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    Results obtained with the Optimal® malaria rapid diagnosis test (MRDT) and polymerase chain reaction PCR on DNA extracted using the Optimal® MRDT. PV = Plasmodium vivax; Neg. = negative; PF = P. falciparum. In cases of a positive reaction for P. falciparum in the Optimal® MRDT, the possibility of a mixed infection with P. vivax, P. ovale, or P. malariae cannot be excluded (see case 4, which was a mixed infection of P. falciparum and P. vivax). Case 6 was negative with the Optimal® MRDT but positive for P. falciparum and P. vivax when DNA isolated with the Optimal® MRDT was used. This figure appears in color at www.ajtmh.org.

  • 1

    Kimura M, Kaneko O, Ishii A, Tanabe K, 1995. Amplification by polymerase chain reaction of Plasmodium falciparum DNA from Giemsa-stained thin blood smears. Mol Biochem Parasitol 70 :193–197.

    • Search Google Scholar
    • Export Citation
  • 2

    Ekala MT, Lekoulou F, Djikou S, Dubreuil G, Issifou S, Ntoumi F, 2000. Evaluation of a simple and rapid method of Plasmodium falciparum DNA extraction using thick blood smears from Gabonese patients. Bull Soc Pathol Exot 93 :8–11.

    • Search Google Scholar
    • Export Citation
  • 3

    Aubouy A, Carme B, 2004. Plasmodium DNA contamination between blood smears during Giemsa staining and microscopic examination. J Infect Dis 190 :1335–1337.

    • Search Google Scholar
    • Export Citation
  • 4

    Moody AH, Chiodini PL, 2002. Non-microscopic method for malaria diagnosis using OptiMAL IT, a second-generation dip-stick for malaria pLDH antigen detection. Br J Biomed Sci 59 :228–231.

    • Search Google Scholar
    • Export Citation
  • 5

    Makler MT, Palmer CJ, Ager AL, 1998. A review of practical techniques for the diagnosis of malaria. Ann Trop Med Parasitol 92 :419–433.

  • 6

    Cooke AH, Chiodini PL, Doherty T, Moody AH, Ries J, Pinder M, 1999. Comparison of a parasite lactate dehydrogenase-based immunochromatographic antigen detection assay (OptiMAL) with microscopy for the detection of malaria parasites in human blood samples. Am J Trop Med Hyg 60 :173–176.

    • Search Google Scholar
    • Export Citation
  • 7

    Kain KC, Wirtz RA, Fernandez I, Franke ED, Rodriguez MH, Lanar DE, 1992. Serologic and genetic characterization of Plasmodium vivax from whole blood-impregnated filter paper discs. Am J Trop Med Hyg 46 :473–479.

    • Search Google Scholar
    • Export Citation
  • 8

    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 polymerisation chain reaction. Mol Biochem Parasitol 61 :315–320.

    • Search Google Scholar
    • Export Citation
  • 9

    Pattanasin S, Proux S, Chompasuk D, Luwiradaj K, Jacquier P, Looareesuwan S, Nosten F, 2003. Evaluation of a new Plasmodium lactate dehydrogenase assay (OptiMAL-IT) for the detection of malaria. Trans R Soc Trop Med Hyg. 97 :672–674.

    • Search Google Scholar
    • Export Citation
  • 10

    Farnert A, Arez AP, Correia AT, Bjorkman A, Snounou G, do Rosario V, 1999. Sampling and storage of blood and the detection of malaria parasites by polymerase chain reaction. Trans R Soc Trop Med Hyg 93 :50–53.

    • Search Google Scholar
    • Export Citation
  • 11

    Chaorattanakawee S, Natalang O, Hananantachai H, Nacher M, Brockman A, Nosten F, Looareesuwan S, Patarapotikul J, 2003. Storage duration and polymerase chain reaction detection of Plasmodium falciparum from blood spot on filter paper. Am J Trop Med Hyg 69 :42–44.

    • Search Google Scholar
    • Export Citation

 

 

 

 

RECOVERY AND USE OF PLASMODIUM DNA FROM MALARIA RAPID DIAGNOSTIC TESTS

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  • 1 Medical Parasitology and Mycology Laboratory, Research Team EA 3593, Referral Hospital and School of Medicine, University of West Indies and French Guiana, Cayenne, French Guiana

The purpose of this study was to show that individual malaria rapid diagnosis tests (MRDTs) could also be used to isolate Plasmodium DNA for genetic studies. We extracted and amplified Plasmodium DNA using two commercial MRDT kits. Phenol/chloroform extraction followed by a nested polymerse chain reaction (PCR) can be used to identify Plasmodium falciparum and Plasmodium vivax from MRDTs. The PCR on MRDT-isolated DNA was more sensitive than antigen capture by MRDT. Satisfactory results were also obtained if older MRDT tests were used, even after long periods of storage at ambient temperature, with no special preservation.

The recovery of Plasmodium DNA from routine Giemsa-stained blood smears has been previously described.1,2 DNA extraction from blood smears is technically feasible and may be used to confirm a diagnosis or for retrospective and prospective molecular studies. However, there are risks of contamination during staining, microscopic examination, and storage of the smears.3 Malaria rapid diagnosis tests (MRDTs) are very useful in isolated places with no available microscope46 and the risk of contamination between tests is minimal. The purpose of this study was to demonstrate that MRDTs conducted for individual diagnosis could also be used, without particular preservation, to conserve Plasmodium DNA for later genetic studies.

Plasmodium DNA was extracted and amplified using two commercial MRDT kits: Optimal® (Diamed AG, Cressier sur Morat, Switzerland) and ICT Now® (Binax Inc., Portland, ME). These immunochromatographic tests are based on blood hemolysis and the subsequent reaction between monoclonal antibodies and antigens such as parasite lactate dehydrogenase (Optimal®) and histidine-rich protein II (ICT Now®). Capillary forces produced by blotting paper located at the top of a nitrocellulose membrane capture the blood sample. After migration on this membrane, colloidal gold red bands appear if the sample is positive.

We first tested the recovery of Plasmodium DNA. We extracted DNA from MRDT blotting paper. We tested three different extraction methods: the saponin/Chelex 100 method,7 the QIAamp DNA mini blood kit (Qiagen, Valencia, CA), and the phenol/chloroform method.2 For each extraction method, we used 10 MRDT positive samples with the same malaria-positive blood solution (50,000 Plasmodium falciparum/μL). According to the manufacturer’s instructions, 10–20 μL of blood should be used for the MRDT. The part of the blotting paper impregnated with blood was cut into small pieces (3 mm × 1 mm) and immersed in 600 μL of TNE buffer (0.15 M NaCl, 10 mM Tris HCl, 1 mM EDTA, pH 8) supplemented with 0.5% Triton X-100, 0.5% sodium dodecyl sulfate, and 5 mg/mL of proteinase K. Samples were incubated for one hour at 37°C, tubes were periodically shaken and centrifugated (4,000 × g for 30 seconds at ambient temperature). DNA was extracted from the supernatant and precipitated at −20°C in absolute ethanol containing 400 mM sodium acetate, pH 5.2. The nested polymerase chain reaction (PCR) used to identify Plasmodium species has been previously described;8 amplifications were performed in duplicate. We determined the size of the amplicons by agarose gel electrophoresis in the presence of 0.5 μg/mL of ethidium bromide. We found that the phenol/chloroform method was the most effective.

Five negative MRDT (three Optimal® and two ICT Now®) and 35 positive MRDT (25 Optimal® and 10 ICT Now®) samples, all initially used for routine individual diagnosis, were used. All patients were living in French Guiana and the MRDTs were obtained from various health centers in French Guiana. Microscopy was carried out to confirm the diagnosis of malaria in all positive cases (23 were positive for P. falciparum, 12 for P. vivax, and 5 were negative). The corresponding infected patients had parasite densities of 400–100,000 Plasmodium/μL.

We compared the sensitivity thresholds of MRDTs and PCR on MRDT-isolated DNA by serially diluting blood specimens from two patients, one infected with P. falciparum and another infected with P. vivax, in blood from a malaria-negative subject of the same ABO blood group. Seven dilutions, ranging from 50,000 to 1.2 parasites/μL of blood, were used for P. falciparum and P. vivax. Microscopy was used as a control for each dilution. The PCR on blood and on DNA isolated with the MRDT was performed in duplicate.

We investigated the effect of storage duration by also investigating 10 old MRDTs: two Optimal® MRDTs used six months earlier, three Optimal® MRDTs, and four ICT Now® MRDTs used 12 months earlier, and one ICT Now®MRDT used three years earlier. All 10 MRDTs had been stored in ambient conditions (temperature = 25–30°C, humidity = 70–85%).

The phenol/chloroform method gave the highest extraction yields in malaria-positive PCR tests for all (10 of 10) MRDT (Optimal) tests conducted with the same malaria-positive blood solution. With the Chelex and Qiagen extraction methods, only 4 of 10 and 2 of 10 tests, respectively, were positive. We therefore used the phenol chloroform method for all subsequent extractions.

The results obtained with MRDT initially used for routine individual diagnosis and for PCR on MRDT-extracted DNA are shown in Figure 1. Four of the five negative MRDT samples did not yield an amplicon for P. falciparum or P. vivax. The fifth showed positive amplification for P. falciparum and P. vivax (patient 6, Figure 1). Microscopy-based diagnosis was performed for all of these patients in peripheral health centers, where it was only possible to detect cases in which parasitemia levels exceeded 50–100 Plasmodium/μL. Twenty-three patients tested positive by both the Optimal® MRDT and PCR: 14 tested positive for P. falciparum, 8 for P. vivax, and one Optimal® MRDT was positive for P. falciparum, whereas the corresponding PCR amplification was positive for P. falciparum and P. vivax (patient 4, Figure 1). However, if a positive reaction for P. falciparum is obtained in the Optimal® MRDT, the possibility of a mixed infection with P. vivax, P. ovale, or P. malariae cannot be excluded.9 Two samples (one for P. falciparum and one for P. vivax) positive with the Optimal® MRDT showed negative results with the PCR. In these two cases, the person carrying out the test may not have followed the Optimal® MRDT protocol correctly. The blood on the blotting paper in these cases was much paler than would normally be expected (pale red rather than dark red).

We carried out PCR tests on DNA from 10 positive ICT Now® MRDT samples. The seven that were positive for P. falciparum were also positive for P. falciparum by PCR amplification. The three that were positive for P. vivax were also positive by P. vivax PCR amplification.

We compared the sensitivities of microscopy, the Optimal® MRDT, PCR on Optimal® MRDT-isolated DNA, and PCR on whole blood DNA (Table 1). Microscopy detected a minimum parasitemia level of approximately 5 parasites/μL of blood in our assays, whereas the examination result was considered negative only after analysis of a thick blood smear sample including at least 1,200 leukocytes (i.e., ~0.20 μL). The threshold for positive results was the same for P. falciparum and P. vivax amplifications. The Optimal® MRDT also showed no difference in the sensitivity thresholds for P. falciparum and P. vivax. The PCR on Optimal® MRDT-isolated DNA appeared to be 10 times more sensitive than Optimal® MRDT itself. This low sensitivity of the MRDT may account for the positive PCR results obtained with certain negative Optimal® MRDT results. The amplification of DNA from 15 μL of whole blood could be used to detect parasitemia levels as low as 3.7 × 10−5% (approximately 1.2 parasites/μL of blood in our assays). The sensitivities recorded here were similar to those reported in a previous study using the same amplification protocol and DNA extracted from whole blood.7 The detection threshold of the PCR on blood was 40 times higher than that of the control PCR on Optimal® MRDT-isolated DNA. Before migration on the nitrocellulose membrane, blood is lysed and diluted and the blotting paper does not capture the entire volume. Moreover, molecular examination of membranes confirmed that some parasite DNA remained on the membrane at the end of the test. This may account for the observed loss of sensitivity. Lysis and dilution buffer incubated with 10 μL of parasitized blood (50,000 P. falciparum/μL) were collected and used for amplification. Solutions were tested separately and together, with and without migration on the MRDT. No PCR inhibitors were found in the lysis or dilution buffers used for the Optimal® and ICT® MRDTs.

The MRDT can be stored in ambient conditions with no risk of contamination. Each test is stored in a plastic (Optimal® MRDT) or cardboard (ICT Now® MRDT) box, which is closed after use, making it possible to store the test safely for long periods. However, the amplification of Plasmodium DNA from fresh blood was 100 times more sensitive than amplification from dried blood on blotting paper.10 We investigated the impact of storage duration on DNA extraction yield. All amplifications of DNA from positive Optimal® MRDT samples and positive ICT Now® MRDT samples stored for 6–36 months before use were positive (10 kits tested). These results are consistent with those of a previous study showing that storage for four years does not affect DNA quality.11 Overall, our results suggest that positive MRDT (such as Optimal® and ICT Now®) samples are a good source of malaria parasite DNA for retrospective and prospective genetic studies without additional population sampling.

Table 1

Comparison of the sensitivities of microscopy, Optimal® MRDT, PCR from Optimal® MRDT, and PCR from blood with different levels of parasitemia*

Parasites/μL of blood
50,0005,0005005052.51.2
* Blood specimens were obtained from two patients, one infected with P. falciparum (Pf) and another infected with P. vivax (Pv), were serially diluted in blood from a malaria-negative subject from the same ABO blood group. Seven dilutions from 50,000 to 1.2 parasite/μL of blood were performed for P. falciparum and P. vivax. MRDT = malaria rapid diagnostic test; PCR = polymerase chain reaction.
† PCR was performed with approximately the same amount of blood used for MRDT (15 μL)
Microscopic examination for Pf+++++
Microscopic examination for Pv+++++
Optimal® with Pf+++
Optimal® with Pv+++
PCR for Pf from Optimal® with Pf++++
PCR for Pv from Optimal® with Pv++++
PCR for Pf from whole blood with Pf+++++++
PCR for Pv from whole blood with Pv+++++++
Figure 1.
Figure 1.

Results obtained with the Optimal® malaria rapid diagnosis test (MRDT) and polymerase chain reaction PCR on DNA extracted using the Optimal® MRDT. PV = Plasmodium vivax; Neg. = negative; PF = P. falciparum. In cases of a positive reaction for P. falciparum in the Optimal® MRDT, the possibility of a mixed infection with P. vivax, P. ovale, or P. malariae cannot be excluded (see case 4, which was a mixed infection of P. falciparum and P. vivax). Case 6 was negative with the Optimal® MRDT but positive for P. falciparum and P. vivax when DNA isolated with the Optimal® MRDT was used. This figure appears in color at www.ajtmh.org.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 74, 6; 10.4269/ajtmh.2006.74.941

*

Address correspondence to Vincent Veron or Bernard Carme, Laboratoire de Parasitologie et Mycologie Médicale, EA 3593, Centre Hospitalier de Cayenne, Rue des Flamboyants, BP 6006, F-97 306 Cayenne, French Guiana. E-mails: veronivi@voila.fr or b.carme@wanadoo.fr

Authors’ address: Vincent Veron and Bernard Carme, Laboratoire de Parasitologie et Mycologie Médicale, EA 3593, Centre Hospitalier de Cayenne, Rue des Flamboyants, BP 6006, F-97 306 Cayenne, French Guiana, E-mails: veronvi@voila.fr and b.carme@wanadoo.fr.

Acknowledgments: We thank Brice Rotureau and Mathieu Nacher for assistance in revising the manuscript.

Financial support: This study was supported by the Ministère Français de la Recherche Scientifique Research Team EA 3593.

REFERENCES

  • 1

    Kimura M, Kaneko O, Ishii A, Tanabe K, 1995. Amplification by polymerase chain reaction of Plasmodium falciparum DNA from Giemsa-stained thin blood smears. Mol Biochem Parasitol 70 :193–197.

    • Search Google Scholar
    • Export Citation
  • 2

    Ekala MT, Lekoulou F, Djikou S, Dubreuil G, Issifou S, Ntoumi F, 2000. Evaluation of a simple and rapid method of Plasmodium falciparum DNA extraction using thick blood smears from Gabonese patients. Bull Soc Pathol Exot 93 :8–11.

    • Search Google Scholar
    • Export Citation
  • 3

    Aubouy A, Carme B, 2004. Plasmodium DNA contamination between blood smears during Giemsa staining and microscopic examination. J Infect Dis 190 :1335–1337.

    • Search Google Scholar
    • Export Citation
  • 4

    Moody AH, Chiodini PL, 2002. Non-microscopic method for malaria diagnosis using OptiMAL IT, a second-generation dip-stick for malaria pLDH antigen detection. Br J Biomed Sci 59 :228–231.

    • Search Google Scholar
    • Export Citation
  • 5

    Makler MT, Palmer CJ, Ager AL, 1998. A review of practical techniques for the diagnosis of malaria. Ann Trop Med Parasitol 92 :419–433.

  • 6

    Cooke AH, Chiodini PL, Doherty T, Moody AH, Ries J, Pinder M, 1999. Comparison of a parasite lactate dehydrogenase-based immunochromatographic antigen detection assay (OptiMAL) with microscopy for the detection of malaria parasites in human blood samples. Am J Trop Med Hyg 60 :173–176.

    • Search Google Scholar
    • Export Citation
  • 7

    Kain KC, Wirtz RA, Fernandez I, Franke ED, Rodriguez MH, Lanar DE, 1992. Serologic and genetic characterization of Plasmodium vivax from whole blood-impregnated filter paper discs. Am J Trop Med Hyg 46 :473–479.

    • Search Google Scholar
    • Export Citation
  • 8

    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 polymerisation chain reaction. Mol Biochem Parasitol 61 :315–320.

    • Search Google Scholar
    • Export Citation
  • 9

    Pattanasin S, Proux S, Chompasuk D, Luwiradaj K, Jacquier P, Looareesuwan S, Nosten F, 2003. Evaluation of a new Plasmodium lactate dehydrogenase assay (OptiMAL-IT) for the detection of malaria. Trans R Soc Trop Med Hyg. 97 :672–674.

    • Search Google Scholar
    • Export Citation
  • 10

    Farnert A, Arez AP, Correia AT, Bjorkman A, Snounou G, do Rosario V, 1999. Sampling and storage of blood and the detection of malaria parasites by polymerase chain reaction. Trans R Soc Trop Med Hyg 93 :50–53.

    • Search Google Scholar
    • Export Citation
  • 11

    Chaorattanakawee S, Natalang O, Hananantachai H, Nacher M, Brockman A, Nosten F, Looareesuwan S, Patarapotikul J, 2003. Storage duration and polymerase chain reaction detection of Plasmodium falciparum from blood spot on filter paper. Am J Trop Med Hyg 69 :42–44.

    • Search Google Scholar
    • Export Citation
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