Detection of remaining Plasmodium DNA and gametocytes during follow up after curative malaria treatment among returned travellers in Norway

Christel Gill Haanshuus,Kristine Mørch

doi:10.1186/s12936-020-03367-6

Christel Gill Haanshuus, Kristine Mørch

Open Access

https://doi.org/10.1186/s12936-020-03367-6

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Abstract

BackgroundPCR can be positive weeks after effective malaria treatment, potentially leading to over diagnose of recrudescence and re-infections. The DNA detected by PCR post-treatment might stem from residuals of destroyed asexual parasites, or from live gametocytes. The objective of this clinical observational study was to describe the presence of positive PCR for Plasmodium falciparum and Plasmodium vivax in follow-up samples post-treatment from returned travellers, and the proportion of positive PCR due to gametocytes.MethodsWhole blood was collected during hospitalization and outpatient routine follow-up from 13 patients with imported malaria. DNA was extracted applying QIAamp DNA Blood Mini Kit, while mRNA was collected and extracted applying PAXgene Blood RNA Tubes and Kit. All DNA samples (N = 25) were analysed with a genus-specific cytb real-time SYBR PCR, and P. falciparum DNA samples (N = 22) were also analysed with a falciparum-specific varATS real-time TaqMan PCR. All the mRNA samples (N = 18) were analysed with both a genus-specific 18S rRNA RT-PCR and a gametocyte-specific Pfs25 (P. falciparum)/Pvs25 (P. vivax) RT-PCR.ResultsLatest samples were collected at day 1 (n = 2) and from day 11–54 (n = 11) after treatment. Genus DNA cytb PCR was positive up to 49 days after effective treatment, and 18S rRNA transcripts from active P. falciparum parasites were detectable for at least 11 days. Gametocyte-specific mRNA was detected at latest only two days after treatment. Among six patients with late positive PCR for P. falciparum, four had high parasitaemia at admittance (6–30%), while two had parasitaemia < 2%. Late detection of P. vivax was not found by any of the PCR methods.ConclusionsDNA-based PCR can be positive up to at least seven weeks after curative malaria treatment, potentially leading to over-diagnose of recrudescence and re-infections. Based on the observations in this study, it is unclear if the DNA origins from residuals of destroyed parasites or live gametocytes, warranting further investigations.

Highlights

Polymerase chain reaction (PCR) can be positive weeks after effective malaria treatment, potentially leading to over diagnose of recrudescence and re-infections
In Plasmodium falciparum, immature gametocytes sequester in internal host organs, in the bone marrow, and undergo five morphological development stages in the course of 7–12 days, only mature gametocytes circulate in the blood until they die of age [2, 3]
All the mRNA samples (N = 18) were analysed both with a genus-specific 18S rRNA reverse transcript (RT)-PCR, and a gametocyte-specific Pfs25 (P. falciparum)/Pvs25 (P. vivax) RT-PCR detecting female gametocytes, following previously published methods [14]

Summary

Introduction

PCR can be positive weeks after effective malaria treatment, potentially leading to over diagnose of recrudescence and re-infections. In Plasmodium falciparum, immature gametocytes sequester in internal host organs, in the bone marrow, and undergo five morphological development stages in the course of 7–12 days, only mature gametocytes circulate in the blood until they die of age [2, 3]. It has been estimated from mathematical modelling that P. falciparum gametocyte carriage may persist for up to 55 days after treatment [4]. They mature much faster than P. falciparum gametocytes, they are commonly present before symptoms and before parasite detection by microscopy, and live only for up to three days [2, 8]

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