1,721,100 research outputs found
Molecular epidemiology of asymptomatic Plasmodium species infections in Papua New Guinea
Full text linkAsymptomatic malaria infections present a major challenge to malaria control and elimination in endemic settings, as they contribute to maintaining malaria transmission but are missed during clinical surveillance. Little is known on the true extent and basic epidemiological parameters of asymptomatic malaria infections, such as the infection rate or the duration of infection. In case of P. vivax, an additional challenge is its ability to form dormant liver stages that evade diagnostic tools.
The aim of this thesis was to advance our basic understanding of asymptomatic P. falciparum and P. vivax infections and their relevance for malaria transmission. To this end, the extent of low-density malaria infections and the presence of gametocytes, i.e. the parasite stage responsible for human-to-mosquito transmission, is examined using highly sensitive molecular tools in Papua New Guinea and Tanzania. Longitudinal tracking of P. falciparum and P. vivax clones in a cohort study in Papua New Guinean children provides novel insights into the molecular epidemiology of malaria infections, allowing to explore small-scale geographical heterogeneity in malaria exposure, the relationship of exposure and clinical disease, the duration of untreated asymptomatic P. falciparum infections, and the contribution of P. vivax relapses from dormant liver stages to infection and disease
Infection and transmission dynamics of plasmodium vivax in Papua New Guinea
Full text linkBackground
Plasmodium vivax mainly affects Asia, Central and South America and is responsible for 350-450 millions cases per year, hence 25-40% of annual infections of malaria worldwide. In Papua New Guinea (PNG) P. vivax prevalence is among the highest worldwide. The biggest challenge for the control of P. vivax infections is the formation of dormant liver stages, which have the ability to relapse and cause disease even after successful clearance of asexual stages in the blood circulation. P. vivax strains in PNG relapse frequently and fast, and one of the highest doses of Primaquine is necessary to reduce the relapse rate in this region.
Aims and objectives
The overall aim of this thesis was to deepen our understanding of P. vivax molecular epidemiology, infections, transmission and its contribution to the infectious reservoir of P. vivax malaria in PNG. The specific objectives pursued can be summarized as follows. First, to assess P. vivax infection dynamics and transmission dynamics in semi-immune children and to contribute to the understanding of the biology of relapses by comparing two treatment arms. Second, to identify the best RNA sampling strategy for field surveys and improve molecular detection and quantification of P. falciparum and P. vivax gametocytes in field samples. Third, to develop genotyping tools to better study the dynamics of gametocyte production in multi-clone infections in consecutive samples.
Methods
For the above objectives, the laboratory work of this thesis was split into three parts.
(i) P. vivax PCR-positive blood samples from a treatment-to-reinfection survey were genotyped by the marker msp1F3 and analyzed for gametocyte carriage by pvs25 qRT-PCR. These samples were collected from 524 children aged 5-10 years and actively and passively followed-up over 8 months in PNG. The children were randomly attributed to two treatment arms consisting of blood-stage clearing drugs and either PQ or placebo. Genotyping data and gametocyte positivity were used to investigate the contribution of relapses to the infectious reservoir of P. vivax malaria. The molecular epidemiology of relapses was assessed by comparing the investment in gametocytogenesis, the molecular force of blood-stage infections (molFOB, number of distinct blood-stage infections per child and year) and the duration of infections in both trial arms using a Bayesian approach that allows for imperfect detection of blood-stage infections.
(ii) In a cross-sectional survey in 315 5-10 years old children from Papua New Guinea, the optimal strategy for gametocyte detection in field studies was assessed. Gametocytes need to be detected by amplification of stage-specific transcripts, which requires RNA-preserving blood sampling. The efficiency of sampling and storage on filter paper versus in solution were compared.
(iii) 111 archived DNA samples from PNG were genotyped by capillary electrophoresis for 6 length-polymorphic and gametocyte-specific markers and their diversity was determined. Serial dilution of gametocyte enriched culture of P. falciparum 3D7 strain permitted to establish the detection limit of each marker in vitro. The two most promising markers, pfg377 and pfs230, were then tested to simultaneously genotype paired RNA and DNA samples from 46 individuals from Burkina Faso.
Results
(i) In the randomized treatment-to-reinfection trial, children who received PQ showed 82% lower risk of experiencing at least one P. vivax infection. The estimated duration of infection, the parasite density and the probability of detection of individual infecting clone (detectability) was similar in both arms of the trial. Gametocyte densities and carriage in positive samples also did not differ between trial arms. Durations increased by age, whereas parasite density and detectability decreased by age. Over the 8 months follow-up, molFOB in placebo arm was 9.9 infections per child and year and almost three times as high as molFOB in PQ arm with 3.5. About 2 relapses were observed per each new infection at all villages. The increasing individual exposure of participants, as measured by molFOB, translated proportionally into an increased relapse burden.
(ii) In the cross-sectional survey in PNG, RNAprotect resulted in the highest proportion of gametocyte positive samples and gametocyte-specific transcript yields. The RNA-based detection resulted in a P. falciparum positivity of 24.1%; of these 40.8% carried gametocytes. P. vivax positivity was 38.4% with 38.0% carrying gametocytes. Most of the gametocyte carriers were also positive by DNA-based detection.
(iii) Analysis of genotyping markers of P. falciparum gametocytes revealed highest discrimination power for pfs230 with 18 alleles, followed by pfg377 with 15 alleles. When assays were performed in parallel on RNA and DNA, only 85% pfs230 samples and 60% pfg377 samples contained at least one matching genotype in DNA and RNA.
Conclusion
This thesis was the first attempt to fill the gaps in the knowledge of infection dynamics of relapses and new infections in semi-immune children. By comparing two trial arms results demonstrated how relapses and new infections have similar duration, parasite density, detectability and investment in gametocytogenesis. The mathematical model applied to genotyping data from the two treatment arms proved very useful to evaluate the infection dynamics of P. vivax. This was achieved despite a major complication of P. vivax molecular epidemiology, namely the unknown history of infections in our participants giving rise to relapses.
Data generated during the course of this thesis was used to highlight that relapses are the major contributors to P. vivax infections and transmission in PNG.
The molecular genotyping tools developed to study P. falciparum gametocyte transmission dynamics will open up new investigations of clone interaction, within-host competition, and clonal fitness. So far, very little is known on gametocyte dynamics in natural infections, where concurrent clonal infections might contribute to transmission equally or in competition with each other. This determines parasite recombination in mosquitoes, which in turn has major consequences for development of multi-locus drug resistance phenotypes or antigenic diversity
Molecular community surveillance of Plasmodium falciparum in 6 sites of different malaria endemicity in Tanzania
Full text linkMalaria prevalence estimates in Tanzania have been documented to decline in the recent years. National malaria data shows prevalence rates have been reduced by half from 18% in 2008 to 9% in 2012 (THMIS 2009; 2013). This decline has been attributed to countrywide implementation of malaria interventions, including indoor residual spraying (IRS), mass distribution of insecticide treated nets (ITNs), long-lasting ITNs and the use of artemisinin combination therapy (ACT), which aim at transmission reduction. Monitoring and evaluation of malaria interventions requires accurate information on the remaining malaria burden in the community. The rapid diagnostic tests (RDTs) and light microscopy (LM) are the commonly used diagnostic tools for parasite detection and estimation of parasite prevalence rates in many resource-limited areas such as Tanzania. However, owing to the low detection limit of LM and RDTs of about 50-100 parasites/µL, their ability to capture low density infections is limited (Moody 2002; MalEra 2011). The use of molecular techniques to detect malaria parasites has been advocated to improve the accuracy of parasite prevalence estimates, especially in moderate to low endemic settings. This is because in areas of reduced endemicity, most infections occur at low densities and cannot be detected by the routine diagnostic tools. With a detection limit of about 0.034 parasites/µL of blood, molecular diagnostics are more reliable for parasite detection. In Tanzania, most of the parasites prevalence estimates have been performed by LM and RDTs, hence the most of the low density infections may remain undetected. Thus this thesis aimed to assess the usefulness of diagnostic methods for epidemiological studies by comparing the performance of routine and molecular diagnostics in parasite and gametocytes detection in community samples from Tanzania. Furthermore, the thesis investigated the occurrence of submicroscopic infections at different endemic sites in Tanzania.
For the above aims we conducted community surveys at 6 sites in Tanzania between 2011 and 2013. These sites were classified as low (Iringa), low urban (Dar-Es Salaam), moderate (coastal Tanga and Lugoba) and high (Rufiji and Morogoro) endemic sites according to district prevalence data recorded by the Tanzania HIV and Malaria indicator surveys of 2008 (THMIS 2009): A total of 2046 volunteers of all ages with signed consent forms were recruited. Finger prick blood was drawn from all individuals for parasite detection by LM, RDT and 18S rRNA qPCR. Gametocytes were detected by both LM and qRT-PCR targeting transcripts of the gametocyte specific expressed marker pfs25.
Generally, high P. falciparum Prevalence rates of 20% (416/2046; 95% CI 18-22%) by 18S rRNA qPCR, 17% (349/2046; 95% CI 15.4-18.7%) by RDT and 11% (229/2046; 95% CI 9.8-12%) by LM were recorded in Tanzania. A substantial variation in molecular prevalence rates from geographically different sites was observed varying from 50% in the high endemic site, Rufiji, to 0.6% in the low endemic site, Iringa. These observed differences highlight the heterogeneity of transmission patterns in Tanzania attributed to geographical differences. Molecular parasite diagnostics unveiled that more than a half, 60% (249/416) of P. falciparum positive samples carried submicroscopic infections. Submicroscopic carriage was prevalent in all endemic settings. However, very few positive samples from areas of low and moderate endemicity impede a firm conclusion on the association of endemicity and submicroscopic carriage to be drawn from our samples. Molecularly determined Gametocyte prevalence was 15.3% (312/2046; 95% CI 13.6-16.8%) when data from all sites were combined. On the other hand, LM detected only 0.88% (18/2046; 95% CI 0.47-1.2%) of all samples implying only about 5% of the total gametocytes detected by molecular assay.
In conclusion molecular parasite detection revealed high parasite prevalence in Tanzania, such precise point prevalence molecular data obtained from community sampling may provide a more reliable basis of planning new tools of interventions or monitoring and evaluating the performance of existing tools in the country. Furthermore, high submicroscopic carriage of >50% in Tanzania, particularly in adults is key indicator of transmission potential of asymptomatic infections in Tanzania community and thus it is relevant for control strategies to focus on identifying submicroscopic carriers in order to successfully interrupt transmission
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Molecular epidemiology and population genetics of "Plasmodium vivax" in Papua New Guinea
Full text linkAbstract: In recent years a new focus has been put on malaria, and elimination and eradication of this disease has been brought back to the agenda. While Plasmodium falciparum causes most of the disease in Africa, P. vivax is predominant in malaria endemic regions in Latin America, Asia and the South Pacific. In the last 10 years increasing reports of severe disease and even mortality caused by P. vivax raised new awareness for this parasite. Forty percent of the world population lives in areas of P. vivax transmission, and severe disease is caused especially in infants and young children. P. vivax has the ability to form semi-dormant liver hypnozoites that can relapse and lead to clinical disease after an extended period of time. As most currently applied drugs are not effective against hypnozoites, they present a mayor obstacle towards malaria control.
In the lowlands of Papua New Guinea (PNG), malaria prevalence reaches levels similar to those in sub-Saharan Africa. Both P. falciparum and P. vivax are frequent, and P. vivax reaches prevalence higher than anywhere else in the world. Individuals from these regions are often co-infected by both parasite species. Multiple concurrent infections with different strains of P. falciparum or P. vivax are common. For this thesis I have genotyped over 3000 P. vivax positive blood samples collected in a cohort study of 264 children. These children, aged 1 to 4.5 years, were followed over 16 months and visited every 8 weeks for collection of two blood samples taken 24 hours apart. Additional samples were collected whenever the children presented sick to the local health centre. In parallel morbidity data was collected. P. vivax was genotyped using two size polymorphic markers, which both distinguished individual parasite clones. Genotyping data of P. falciparum clones was available from a previous study.
Extensive diversity of the genotyping markers and high multiplicity of infection (MOI, the number of co-occurring clones per carrier) was observed. Each P. vivax positive child carried a mean of 2.7 concurrent infections, mean MOI was nearly twice as high as P. falciparum multiplicity in the cohort. We did not detect a seasonal trend in MOI, and only a moderate increase of MOI with age, most pronounced in very young children up to 3 years. Most likely relapses increase P. vivax mean MOI, and they also lead to high MOI during seasons of less transmission.
The detection of parasitemia in field surveys is imperfect owing to parasite densities below or fluctuating around the detection limit of light microscopy or PCR. We have compared >1000 pairs of samples collected 24 hours apart. The collection of a second sample was found to have a limited effect on parasite prevalence: a single PCR missed only 6 to 9% of the combined parasite positivity. The proportion of individual clones missed was more pronounced. Depending on the marker, 19% or 31% of all P. vivax clones were missed when sampling on a single day. As a consequence, mean MOI increased from 2.7 to 3.4 when combining results from paired samples. Detectability does not differ between P. vivax and P. falciparum or between age groups. Thus comparisons of prevalence and MOI between species and age trends are not biased by detectability.
To investigate structuring of P. vivax population in PNG, we have typed a subset of samples with 13 additional markers. Allele frequencies and haplotypes were compared to those from samples collected at other sites in PNG. No differences were detected between parasite populations, most likely because of high gene flow between sites. This is in contrast to the situation in countries of lower endemicity, and differs from the earlier observed moderate structuring in P. falciparum populations from PNG.
In this cohort study, the incidence of P. vivax malaria decreased with age, whereas the incidence of P. falciparum peaked later at the age of 3.5 years. These contrasting trends are in line with results from other countries were both parasites are co-endemic. Differences in the parasite biology, e.g. a more limited reservoir of surface antigens of P. vivax compared to P. falciparum, might cause faster immunization against P. vivax and thus the different age trends in incidence. But also differences in transmission intensity might be responsible. Our typing of clones over an extended period of time allowed estimations of the molecular force of infection (molFOI), i.e. the number of infections per child per year. Each child acquired 14 P. vivax clones per year, but only 6 P. falciparum clones. molFOI did not change with age, suggesting that acquired immunity builds up gradually with each new clone, leading to a decrease in P. vivax incidence over time. This does not rule out that biological differences between P. vivax and P. falciparum also play a role.
PNG is among the countries with the poorest health infrastructure and the highest prevalence of malaria. P. vivax prevalence is in some regions as high as P. falciparum prevalence. High MOI and molFOI, as well as high levels of gene flow imply great resilience of P. vivax towards antimalarial interventions. Fast acquisition of semi-immunity leads to a large number of asymptomatic P. vivax carriers, potentially contributing to transmission, and control is further complicated by relapses, occurring in the blood weeks or months after transmission from the mosquito. The high proportion of P. vivax will be a major challenge towards malaria control and elimination in PNG. ---------- Zusammenfassung: In den letzten Jahren erhielt die Krankheit Malaria neue Aufmerksamkeit, und ihre Ausrottung in einzelnen Ländern oder gar weltweit wird wieder als realistisches Ziel angesehen. Plasmodium falciparum verursacht die meisten Krankheitsfälle in Afrika, dagegen ist P. vivax der vorherrschende Parasit in Malariagebieten in Lateinamerika, Asien und dem Südpazifik. In den letzten zehn Jahren haben Forschungen gezeigt, dass P. vivax häufig schwere Krankheit bis hin zum tödlichen Verlauf verursachen kann, insbesondere bei Kleinkindern. Dadurch geriet der Parasit nach Jahren mit wenig Beachtung erneut in den Fokus der Forschung. Vierzig Prozent der Weltbevölkerung leben in Gebieten mit P.vivax-Übertragung. P. vivax hat die Fähigkeit, Ruhestadien zu bilden, welche längere Zeit nach der Übertragung zum erneuten Krankheitsausbruch führen können. Die meisten der heute vorhandenen Medikamente sind wirkungslos gegen Leberstadien, darum sind diese eine grosse Hürde auf dem Weg zur Bekämpfung von Malaria.
In tief liegenden Gebieten von Papua Neuguinea erreicht die Malariaprävalenz Werte ähnlich der in Afrika südlich der Sahara. P. falciparum wie auch P. vivax sind häufig, und die P. vivax Prävalenz ist höher als irgendwo sonst auf der Welt. Bewohner dieser Gegenden sind häufig gleichzeitig mit beiden Spezies infiziert, sowie mit verschiedenen Linien derselben Art. Für diese Arbeit wurden die P. vivax Stämme in über 3'000 positiven Blutproben analysiert, die im Rahmen einer Kohortenstudie mit 264 Kindern gesammelt wurden. Diese Kinder im Alter von 1 bis 4.5 Jahren wurden während 16 Monaten alle 2 Monate besucht um im Abstand von 24 Stunden zwei Blutproben zu sammeln. Zusätzliche Proben wurden gesammelt, wann immer ein Kind eines der Gesundheitszentren aufsuchte. Parallel dazu wurden alle Erkrankungen der Kinder registriert. P. vivax Stämme wurden aufgrund von zwei molekularen Markern, bei welchen sich die Allele in der Grösse unterscheiden, typisiert. Im Rahmen einer vorhergehenden Studie wurden die P. falciparum Stämme in der Kohorte typisiert.
Die gewählten Marker wiesen eine hohe Diversität auf, und die Multiplizität (die Anzahl verschiedener Stämme pro Träger) war hoch. Jedes infizierte Kind trug im Schnitt 2.7 P. vivax Stämme, dieser Wert ist fast doppelt so hoch wie derjenige von P. falciparum. Es gab keine saisonalen Unterschiede in der Multiplizität, und nur einen geringen Anstieg der Multiplizität mit dem Alter der Kinder; dieser war am deutlichsten in sehr jungen Kindern unter 3 Jahren. Es ist davon auszugehen, dass wiederausbrechende Leberstadien die Multiplizität erhöhen, dies auch während Jahreszeiten mit geringerer Übertragung durch Mücken.
In Feldstudien ist die Detektion von Parasiten häufig unvollständig, d.h. die Dichte eines Teils der Parasiten liegt unter der Nachweisgrenze von Mikroskopie oder molekularen Methoden wie PCR. Wir haben über 1000 Blutproben-Paare verglichen, welche im Abstand von 24 Stunden gesammelt wurden. Die zusätzliche zweite Blutprobe hatte einen eher geringen Einfluss auf die Prävalenz, mittels PCR wurden nur 6 bis 9% der Proben falsch negativ diagnostiziert. Der Anteil der Stämme, die in einer einzelnen Proben nicht detektiert wurden, war dagegen ausgeprägter. Je nach Marker wurden 19 oder 31% aller P. vivax Stämme, die an mindestens einem der 2 Tage nachgewiesen wurden, am Tag 1 nicht entdeckt. Als Folge stieg die Multiplizität von 2.7 auf 3.4 wenn die Resultate von beiden Tagen kombiniert wurden. Die Wahrscheinlichkeit, einen Stamm zu detektieren, unterscheidet sich nicht zwischen P. vivax und P. falciparum oder in verschiedenen Altersklassen, folglich werden Vergleiche der Prävalenz und Multiplizität nicht beeinflusst.
Um die Populationsstruktur von P. vivax in Papua Neuguinea zu untersuchen haben wir einen Teil der Proben aus unserer Kohorte mit 13 weiteren molekularen Markern untersucht. Die Allele-Frequenzen und die Haplotypen wurden verglichen mit den-jenigen von drei anderen Orten in Papua Neuguinea. Es wurden keine Unterschiede zwischen diesen Parasitenpopulationen festgestellt, sehr wahrscheinlich aufgrund eines häufigen genetischen Austauschs zwischen den Populationen. Im klaren Gegensatz dazu wurden in Gebieten mit geringer P. vivax Prävalenz ausgeprägte Unterschiede zwischen Populationen beobachtet, und in früheren Studien wurde auch eine moderat ausgeprägte Populationsstruktur von P. falciparum in Papua Neuguinea festgestellt.
Die Inzidenz von P. vivax, also die Anzahl Erkrankungen, nahm in der Kohorte mit dem Alter deutlich ab, dagegen stieg die P. falciparum Inzidenz bis zu einem Alter von 3.5 Jahren. Diese gegensätzlichen Trends bestätigen Beobachtungen aus anderem Gebieten, wo beide Parasiten endemisch sind. Als mögliche Ursache kommen Unterschiede in der Biologie der Parasiten in Frage, z.B. eine kleinere Diversität von Oberflächenproteinen von P. vivax, wodurch die Immunisierung schneller verlaufen sollte. Auch sind Unterschiede in der Übertragungsrate von P. vivax und P. falciparum möglich. Unsere Typisierung von Stämmen über einen längeren Zeitraum ermöglicht, die „molekulare Infektionshäufigkeit“ zu bestimmen, also die Anzahl Infektionen pro Kind pro Jahr. Jedes Kind wurde im Schnitt mit über 14 P. vivax-Stämmen pro Jahr infiziert, aber nur mit etwa 6 P. falciparum-Stämmen. Die Infektionshäufigkeit änderte sich nicht mit dem Alter, dies lässt darauf schliessen, dass die Kinder mit der Zeit allmählich Immunität erlangten, und dadurch die Anzahl Erkrankungen zurückging. Dies schliesst natürlich nicht aus, dass auch Unterschiede in der Biologie eine Rolle spielt im Erlangen von Immunität.
Papua Neuguinea ist eines der Länder mit dem schlechtesten Gesundheitswesen und gleichzeitig einem hohen Auftreten von Malaria. Die Prävalenz von P. vivax ist manchen Gegenden gleich hoch wie diejenige von P. falciparum. Die hohe Multiplizität und Infektionshäufigkeit, wie auch der hohe Grad von genetischem Austausch zwischen Parasitenpopulationen, lässt auf einen hohen Widerstandsgrad gegenüber Massnahmen zur Malariakontrolle schliessen. Die schnelle Immunisierung gegen P. vivax führt dazu, dass es eine grosse Zahl von asymptomatischen Trägern gibt, die vermutlich immer noch zur Übertragung beitragen. Die Bekämpfung von P. vivax wird weiter erschwert durch die Leberstadien, welche noch Wochen oder Monate nach der Übertragung zu Krankheitsausbrüchen führen können. Der hohe Anteil von P. vivax an allen Malariafällen wird Papua Neuguinea vor grosse Herausforderungen stellen auf dem Weg zur Kontrolle und Elimination von Malaria
Infection Dynamics of Plasmodium falciparum in Papua New Guinea
Full text linkMalaria is one of the leading causes of illness and death in Papua New Guinea (PNG), mainly affecting children under 5 years of age. The current first line treatment for uncomplicated malaria is a combination therapy of sulfadoxine-pyrimethamine and chloroquine (CQ) or amodiaquine, however, frequent treatment failures have been reported shortly after its implementation. Drug resistance has mainly been associated with single nucleotide polymorphisms in five different genes. Different studies have indicated that mutations associated with drug resistance incur fitness costs to the parasite in absence of drug pressure. Among these are reports from different countries where a decreasing prevalence of mutations associated with CQ resistance was observed after CQ has been suspended as first line treatment. As it is experimentally difficult to estimate the fitness of Plasmodium parasites, a surrogate marker is needed to quantify fitness costs associated with drug resistance mutations. We hypothesised that the parasites’ survival within the human host can be used as surrogate marker for parasite fitness. In a pilot study we determined the drug resistance-associated haplotypes of parasite clones at 25 loci on 4 marker genes, and compared their prevalence between newly acquired and chronic infections. A reduced frequency of a 7-fold mutated haplotype and increased frequency of a 5-fold mutated haplotype in long term persistent infections indicated an impaired survival of highly mutated parasites and suggested that the duration of infections is a promising marker for parasite fitness that deserved further investigation. A further approach to define duration of infections more precisely was undertaken with samples from a longitudinal field survey in PNG. A cohort of 269 1-4.5 years old children was followed over a period of 16 months. This provided consecutive blood samples collected in 2-monthly follow-up visits plus a blood sample from each morbid episode. All samples were genotyped for the polymorphic marker gene merozoite surface protein 2 (msp2) in order to distinguish individual parasite clones within a host. The persistence of genotypes in consecutive blood samples of each child was determined. Unexpected high numbers of antimalarial treatments given in the course of this study led to a high turn-over rate of parasite clones and prevented the establishment of asymptomatic long-term infections. The shortage of long untreated periods in our study participants hampered the determination of a novel molecular parameter termed “duration of infection” for each individual parasite clone. This parameter could have been useful for modelling the transmission success and fitness of drug resistant versus drug sensitive parasite clones. We concluded that this approach requires a cohort of semi-immune individuals where treatment is given rarely, e.g. older children or adults, where P. falciparum infections less frequently cause morbid episodes. The high incidence of morbidity in our study cohort allowed us to investigate molecular parameters that have an impact on the development of a subsequent clinical episode. We found that in children >3 years, a higher multiplicity of infection (MOI) at baseline reduced the risk of a P. falciparum episode. These results are in agreement with previous reports and support the concept of premunition. Furthermore, we studied the impact of co-infecting Plasmodium species on MOI and found an increased MOI in the presence of a heterologous species. This can be explained by the observation of reduced parasite densities in mixed-species infections, leading to a decreased need for treatment and thus facilitating the accumulation of multiple clones. Our results provide further evidence for interactions among co-infecting P. falciparum clones as well as among co-infecting Plasmodium species. During the 16 months field survey all morbid episodes were treated with Coartem®. Analysis of msp2 genotyping results revealed an unexpected high number of Coartem® treatment failures. After confirmation of recrudescent parasites with 2 additional marker genes and exclusion of host genetic factors to be responsible for treatment failures, the most likely explanation for the frequently observed Coartem® failures was a combination of poor adherence to the treatment regimen and a lack of fat supplementation which is required for absorption of the drug. Our results disagree with findings from a clinical trial reporting Coartem® to be highly effective in PNG. In contrast, our observations from a non-trial setting highlight potential problems of Coartem® usage in routine clinical practice. In the course of this thesis, genotyping techniques for merozoite surface proteins (msp) 1 and 2 were optimized and applied. Using these high resolution typing techniques based on capillary electrophoresis, we investigated the effect of transmission intensity on diversity and complexity of msp1 and msp2 in samples from PNG and Tanzania. We observed a greater MOI and a greater number of distinct alleles in samples from Tanzania. Genetic diversity was greater for msp2 than for msp1. In both these areas of different malaria endemicity msp2 was found to be superior for distinguishing individual parasite clones. The probability of two infections carrying by chance the same msp2 allele was lower than this probability calculated for msp1. Based on the frequency distribution of msp2 alleles and on the distribution of observed numbers of infections, we estimated the true MOI adjusted for the probability of multiple infections sharing the same allele. For our high resolution typing technique this adjustment made little difference to the estimated mean MOI compared to the observed mean MOI. A central aim of this thesis was to measure molecular parameters of infection dynamics. These can be determined from genotyping longitudinal sets of samples. Some of these parameters were successfully determined: MOI, force of infection (FOI), and detectability. Other parameters such as natural elimination rates and persistence of infections could not be determined due to frequent treatments. We estimated detectability of parasite clones based on samples collected 24 hours apart and investigated its impact on the MOI and FOI. Imperfect detection of parasites occurs as a consequence of sequestration or when parasite densities fluctuate around the detection limit. We found that in our study participants detectability was high. This was likely an effect of high parasite densities in children of this age. The benefit of short-term sampling on measures of MOI and FOI was marginal. We concluded that in future studies carried out in this age group, taking repeated samples 24 hours apart has limited benefit and does not justify the additional costs, work load and discomfort for the study participants. This project contributed to our understanding of the infections dynamics of P. falciparum and the interactions between parasites clones and Plasmodium species. We provided further insights into determinants of malaria episodes and highlighted the potential usefulness of the parameter “duration of infection” as surrogate marker to estimate fitness costs of drug resistance. This thesis provided findings that are relevant for malaria control strategies and treatment guidelines
Methods for analysis of deep sequencing data from mixtures of Plasmodium falciparum clones or stage-specific transcriptomes
Full text linkMalaria is a life-threatening infectious disease caused by Plasmodium parasites transmitted to humans through bites of infected Anopheles mosquitos. An estimated 445,000 people die every year by an infection with Plasmodium parasites, most of them children living in sub-Saharan Africa. As a result of increased malaria control, the mortality was greatly reduced in the last decades. To develop new tools for elimination and to evaluate the impact of control, a good understanding of the epidemiology and biology of malaria parasites is required.
Studies of infection and transmission dynamics of Plasmodium parasites were greatly improved by distinguishing individual parasite clones and monitoring their infection dynamics over time. In regions with high transmission of Plasmodium parasites, individuals are often infected with several clones concurrently. Individual parasites clones can be identified by genotyping. The current standard method used for genotyping is amplification of highly length-polymorphic merozoite surface protein 2 (msp2) or other antigen genes followed by sizing of the amplicon by capillary electrophoresis (CE). The sensitivity to detect low-abundant clones (minority clones) of msp2-CE genotyping is however limited, resulting in an underestimation of multiplicity of infection (MOI). A shortfall of this genotyping method is that frequency of individual clones within a sample cannot be determined. This urges the search for new genotyping methods that rely on sequencing of genomic fragments with extensive single nucleotide polymorphism (SNP).
Improvement in next generation sequencing (NGS) technologies permitted the use of amplicon sequencing (Amp-Seq) in epidemiological studies. Genotyping by amplicon sequencing has a higher sensitivity to detect minority clones, can quantify the frequency of each clone within a sample, and allows the use of SNP polymorphic markers. In the frame of this thesis, a new Amp-Seq genotyping assay was developed, including known SNP polymorphic markers, and novel marker ‘cpmp’, as well as a bioinformatic analysis workflow. This genotyping assay was applied on field samples from a longitudinal study conducted in Papua New Guinea. A comparison to msp2-CE genotyping confirmed the higher sensitivity to detect minority clones by Amp-Seq genotyping method and showed a significant underestimation of MOI by classical size polymorphic marker. However, no significant increase in molecular force of infection (molFOI), i.e. number of new infections per individual per year, was observed.
Quantification of the frequency of individual clones in longitudinal samples permitted to infer multi-locus haplotypes. Multi-locus haplotypes increased discriminatory power of genotyping and robustly distinguished new infections from those detected in an individual earlier. For calculating the density of clones from multi-clone infections the within-host clone frequency is multiplied by parasitaemia of this infection determined by quantitative PCR. Density of individual parasites clones in multi-clone infections over time is a new parameter for epidemiological studies. It will permit to study the dynamics, and thus fitness, of parasite clones exposed to within-host competition or to acquired natural immunity.
NGS also gained great importance in gene expression studies of Plasmodium parasites in patient samples. Transcriptome studies are complicated by the mixture of different developmental stages present concurrently in samples collected from patients. Even in in vitro cultured samples after tight synchronisation or enrichment of a specific developmental stage, small fractions of other development stages are still found. This problem is of particular relevance for P. vivax, as the absence of continuous in vitro culture so far has hampered the study of isolated parasite stages. For example, the transcriptome of P. vivax gametocytes, one of the stages found in peripheral blood and infective to mosquitos, has not yet been described.
A solution for differentiating mixed transcription may come from deconvolution methods, which either infer the stage proportion in samples or stage-specific transcriptome signatures. A large selection of different deconvolution methods has been developed for the analysis of heterogeneous tissues, e.g. cancer tissues or hematopoietic cell, but these methods have rarely been applied to mixed stages of malaria parasites. The best suited combination of normalisation and deconvolution methods for analysis of RNA sequencing (RNA-Seq) data from mixed-stage samples of Plasmodium parasites was evaluated based on experimentally mixed highly synchronised blood stages. Normalisation by count per million and deconvolution with a negative binomial regression model followed by selection of genes with significant fold change resulted in the best agreement with transcriptomes as observed in single stages. This strategy can easily be transferred to Plasmodium field samples with known stage proportions. This analysis performed in cultured parasites of defined mixed stages served as proof-of-concept and confirmed that identification of stage-specific genes is feasible also in field samples, notably in species that cannot be cultivated, such as P. vivax.
NGS permits fundamentally new approaches to study Plasmodium parasites. This thesis presents a novel marker and data analysis platform for highly sensitive P. falciparum genotyping. Furthermore, a best practice workflow was identified to infer stage-specific gene expression from parasite infections consisting of mixed developmental stages. This provides a crucial tool for the analysis of gene expression data generated from Plasmodium field samples
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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