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Detection of a substantial number of sub-microscopic Plasmodium falciparum infections by polymerase chain reaction: a potential threat to malaria control and diagnosis in Ethiopia
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
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2013 (English)In: Malaria Journal, ISSN 1475-2875, Vol. 12, 352- p.Article in journal (Refereed) Published
Abstract [en]

Background: Prompt and effective malaria diagnosis not only alleviates individual suffering, but also decreases malaria transmission at the community level. The commonly used diagnostic methods, microscopy and rapid diagnostic tests, are usually insensitive at very low-density parasitaemia. Molecular techniques, on the other hand, allow the detection of low-level, sub-microscopic parasitaemia. This study aimed to explore the presence of sub-microscopic Plasmodium falciparum infections using polymerase chain reaction (PCR). The PCR-based parasite prevalence was compared against microscopy and rapid diagnostic test (RDT). Methods: This study used 1,453 blood samples collected from clinical patients and sub-clinical subjects to determine the prevalence of sub-microscopic P. falciparum carriages. Subsets of RDT and microscopy negative blood samples were tested by PCR while all RDT and microscopically confirmed P. falciparum-infected samples were subjected to PCR. Finger-prick blood samples spotted on filter paper were used for parasite genomic DNA extraction. Results: The prevalence of sub-microscopic P. falciparum carriage was 19.2% (77/400) (95% CI = 15.4-23.1). Microscopy-based prevalence of P. falciparum infection was 3.7% (54/1,453) while the prevalence was 6.9% (100/1,453) using RDT alone. Using microscopy and PCR, the estimated parasite prevalence was 20.6% if PCR were performed in 1,453 blood samples. The prevalence was estimated to be 22.7% if RDT and PCR were used. Of 54 microscopically confirmed P. falciparum-infected subjects, PCR detected 90.7% (49/54). Out of 100 RDT-confirmed P. falciparum infections; PCR detected 80.0% (80/100). The sensitivity of PCR relative to microscopy and RDT was, therefore, 90.7% and 80%, respectively. The sensitivity of microscopy and RDT relative to PCR was 16.5 (49/299) and 24.2% (80/330), respectively. The overall PCR-based prevalence of P. falciparum infection was 5.6- and 3.3 fold higher than that determined by microscopy and RDT, respectively. None of the sub-microscopic subjects had severe anaemia, though 29.4% had mild anaemia (10-11.9 g/dl). Conclusions: Asymptomatic, low-density malaria infection was common in the study area and PCR may be a better tool for measuring Plasmodium prevalence than microscopy and RDT. The inadequate sensitivity of the diagnostic methods to detect substantial number of sub-microscopic parasitaemia would undoubtedly affect malaria control efforts, making reduction of transmission more difficult. RDT and microscopy-based prevalence studies and subsequent reports of reduction in malaria incidence underestimate the true pictures of P. falciparum infections in the community. PCR, on the other hand, seems to have reasonable sensitivity to detect a higher number of infected subjects with low and sub-microscopic parasite densities than RDTs or microscopy.

Place, publisher, year, edition, pages
2013. Vol. 12, 352- p.
Keyword [en]
Sub-microscopic carriage, Asymptomatic malaria, Microscopy, RDT, PCR, Ethiopia
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-216887DOI: 10.1186/1475-2875-12-352ISI: 000329097000001OAI: diva2:691332
Available from: 2014-01-27 Created: 2014-01-27 Last updated: 2015-01-22Bibliographically approved
In thesis
1. Dynamics of Resistant Plasmodium falciparum Parasites
Open this publication in new window or tab >>Dynamics of Resistant Plasmodium falciparum Parasites
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Persistence of drug resistant Plasmodium falciparum is a major problem to management and control malaria in endemic areas. The focus of this thesis was to study the dynamics of resistant P. falciparum parasites. The study was performed in two African countries: 1) Sudan: Asar village in eastern Sudan, here we examined the persistence of drug sensitive and resistant P. falciparum genotypes among individuals with single-clone and multiple clones infection during the dry season. We genotyped microsatellite loci in the vicinity of the dihydrofolate reductase gene (dhfr) and the dihydropteroate synthase gene (dhps). Microsatellite investigation showed that asymptomatic parasitemia persisted in some patients for several months throughout the dry season and into the next transmission season. In some samples mixed infections were detected, and we noted several cases where the microsatellite haplotype varied from month to month, suggesting turnover of different parasite populations in the blood. This demonstrates that even during asymptomatic infections there can be dynamics within the parasite population in an individual. In addition, we calculated the parasite density throughout the dry season to the next transmission season by using allele-specific quantitative PCR. Parasite density during the dry season fluctuated, but was generally lower than in the first transmission season. A significant difference (P<0.05) between dry and first transmission season was found in regard to the parasite density, whereas no significant difference was observed when dry and second transmission season were compared (P>0.05). 2) Ethiopia: West Arsi zone, one of the malaria endemic zones of the Oromia region. In the first study we determined the prevalence of asymptomatic malaria carriages from November-December 2012. According to PCR the prevalence of sub-microscopic P. falciparum carriage was 19.2%, microscopy-based prevalence was 3.7% while the prevalence was 6.9% using RDT. Based on this, PCR was considered a better tool for measuring Plasmodium prevalence than microscopy and RDT. A second study addressed the genetic diversity of chloroquine resistance (CQR) in P. falciparum by analysing four microsatellite markers in and around the pfcrt gene. Although CQ was withdrawn for more than a decade, 100% of the parasites still carried the Pfcrt K76T mutation. Only the CVIET haplotype was identified. Based on combinations of MS markers, seven different Ethiopian CQR variants (E1-E7) were identified. Both intronic and MS flanking the pfcrt gene showed low levels of diversity.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. 49 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1020
National Category
Other Basic Medicine
Research subject
Molecular Genetics
urn:nbn:se:uu:diva-230224 (URN)978-91-554-9007-2 (ISBN)
Public defence
2014-11-12, C8:301, BMC, Husargatan 3, Uppsala, 09:15 (English)
Available from: 2014-10-22 Created: 2014-08-20 Last updated: 2015-01-22

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