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A Simple Method to Detect Plasmodium falciparum Directly from Blood Samples Using the Polymerase Chain Reaction
Abstract
We have developed a simple method for treating blood samples permitting direct detection of Plasmodium falciparum parasites using the P. falciparum-specific DNA probe pPF14 after polymerase chain reaction (PCR) amplification of target DNA sequences, and have compared this method with microscopic examination of thick blood smears. For PCR amplification, blood samples were lysed, then filtered onto filter paper. After drying, a piece of the filter paper was added directly to the PCR mixture for amplification. The presence of PCR products was detected using nonisotopically labeled probe. This method permits detection of less than than 10 parasites in a 20-microliters sample, and minimizes the effects of PCR inhibitors generally found in blood. Samples were collected from patients presenting at malaria clinics in Mae Sod and Mae Ramat, Thailand, and 626 were analyzed both by the PCR method and by conventional microscopy. Of these, 157 were positive both by microscopy and by PCR, while 297 were negative by both methods. PCR detected 131 samples that were negative by microscopy, and failed to detect 41 samples identified as positive by microscopy. All discordant samples were re-analyzed by microscopy and by PCR. Upon re-examination at a higher sensitivity, microscopy identified five additional positive cases, while six previously positive cases were found to be negative. This method of treating blood samples for PCR may also be useful in other diagnostic assays.
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“PCR methods to detect malaria infection were described more than two decades ago, and are generally more sensitive for detecting parasite DNA than thick BS is for detecting parasites [27,28].”
From Diagnosing malaria in pregnancy: An update (2012) · cites this paper as [27]
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“DNA extraction was performed as previously described (Barker et al., 1992).”
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“The cycling conditions were as follows: [1] denaturation at 94 C for 5 min, [2] 40 cycles 94 C for 1 min, [3] 1 min at 62 C, [4] 1 min at 72 C, and [5] incubation at 72 C for 5 min (Barker et al., 1992; Brown et al., 1992; Kain et al., 1993; Tirasophon et al., 1994).”
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“The blood samples for PCR were centrifuged at 1000 g for five minutes and plasma was discarded and cells were separately stored at –20°C.The stored samples were subsequently thawed and the DNA extraction and PCR assay was carried out as described by Barker and Snounou et al [4,5].”
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Metadata
- Authors
- Barker, R. H., Banchongaksorn, T., Courval, J. M., Suwonkerd, W., Rimwungtragoon, K., & Wirth, D. F.
- Affiliations
- Harvard University (US)
- ORCID
- Wirth, Dyann F.
- Venue
- The American Journal of Tropical Medicine and Hygiene
- Published
- 1992-04
- Volume
- 46
- Issue
- 4
- Pages
- 416-426
- ISSN
- 0002-9637 (ISSN-L)
- OpenAlex
- W2411788447
- Cited by
- 148
- References
- 0
- Citations by year
- 2023: 1 · 2021: 2 · 2020: 1 · 2019: 1 · 2018: 2 · 2016: 3 · 2015: 4 · 2014: 4 · 2013: 7 · 2012: 3
- FWCI
- 13.83
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- American Journal of Tropical Medicine and Hygiene (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Health Sciences › Medicine › Public Health, Environmental and Occupational Health › Malaria Research and Control
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
- Physical Sciences › Engineering › Biomedical Engineering › Biosensors and Analytical Detection
- Keywords
- Plasmodium falciparum, malaria diagnosis, PCR inhibitors, sensitivity and specificity
- MeSH terms
- Animals, Base Sequence, DNA, Humans, Molecular Sequence Data, Nucleic Acid Hybridization, Plasmodium falciparum, Sensitivity and Specificity, Blotting, Southern, DNA Probes, DNA, Protozoan, Polymerase Chain Reaction, Malaria, Falciparum
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