = sample-to-cutoff ratio; TTM = transfusion-transmitted malaria. From the 1Department of Haematology, University of Cambridge, Cambridge, United Kingdom; and the 2Department of Transfusion Medicine, Azienda Ospedaliera della Provincia di Lecco, A. Manzoni Hospital, Lecco, Italy. Address reprint requests to: Jean-Pierre Allain, Department of Haematology, Cambridge Blood Centre, Long Road, Cambridge CB2 0PT, Cambridge, UK; e-mail: [email protected]. Received for publication December 4, 2013; revision received February 17, 2014, and accepted February 17, 2014. doi: 10.1111/trf.12650 © 2014 AABB TRANSFUSION 2014;54:2419-2424. Volume 54, October 2014 TRANSFUSION
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TABLE 1. Demographics of 97 candidate blood donors Category and items Gender Male Female Age (years) 19-29 30-39 40-49 50-59 60-65 Country of birth Ivory Coast Senegal Benin Burkina Faso Togo Cameroon Madagascar Sierra Leone Rwanda Peru Sri Lanka Italy
Candidate donors* 67 (69.1) 30 (30.9) 32 (33) 26 (26.8) 26 (26.8) 8 (8.2) 5 (5.2) 33 (34) 24 (24.7) 11 (11.3) 12 (12.4) 4 (4.2) 4 (4.2) 2 (2.1) 1 (1.0) 1 (1.0) 1 (1.0) 1 (1.0) 3 (3.1)
* Data are reported as number (%).
period of 6 months or 3 years depending on the risk of exposure for prospective blood donors at risk of malaria. In many countries like the United Kingdom and France, this period could be reduced to 4 months if an immunologic or molecular genomic test is negative at each donation, but this provision has not been adopted in Italy, because national guidelines do not recognize any diagnostic method reducing the deferral time after a visit to an endemic area. In West Europe several malaria testing strategies have been developed by blood services, including serologic and nucleic acid tests on new donors at risk of malaria that could contribute to safer blood donations, significantly decreasing the risk of TTM.8,9 In this study we investigated malaria infection in a cohort of 97 at-risk blood donors in Northern Italy. Plasmodium antibodies and qualitative as well as quantitative molecular assays were performed.
MATERIALS AND METHODS Samples Ninety-seven whole blood samples from candidate blood donors at risk of malaria were collected in the “A. Manzoni” Hospital Blood Center in Lecco, Italy, between April 15, 2011, and September 18, 2013. They originated from sub-Saharan Africa, except one donor from South America, one from Sri Lanka, and three from Italy who had recently traveled to endemic areas (Table 1). At the Lecco blood center that collects 13,000 units/year, those from sub-Saharan Africa represent 0.5%. As requested by Italian regulation, all blood donors were interviewed at enroll2420
TRANSFUSION Volume 54, October 2014
ment and before repeat donation for history of malaria and other transfusion-transmitted infectious diseases. All donors are informed of the importance of providing reliable answers regarding their medical history. Eighty samples of plasma from random Italian donors with no known history of being born in or having traveled to endemic areas have been included in this study as negative controls for serologic testing.
Commercial malaria enzyme immunoassay All plasma samples from 97 candidate donors and 80 controls were tested for malarial antibodies using an enzyme immunoassay kit (malaria enzyme immunoassay [EIA] Ab, Bio-Rad, Marnes la Coquette, France) as recommended by the manufacturer. This malaria EIA kit uses a mixture of P. falciparum and P. vivax recombinant antigens with cross-reactivity with P. ovale and P. malariae. Results were expressed as sample-to-cutoff ratio (S/CO). The test package insert indicates a gray zone between 0.8 and 1.0, which was extended to 0.5 to 1.0 by the cautious testing laboratory.
Nucleic acid extraction Nucleic acids were extracted from 0.5 mL of whole blood using a DNA blood mini kit (QIAamp, Qiagen, Manchester, UK) according to the manufacturer’s instructions. For the control group, nucleic acids were extracted from 0.2 mL of plasma using a viral kit (High Pure, Roche, Mannheim, Germany).
Plasmodium molecular testing Whole blood sample nucleic acid extracts were tested by quantitative, real-time polymerase chain reaction (qPCR; MX3000 Stratagene, La Jolla, CA). Two conserved regions of the Plasmodium genome, the 18S rRNA gene and the mitochondrial gene, region were amplified as previously described.10 Amplification was performed in duplicate using a PCR kit (Brilliant III Ultra-Fast Q, Stratagene) according to the manufacturer’s instructions. First, all samples were tested by a qualitative qPCR and only the samples giving a reactive signal with both replicates were quantified. Quantification of Plasmodium genome was performed as previously described11 using the First WHO International Standard for P. falciparum DNA (NIBSC, Code 04/176) appropriately diluted to construct a reference curve. Cycling conditions were as follows: 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds, and 60°C for 1 minute.
Identification of Plasmodium species Oligonucleotide primers for the nested PCR assay were obtained from Sigma-Aldrich (St Louis, MO) and designed
PLASMODIUM GENOME IN ITALIAN BLOOD DONORS
based on the Plasmodium small-subunit ribosomal RNA genes.12 Nested PCR was performed as previously described.11
Allele amplification and sequencing All samples positive for species identification by nested PCR were confirmed by amplification of the polymorphic repetitive regions in MSP-1 Block 2, MSP-2 Block 3, and glutamate-rich protein (GLURP) by nested PCR as described.13 This amplification identified allelic variants of K1, MAD20, and RO33 families of MSP-1 Block 2, FC27 and 3D7 of MSP-2 Block 3, and the repetitive regions of GLURP genes from P. falciparum. All amplicons were excised, purified, and sequenced. Samples with more than one genotype were considered polyclonal infection while the presence of a single allele was considered as monoclonal infection.14
Sequence analysis Sequence analysis was carried out with computer software (MacVector Version 10.5, MacVector, Inc., Cary, NC).
formed with primers from the 18S rRNA region: four samples (261, 282, 283, 331) had a cycle threshold value below 36, 60 samples were in a range between 36 and 40 cycles, and 40 samples were negative. To confirm these results, the qPCR targeting the mitochondrial gene was performed. This second qPCR confirmed the positivity of the four low-cycle-threshold samples; the other 100 samples were negative. Therefore four of 97 at-risk samples (4.1%) including three of 83 seropositive samples (3.6%) contained confirmed detectable Plasmodium DNA. In addition, one of 14 gray zone samples contained Plasmodium DNA (Table 2).
Quantification of Plasmodium genome Samples confirmed positive for Plasmodium DNA were quantified for the level of Plasmodium genome. It ranged between 4 × 103 and 8.5 × 104 copies/mL (Table 2). Whether quantified with 18S rRNA or mitochondrial reagents, genome levels were consistent (Table 2). It is recognized that approximately five copies of the genome are present per infected red blood cell.15
Identification of Plasmodium species
RESULTS Serologic screening of potential donors and control group Ninety-seven samples from candidate blood donors testing either positive or in the gray zone for the presence of antibodies to Plasmodium by EIA were analyzed. Eighty-three of 97 samples were reactive (85.6%): 10 had a S/CO value more than 26 and 73 with a S/CO median value of 4.3 (range, 1.04-25.24). Fourteen samples (14.4%) reacted in a “gray zone” of S/CO ranging between 0.5 and 0.80. All 80 plasma samples of the control group were nonreactive.
Using genus-specific primers, Samples 261, 282, 283, and 331 provided a 240-bp amplicon. These four samples were then tested with species-specific nested PCR. The results shown in Fig. 1 identified two samples infected with P. falciparum species (261 and 282) and one (seronegative) with P. malariae (283). The last sample (331) presented a coinfection with P. malariae and P. ovale. Amplicons were excised from the gel, purified, and sequenced. Sequences confirmed the Plasmodium species identified with species-specific primers (data not shown).
Detection of alleles Detection of Plasmodium genome The first molecular screening to investigate the presence of the parasite in the candidate donor blood was per-
Allele-specific nested PCR was carried out to further characterize P. falciparum parasitemia (MSP-1, MSP-2, and GLURP). Two alleles of MSP-1 (MAD20 and K1), two of MSP-2 (3D7 and FC27), and only one of GLURP (GLURP)
TABLE 2. Characteristics of four asymptomatic, candidate blood donors carrying Plasmodium genome
ID 261
Age (years) 23
282 283 331
30 24 22
Sex Male
Time since exposure (months) 4
Anti-Plasmodium (S/CO) >26
18S rRNA 2 × 104
Mitochondrial gene 7.5 × 104
Male Male Male
36 60 60
>26 0.63* 13.34
2.5 × 104 4 × 103 8.5 × 104
4 × 104 4.5 × 103 3.5 × 104
Plasmodium genome (copies/mL)
Plasmodium species P.f. P.f. P.m. P.m. P.o.
Allele sequenced MSP-1 MAD 20 K1 ND ND ND
MSP-2 3D7 FC27 FC27 ND ND
GLURP GLURP GLURP ND ND
* Sample tested four times with malaria EIA kit with S/CO values ranging between 0.28 and 0.63. ND = not detected; P.f. = P. falciparum; P.m. = P. malariae; P.o. = P. ovale.
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Fig. 1. Species-specific nested PCR. The top line indicates species of Plasmodium. The second line indicates the sample number. Samples 261 and 282 show a band of 205 bp when amplified using specific P. falciparum primers, Sample 283 shows a 144-bp band specific for P. malariae, and Sample 331 shows a band of 144 bp when amplified using specific P. malariae primers and a 787-bp band specific for P. ovale. Molecular marker (M) is Hyperladder II (M2).
were amplified from Sample 261. In Sample 282 only one allele in MSP-2 (FC27) and one in GLURP (GLURP) were amplified. With Samples 283 and 331 that carried P. malariae and P. ovale, respectively, no alleles could be amplified possibly because of species-specific sequence divergence. Allele amplification might also be related to a parasite genome level of more than 104 copies/mL.
Donor history All four parasitemic donors were young men (
BACKGROUND: At present, the main risk of transfusion-transmitted malaria (TTM) in nonendemic countries is chronic, asymptomatic immigrants from malaria-endemic areas. Semi-immune donors may carry undetected parasitemia. This study examines Plasmodium infection in at-risk blood donors in Northern Italy. STUDY DESIGN AND METHODS: Plasma samples from 97 candidate donors and 80 controls were tested for malarial antibodies using a commercial enzyme immunoassay. The conserved 18S rRNA and the mitochondrial genes of Plasmodium were amplified to detect and quantify parasite genomes (copies/mL). Plasmodium species were identified with a species-specific nested polymerase chain reaction. Parasitemic samples were further tested by amplification of polymorphic repetitive regions in MSP-1 Block 2, MSP-2 Block 3, and glutamate-rich protein (GLURP) confirmed by sequencing. RESULTS: Three of 83 seropositive (3.6%) and one of 14 seronegative at-risk candidate donors carried Plasmodium genome (4 × 103-8.5 × 104 copies/mL): two P. falciparum, one P. malariae (seronegative sample), and one coinfection with P. malariae and P. ovale. Alleles of MSP-1 (MAD20 and K1), MSP-2 (3D7 and FC27), and GLURP were amplified from Sample 261. In Sample 282 only one allele in MSP-2 (FC27) and GLURP was amplified. No alleles were detected in Samples 283 and 331. CONCLUSIONS: Immigrants from endemic countries might carry infectious Plasmodium after 2 to 5 years of continuous residence in Italy. Serologic screening may miss donors carrying P. malariae. Permanent exclusion or screening for both antibodies and genome are needed to prevent TTM.
G
lobally, malaria presents a significant disease burden with 150 million infections reported every year, mainly in sub-Saharan Africa,1 especially among children and pregnant women,2 and it remains a serious adverse effect of transfusion because of the asymptomatic persistence of parasites in some blood donors.3 Malaria is usually transmitted by the bite of an infected Anopheles mosquito, but cases of transfusion-transmitted malaria (TTM) have been reported,4 even in countries in which malaria is not endemic, such as Italy, where the incidence of TTM ranges from 0 to 2 cases per million blood donations.5 TTM is an important clinical and public health problem especially because, in recipients with no immunity to malaria, it can be rapidly fatal if it is not recognized and treated quickly.6 This can be particularly important in the large proportion of transfused patients in Europe who are immunocompromised. Over the past few years in Western Europe, increasing numbers of potential donors have been immigrants from malaria-endemic areas and, unwittingly, this situation has an impact on the resources of Italian blood centers.7 Italian laws guiding blood donation comply with current European regulations, which require a deferral
ABBREVIATIONS: GLURP = glutamate-rich protein; qPCR = quantitative, real-time polymerase chain reaction; S/CO = sample-to-cutoff ratio; TTM = transfusion-transmitted malaria. From the 1Department of Haematology, University of Cambridge, Cambridge, United Kingdom; and the 2Department of Transfusion Medicine, Azienda Ospedaliera della Provincia di Lecco, A. Manzoni Hospital, Lecco, Italy. Address reprint requests to: Jean-Pierre Allain, Department of Haematology, Cambridge Blood Centre, Long Road, Cambridge CB2 0PT, Cambridge, UK; e-mail: [email protected]. Received for publication December 4, 2013; revision received February 17, 2014, and accepted February 17, 2014. doi: 10.1111/trf.12650 © 2014 AABB TRANSFUSION 2014;54:2419-2424. Volume 54, October 2014 TRANSFUSION
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TABLE 1. Demographics of 97 candidate blood donors Category and items Gender Male Female Age (years) 19-29 30-39 40-49 50-59 60-65 Country of birth Ivory Coast Senegal Benin Burkina Faso Togo Cameroon Madagascar Sierra Leone Rwanda Peru Sri Lanka Italy
Candidate donors* 67 (69.1) 30 (30.9) 32 (33) 26 (26.8) 26 (26.8) 8 (8.2) 5 (5.2) 33 (34) 24 (24.7) 11 (11.3) 12 (12.4) 4 (4.2) 4 (4.2) 2 (2.1) 1 (1.0) 1 (1.0) 1 (1.0) 1 (1.0) 3 (3.1)
* Data are reported as number (%).
period of 6 months or 3 years depending on the risk of exposure for prospective blood donors at risk of malaria. In many countries like the United Kingdom and France, this period could be reduced to 4 months if an immunologic or molecular genomic test is negative at each donation, but this provision has not been adopted in Italy, because national guidelines do not recognize any diagnostic method reducing the deferral time after a visit to an endemic area. In West Europe several malaria testing strategies have been developed by blood services, including serologic and nucleic acid tests on new donors at risk of malaria that could contribute to safer blood donations, significantly decreasing the risk of TTM.8,9 In this study we investigated malaria infection in a cohort of 97 at-risk blood donors in Northern Italy. Plasmodium antibodies and qualitative as well as quantitative molecular assays were performed.
MATERIALS AND METHODS Samples Ninety-seven whole blood samples from candidate blood donors at risk of malaria were collected in the “A. Manzoni” Hospital Blood Center in Lecco, Italy, between April 15, 2011, and September 18, 2013. They originated from sub-Saharan Africa, except one donor from South America, one from Sri Lanka, and three from Italy who had recently traveled to endemic areas (Table 1). At the Lecco blood center that collects 13,000 units/year, those from sub-Saharan Africa represent 0.5%. As requested by Italian regulation, all blood donors were interviewed at enroll2420
TRANSFUSION Volume 54, October 2014
ment and before repeat donation for history of malaria and other transfusion-transmitted infectious diseases. All donors are informed of the importance of providing reliable answers regarding their medical history. Eighty samples of plasma from random Italian donors with no known history of being born in or having traveled to endemic areas have been included in this study as negative controls for serologic testing.
Commercial malaria enzyme immunoassay All plasma samples from 97 candidate donors and 80 controls were tested for malarial antibodies using an enzyme immunoassay kit (malaria enzyme immunoassay [EIA] Ab, Bio-Rad, Marnes la Coquette, France) as recommended by the manufacturer. This malaria EIA kit uses a mixture of P. falciparum and P. vivax recombinant antigens with cross-reactivity with P. ovale and P. malariae. Results were expressed as sample-to-cutoff ratio (S/CO). The test package insert indicates a gray zone between 0.8 and 1.0, which was extended to 0.5 to 1.0 by the cautious testing laboratory.
Nucleic acid extraction Nucleic acids were extracted from 0.5 mL of whole blood using a DNA blood mini kit (QIAamp, Qiagen, Manchester, UK) according to the manufacturer’s instructions. For the control group, nucleic acids were extracted from 0.2 mL of plasma using a viral kit (High Pure, Roche, Mannheim, Germany).
Plasmodium molecular testing Whole blood sample nucleic acid extracts were tested by quantitative, real-time polymerase chain reaction (qPCR; MX3000 Stratagene, La Jolla, CA). Two conserved regions of the Plasmodium genome, the 18S rRNA gene and the mitochondrial gene, region were amplified as previously described.10 Amplification was performed in duplicate using a PCR kit (Brilliant III Ultra-Fast Q, Stratagene) according to the manufacturer’s instructions. First, all samples were tested by a qualitative qPCR and only the samples giving a reactive signal with both replicates were quantified. Quantification of Plasmodium genome was performed as previously described11 using the First WHO International Standard for P. falciparum DNA (NIBSC, Code 04/176) appropriately diluted to construct a reference curve. Cycling conditions were as follows: 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds, and 60°C for 1 minute.
Identification of Plasmodium species Oligonucleotide primers for the nested PCR assay were obtained from Sigma-Aldrich (St Louis, MO) and designed
PLASMODIUM GENOME IN ITALIAN BLOOD DONORS
based on the Plasmodium small-subunit ribosomal RNA genes.12 Nested PCR was performed as previously described.11
Allele amplification and sequencing All samples positive for species identification by nested PCR were confirmed by amplification of the polymorphic repetitive regions in MSP-1 Block 2, MSP-2 Block 3, and glutamate-rich protein (GLURP) by nested PCR as described.13 This amplification identified allelic variants of K1, MAD20, and RO33 families of MSP-1 Block 2, FC27 and 3D7 of MSP-2 Block 3, and the repetitive regions of GLURP genes from P. falciparum. All amplicons were excised, purified, and sequenced. Samples with more than one genotype were considered polyclonal infection while the presence of a single allele was considered as monoclonal infection.14
Sequence analysis Sequence analysis was carried out with computer software (MacVector Version 10.5, MacVector, Inc., Cary, NC).
formed with primers from the 18S rRNA region: four samples (261, 282, 283, 331) had a cycle threshold value below 36, 60 samples were in a range between 36 and 40 cycles, and 40 samples were negative. To confirm these results, the qPCR targeting the mitochondrial gene was performed. This second qPCR confirmed the positivity of the four low-cycle-threshold samples; the other 100 samples were negative. Therefore four of 97 at-risk samples (4.1%) including three of 83 seropositive samples (3.6%) contained confirmed detectable Plasmodium DNA. In addition, one of 14 gray zone samples contained Plasmodium DNA (Table 2).
Quantification of Plasmodium genome Samples confirmed positive for Plasmodium DNA were quantified for the level of Plasmodium genome. It ranged between 4 × 103 and 8.5 × 104 copies/mL (Table 2). Whether quantified with 18S rRNA or mitochondrial reagents, genome levels were consistent (Table 2). It is recognized that approximately five copies of the genome are present per infected red blood cell.15
Identification of Plasmodium species
RESULTS Serologic screening of potential donors and control group Ninety-seven samples from candidate blood donors testing either positive or in the gray zone for the presence of antibodies to Plasmodium by EIA were analyzed. Eighty-three of 97 samples were reactive (85.6%): 10 had a S/CO value more than 26 and 73 with a S/CO median value of 4.3 (range, 1.04-25.24). Fourteen samples (14.4%) reacted in a “gray zone” of S/CO ranging between 0.5 and 0.80. All 80 plasma samples of the control group were nonreactive.
Using genus-specific primers, Samples 261, 282, 283, and 331 provided a 240-bp amplicon. These four samples were then tested with species-specific nested PCR. The results shown in Fig. 1 identified two samples infected with P. falciparum species (261 and 282) and one (seronegative) with P. malariae (283). The last sample (331) presented a coinfection with P. malariae and P. ovale. Amplicons were excised from the gel, purified, and sequenced. Sequences confirmed the Plasmodium species identified with species-specific primers (data not shown).
Detection of alleles Detection of Plasmodium genome The first molecular screening to investigate the presence of the parasite in the candidate donor blood was per-
Allele-specific nested PCR was carried out to further characterize P. falciparum parasitemia (MSP-1, MSP-2, and GLURP). Two alleles of MSP-1 (MAD20 and K1), two of MSP-2 (3D7 and FC27), and only one of GLURP (GLURP)
TABLE 2. Characteristics of four asymptomatic, candidate blood donors carrying Plasmodium genome
ID 261
Age (years) 23
282 283 331
30 24 22
Sex Male
Time since exposure (months) 4
Anti-Plasmodium (S/CO) >26
18S rRNA 2 × 104
Mitochondrial gene 7.5 × 104
Male Male Male
36 60 60
>26 0.63* 13.34
2.5 × 104 4 × 103 8.5 × 104
4 × 104 4.5 × 103 3.5 × 104
Plasmodium genome (copies/mL)
Plasmodium species P.f. P.f. P.m. P.m. P.o.
Allele sequenced MSP-1 MAD 20 K1 ND ND ND
MSP-2 3D7 FC27 FC27 ND ND
GLURP GLURP GLURP ND ND
* Sample tested four times with malaria EIA kit with S/CO values ranging between 0.28 and 0.63. ND = not detected; P.f. = P. falciparum; P.m. = P. malariae; P.o. = P. ovale.
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Fig. 1. Species-specific nested PCR. The top line indicates species of Plasmodium. The second line indicates the sample number. Samples 261 and 282 show a band of 205 bp when amplified using specific P. falciparum primers, Sample 283 shows a 144-bp band specific for P. malariae, and Sample 331 shows a band of 144 bp when amplified using specific P. malariae primers and a 787-bp band specific for P. ovale. Molecular marker (M) is Hyperladder II (M2).
were amplified from Sample 261. In Sample 282 only one allele in MSP-2 (FC27) and one in GLURP (GLURP) were amplified. With Samples 283 and 331 that carried P. malariae and P. ovale, respectively, no alleles could be amplified possibly because of species-specific sequence divergence. Allele amplification might also be related to a parasite genome level of more than 104 copies/mL.
Donor history All four parasitemic donors were young men (
