Dataset Information

Limited Degradation of the Plasmodium falciparum Gametocyte Marker pfs25 mRNA Exposed to Tropical Temperatures: Considerations for Malaria Transmission Field Studies.

ABSTRACT: An important aspect of many malaria molecular epidemiology and transmission studies is RNA-based detection of gametocytes. Ensuring RNA stability represents a challenge in tropical, resource-limited environments, as RNA may quickly degrade when samples are not preserved under adequate conditions. This study investigated the degradation of pfs25 messenger RNA (mRNA), the most widely used Plasmodium falciparum gametocyte marker, in whole blood spiked with cultured P. falciparum gametocytes, exposed to different temperatures for up to 48 hours, and collected with different anticoagulants. The levels of pfs25 mRNA were similar between samples stored at 4°C and 30°C for up to 48 hours before stabilization with RNAprotect (Qiagen, Hilden, Germany). We observed that pfs25 mRNA in heparin-collected blood degraded less than that in ethylenediaminetetraacetic acid (EDTA)-collected blood over the 48-hour period. For field studies aiming for P. falciparum gametocyte detection, immediate stabilization of blood samples is not necessary, as the pfs25 transcript is relatively stable, more so in heparin than EDTA collection tubes.

SUBMITTER: Waltmann A

PROVIDER: S-EPMC4824233 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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Limited Degradation of the Plasmodium falciparum Gametocyte Marker pfs25 mRNA Exposed to Tropical Temperatures: Considerations for Malaria Transmission Field Studies.

Waltmann Andreea A Karl Stephan S Chiu Chris C Mueller Ivo I

The American journal of tropical medicine and hygiene 20160208 4

An important aspect of many malaria molecular epidemiology and transmission studies is RNA-based detection of gametocytes. Ensuring RNA stability represents a challenge in tropical, resource-limited environments, as RNA may quickly degrade when samples are not preserved under adequate conditions. This study investigated the degradation of pfs25 messenger RNA (mRNA), the most widely used Plasmodium falciparum gametocyte marker, in whole blood spiked with cultured P. falciparum gametocytes, expose ...[more]

PMID: 26856913

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Project description:BackgroundA goal of malaria epidemiological interventions is the detection and treatment of parasite reservoirs in endemic areas-an activity that is expected to reduce local transmission. Since the gametocyte is the only transmissible stage from human host to mosquito vector, this study evaluated the pre and post presence of gametocytes during a mass screening and treatment (MST) intervention conducted during 2013 in East Nusa Tenggara, Indonesia.MethodsRT-qPCR targeting pfs25 and pvs25 transcripts-gametocyte molecular markers for Plasmodium falciparum and Plasmodium vivax, respectively, was performed to detect and quantify gametocytes in blood samples of P. falciparum and P. vivax-infected subjects over the course of the MST study. The presence of both asexual and sexual parasites in microscopic and submicroscopic infections was compared from the start and end of the MST, using proportion tests as well as parametric and non-parametric tests.ResultsParasite prevalence remained unchanged for P. falciparum (6% = 52/811 versus 7% = 50/740, p = 0.838), and decreased slightly for P. vivax (24% = 192/811 versus 19% = 142/740, p = 0.035) between the MST baseline and endpoint. No significant difference was observed in gametocyte prevalence for either P. falciparum (2% = 19/803 versus 3% = 23/729, p = 0.353, OR = 1.34, 95%CI = 0.69-2.63), or P. vivax (7% = 49/744 versus 5% = 39/704, p = 0.442, OR = 0.83, 95%CI = 0.52-1.31). Even though there was an insignificant difference between the two time points, the majority of parasite positive subjects at the endpoint had been negative at baseline (P. falciparum: 66% = 29/44, P. vivax: 60% = 80/134). This was similarly demonstrated for the transmissible stage-where the majority of gametocyte positive subjects at the endpoint were negative at baseline (P. falciparum: 95% = 20/21, P. vivax: 94% = 30/32). These results were independent of treatment provided during MST activities. No difference was demonstrated in parasite and gametocyte density between both time points either in P. falciparum or P. vivax.ConclusionIn this study area, similar prevalence rates of P. falciparum and P. vivax parasites and gametocytes before and after MST, although in different individuals, points to a negligible impact on the parasite reservoir. Treatment administration based on parasite positivity as implemented in the MST should be reevaluated for the elimination strategy in the community. Trial registration Clinical trials registration NCT01878357. Registered 14 June 2013, https://www.clinicaltrials.gov/ct2/show/NCT01878357.

Project description:BackgroundMalaria caused by Plasmodium falciparum remains a serious global public health challenge especially in Africa. Interventions that aim to reduce malaria transmission by targeting the gametocyte reservoir are key to malaria elimination and/or eradication. However, factors that are associated with gametocyte carriage have not been fully explored. Consequently, identifying predictors of the infectious reservoir is fundamental in the elimination campaign.MethodsWe cultured P. falciparum NF54 gametocytes (to stage V) and prepared crude gametocyte extract. Samples from a total of 687 participants (aged 6 months to 67 years) representing two cross-sectional study cohorts in Kilifi, Kenya were used to assess IgG antibody responses by ELISA. We also analyzed IgG antibody responses to the blood-stage antigen AMA1 as a marker of asexual parasite exposure. Gametocytemia and asexual parasitemia data quantified by microscopy and molecular detection (QT-NASBA) were used to determine the relationship with antibody responses, season, age, and transmission setting. Multivariable logistic regression models were used to study the association between antibody responses and gametocyte carriage. The predictive power of the models was tested using the receiver operating characteristic (ROC) curve.ResultsMultivariable logistic regression analysis showed that IgG antibody response to crude gametocyte extract predicted both microscopic (OR=1.81 95% CI: 1.06-3.07, p=0.028) and molecular (OR=1.91, 95% CI: 1.11-3.29, p=0.019) P. falciparum gametocyte carriage. Antibody responses to AMA1 were also associated with both microscopic (OR=1.61 95% CI: 1.08-2.42, p=0.020) and molecular (OR=3.73 95% CI: 2.03-6.74, p<0.001) gametocytemia. ROC analysis showed that molecular (AUC=0.897, 95% CI: 0.868-0.926) and microscopic (AUC=0.812, 95% CI: 0.758-0.865) multivariable models adjusted for gametocyte extract showed very high predictive power. Molecular (AUC=0.917, 95% CI: 0.891-0.943) and microscopic (AUC=0.806, 95% CI: 0.755-0.858) multivariable models adjusted for AMA1 were equally highly predictive.ConclusionIn our study, it appears that IgG responses to crude gametocyte extract are not an independent predictor of gametocyte carriage after adjusting for AMA1 responses but may predict gametocyte carriage as a proxy marker of exposure to parasites. Serological responses to AMA1 or to gametocyte extract may facilitate identification of individuals within populations who contribute to malaria transmission and support implementation of transmission-blocking interventions.

Project description:BackgroundFor future eradication strategies of malaria it is important to control the transmission of gametocytes from humans to the anopheline vector which causes the spread of the disease. Sensitive, non-invasive methods to detect gametocytes under field conditions can play a role in monitoring transmission potential.MethodsMicroscopically Plasmodium falciparum-positive patients from Jimma, Ethiopia donated finger-prick blood, venous blood, saliva, oral mucosa and urine samples that were spotted on filter paper or swabs. All samples were taken and stored under equal, standardized conditions. RNA was extracted from the filter paper and detected by real-time QT-NASBA. Pfs16-mRNA and Pfs25-mRNA were measured with a time to positivity to detect gametocyte specific mRNA in different gametocyte stages. They were compared to 18S-rRNA, which is expressed in all parasite stages. Results were quantified via a known dilution series of artificial RNA copies.ResultsNinety-six samples of 16 uncomplicated malaria patients were investigated. 10 (66.7%) of the slides showed gametocyte densities between 0.3-2.9 gametocytes/?l. For all RNA-targets, molecular detection in blood samples was most sensitive; finger-prick sampling required significantly smaller amounts of blood than venous blood collection. Detection of asexual 18S-rRNA in saliva and urine showed sensitivities of 80 and 67%, respectively. Non-invasive methods to count gametocytes proved insensitive. Pfs16-mRNA was detectable in 20% of urine samples, sensitivities for other materials were lower. Pfs25-mRNA was not detectable in any sample.ConclusionsThe sensitivity of non-invasively collected material such as urine, saliva or mucosa seems unsuitable for the detection of gametocyte-specific mRNA. Sensitivity in asymptomatic carriers might be generally even lower. Finger-prick testing revealed the highest absolute count of RNA copies per ?L, especially for Pfs25-mRNA copies. The method proved to be the most effective and should preferably be applied in future transmission control and eradication plans. A rapid test for gametocyte targets would simplify efforts.

Dataset Information

Limited Degradation of the Plasmodium falciparum Gametocyte Marker pfs25 mRNA Exposed to Tropical Temperatures: Considerations for Malaria Transmission Field Studies.

Publications

Limited Degradation of the Plasmodium falciparum Gametocyte Marker pfs25 mRNA Exposed to Tropical Temperatures: Considerations for Malaria Transmission Field Studies.

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