PCR detection of Plasmodium falciparum in human urine and saliva samples

Abstract

Background: 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.

Methods: Urine 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 discussion: Malaria infections exhibited primarily low-grade parasite densities, with a geometric mean of 775 asexual parasites/mul. 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 2x 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.

Conclusion: P. 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.

Figure 1

Showing P. falciparum MSP2 amplicon from saliva (173Qs), urine (173Qu, 173u) and blood (173b) samples of patient 173. Qs and Qu denote saliva and urine samples extracted by Qiagen^® commercial kit (crude cell lysate protocol), while u denotes whole urine sample extracted by the Chelex method. Qs-, Qu-, u- and b-, denote amplicon from corresponding extracts of saliva, urine and blood donated by thick film negative healthy control. 3D7, amplicon from positive control laboratory standard; M, 100 bp DNA ladder. Extraction of urine replicate sample 173u was performed on 20.02.06, while Qiagen extractions were carried out on 09.02.06. Identical MSP2 alleles are apparent in amplicon from urine, saliva and blood samples of patient 173, as compared to 3D7 lab standard.

Figure 2

Amplicon from saliva, urine and blood extracts of field samples 216, 34 and 210. Matching MSP2 alleles are seen in saliva (Qs) and blood (b) amplicon from each individual. In contrast, between-patient polymorphic differences are evident, reflecting diverse infections. Urine samples from these patients did not amplify, except that of 210 (210Qu). Qs, Qu denote Qiagen saliva and urine extracts, respectively, by crude lysate approach; Cu denotes Qiagen urine extracts, by cultured animal cells protocol; Du denotes Chelex direct extraction on whole urine. Qs-, Cu-, b- were corresponding extracts of saliva, urine and blood samples from healthy negative control, while 3D7 was positive control laboratory standard.

Figure 3

Xmn I digest of DHFR F-M4 amplicon from saliva, urine and blood samples of patients 7, 15, 17 and 28. Qs, Qu denotes saliva and urine samples extracted by Qiagen^® kit (crude cell lysate protocol). Blood samples were extracted on different days by Chelex from dry filter paper blots. U is undigested DHFR F-M4 amplicon. Identical restriction fragment patterns are evident on different extracts from each patient. Patients 7, 15 and 28 were infected with parasites carrying the mutated (antifolate resistance associated) allele (coding for DHFR Arg-59). Patient 17 carried mixed wild type (Cys-59) and mutated (Arg-59) variants, of which the mutated form was barely discernible in the urine amplicon. Incomplete digestion is present in some amplications, with a band at 326 bp.