Abstract
BackgroundCurrent detection or screening for malaria infection necessitates drawing blood by fingerprick or venipuncture, which poses risks and limitations for repeated measurement. This study presents PCR detection of Plasmodium falciparum in human urine and saliva samples, and illustrates this potential application in genotyping malaria infections.MethodsUrine and saliva were obtained from 47 thick film positive and 4 negative individuals one day after collection of blood slides and filter paper blood spots. P. falciparum DNA was extracted from blood, urine and saliva, in separate groups, using the Chelex method or Qiagen DNEasy® kit (urine and saliva only). Blood, urine and saliva extracts were subjected to PCR in separate batches. Amplicons from the various sample types were examined for MSP2 polymorphisms and restriction fragment patterns on DHFR amino acid codon 59.Results and discussionMalaria infections exhibited primarily low-grade parasite densities, with a geometric mean of 775 asexual parasites/μl. Regularly matching polymorphic MSP2 genotypes were found between the corresponding urine, saliva and peripheral blood amplicons of each individual, with different inter-individual polymorphic genotypes. Amplicon yields were significantly dependent on DNA extraction method, parasite density and primer set (p < 0.001). A Qiagen® kit extraction had more than 2× higher amplicon yield than the Chelex method, for both urine and saliva. Amplicon yields were 1.6 fold higher from saliva than urine. For each unit increase in log parasite density, the probability of amplicon enhanced 1.8 fold. Highest amplicon yields were obtained from the primer set with the shortest PCR product.ConclusionP. falciparum infection is detectable by PCR on human urine and saliva samples. Subject to further refinement of extraction technique and amplicon yields, large-scale malaria parasite screening and epidemiological surveys could be possible without the need to collect blood and use of needles or sharps.
Highlights
Current detection or screening for malaria infection necessitates drawing blood by fingerprick or venipuncture, which poses risks and limitations for repeated measurement
The present study describes the use of urine and saliva samples for PCR detection of Plasmodium falciparum infection and illustrates potential application in genotyping malaria parasites
MSP2 genotyping of P. falciparum on urine and saliva samples Saliva, urine and blood extracts were subjected to nested PCR in separate batches using MSP2 family-specific primers and loaded in adjacent lanes for each patient during electrophoresis
Summary
Current detection or screening for malaria infection necessitates drawing blood by fingerprick or venipuncture, which poses risks and limitations for repeated measurement. This study presents PCR detection of Plasmodium falciparum in human urine and saliva samples, and illustrates this potential application in genotyping malaria infections. Use of efficient molecular tools is becoming widely integrated to afford more informative and comparable results in programmes such as national drug therapeutic efficacy monitoring [4,5]. Molecular genotyping by national drug therapeutic efficacy monitoring programmes to distinguish post-treatment recrudescence from new infections is becoming universal standard [6]. Tubes, while some were blotted onto filter paper cuttings. Additional blood samples were collected on filter paper. Separate drying boards were used for urine, saliva and blood filter paper blots
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