3 . Principle of nucleic acid sequence-based amplification. Primer 1, including a T7 promoter site, hybridises to the single-stranded target RNA (but not to DNA) and is extended by AMV-RT using free nucleotides from the reaction mixture. The produced cDNA:RNA hybrid is hydrolysed by RNAse H that destroys the original target RNA. Primer 2 anneals to the remaining single stranded DNA copy, is extended by AMV-RT and results in a double stranded DNA sequence with a double stranded, and thus functional, T7 promoter site. New RNA is transcribed from this sequence using T7 RNA polymerase. Each new RNA molecule re-enters the reaction until the primers are finished. From the time of primer depletion, amplification will depend on transcription only, resulting in a linear increase of amplicons. 

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... variability. This may be of benefit for the parasites as it may enhance survival in changing habitats and unfavourable mutations will not accumulate. Considering the evolutionary rule that fitness should be optimised in combination with other parasites from the same suborder that do rely much more on sexual stage development, it remains a question why P. falciparum produces relatively small numbers of transmission stages. Additionally we may question how transmission of the disease can be so efficient despite the relatively low production of transmission stages. Control of malaria has proven to be a difficult task. Despite intense control efforts, the disease remains endemic in many parts of the world. Moreover, malaria mortality is increasing due to lack of an effective vaccine, drug resistance of parasites, insecticide resistance of mosquito vectors and changing socio-economic and environmental factors [26]. As measures to prevent infection are not fully efficient, early diagnosis and drug treatment form the fundaments of malaria control strategies. Several types of drugs are available for malaria treatment, most of which are solely directed against the asexual parasite stages that cause disease symptoms. Some antimalarial drugs, such as artemisinin derivatives, also affect the sexual transmission stages [27, 28], while in contrast chloroquine and sulphadoxine-pyrimethamine (SP) induce development of gametocytes [29-32]. Such drug- induced effects on gametocytes may affect transmission and therefore also the spread of drug resistance. Evaluation of drug treatment policies is needed [33] to optimise the use of available drugs, to decrease the spread and development of drug resistance and to reduce the risk of compulsory switching to drugs that are still effective but more expensive. Accurate quantification of transmission stages is of great importance to monitor the spread of malaria over a population, including the spread of drug resistance. Furthermore, quantification of gametocytes is valuable for epidemiological studies and studies that evaluate control measures, especially those aimed at reducing the transmission of malaria. Currently, such studies mostly rely on microscopical detection of gametocytes. Microscopic analysis is routinely used for diagnosis of malaria but with its detection limit of 1-20 parasites and about 16 gametocytes per microlitre of blood is not always sensitive enough and too laborious for the purpose of these studies. Therefore, sexual stages are rarely included in drug resistance monitoring or in evaluation of interventions targeting transmission. As transmission is an essential part of the P. falciparum life cycle, with potential targets for control, it is of major importance to provide quantitative, sensitive tools to adequately study this part of the life cycle. Newly developed molecular techniques for P. falciparum detection based on DNA or RNA amplification are more sensitive than microscopy but also have their limitations and encounter problems with either quantification or stage-specific detection and are therefore not applied routinely. Polymerase Chain Reaction (PCR) can detect parasite DNA with a detection limit of up to 20 parasites/ml of blood [34-37] but is semi-quantitative and cannot accurately quantify the number of parasites present. Quantitative real-time PCR (Real-time Q-PCR) was developed for detection of Plasmodium falciparum 18S small subunit ribosomal RNA genes and can detect 20 parasites/ml of blood [38]. This technique however, requires relatively large quantities of venous blood (0.5 ml) and leukocytes need to be filtered from the blood samples to prevent processing problems, caused by otherwise gelatinous DNA preparations [38]. Additionally, PCR techniques based on amplification of DNA cannot detect various developmental stages of the parasites, including gametocytes. Reverse- transcriptase PCR (RT-PCR) avoids this problem and can detect various developmental stages in a quantitative and sensitive assay based on stage-specific gene expression [39]. However, since most Plasmodium genes have no introns (i.e. the DNA is identical to the RNA sequence), this method cannot unambiguously detect RNA without complete removal of genomic DNA from the sample. As DNAse treatment of the isolated RNA frequently does not provide absolute removal of genomic DNA, this affects accurate quantification using RT- PCR. Alternative techniques are needed for sensitive and accurate quantification of both total parasite load and gametocytes in large numbers of samples. One technique that may avoid the above-described problems is quantitative nucleic acid sequence-based amplification (QT-NASBA). QT-NASBA utilises the activities of three enzymes (AMV-RT, RNase H and T7 RNA polymerase) and two target-specific primers, of which one includes a T7 polymerase- promoter, for continuous and direct amplification of RNA molecules in a single mixture at a temperature of 41 ° C [40, 41] (figure 1.3). The low temperature ensures that primers only anneal to single stranded target RNA and prevents amplification of genomic DNA that may be present in the sample.This allows for specific detection of living, metabolically active cells and organisms. The amplification process is part of a system, which includes a nucleic acid isolation procedure, and powerful detection methodology. QT-NASBA can detect various developmental stages of the same organism through measurement of stage-specific gene- expression and can be performed on small (50 μ l) finger prick blood samples. The high accuracy, sensitivity and quick reaction times that allow for high throughput of samples, make QT-NASBA extremely suitable for large-scale epidemiological investigations. NASBA has been successfully used for the detection and quantification of several disease agents such as HIV-1 [42], Hepatitis A [43], B [44] and C viruses [45, 46]. The ability of NASBA to detect treatment efficacy has been shown for e.g. Mycobacteria [47, 48] and HIV-1 [49, 50]. Frequently, NASBA proved to be more sensitive and specific than other available molecular techniques, particularly for RNA targets [51]. QT-NASBA for detection of Plasmodium falciparum parasite loads has a detection limit of 10 parasites/ml of blood [52, 53]. This technique is based on specific amplification of 18S rRNA of the parasites and detection using electrochemiluminescence (ECL). Due to the possibility of QT-NASBA to accurately quantify RNA in a DNA background, it can be applied to detect various parasite developmental stages using their stage-specific differences in gene expression. For Plasmodium falciparum this may enable separate quantification of gametocytes in the blood sample. The primary objective was to study the prevalence and dynamics of P. falciparum sexual stages in the field. More detailed knowledge on this subject may benefit malaria control that aims to reduce transmission of malaria. A more sensitive tool for the detection of P. falciparum gametocytes, compared to standard microscopic detection, was required. Therefore, a molecular technique based on RNA amplification (QT-NASBA) was developed for quantification of P. falciparum parasites, sexually committed parasites and mature gametocytes [chapter 2]. Subsequently, these QT- NASBAs were improved with real-time detection using molecular beacon technology and compared to other techniques used for parasite quantification [chapters 3 and 5]. To facilitate the collection of samples for QT-NASBA during field studies, the protocol for storage of samples at -70oC was adapted for storage at -20oC [chapter 4]. The quantitative NASBAs were then used to study (sub)microscopic asexual and sexual stage parasite dynamics in adult volunteers that were experimentally infected with P. falciparum [chapter 2], in Kenyan children after drug treatment for uncomplicated symptomatic P. falciparum infection [chapter 5] and in individuals of all ages with asymptomatic P. falciparum in Burkina Faso [chapter 6]. The potential of submicroscopic gametocytaemia to be transmitted from the human host to the mosquito vector was determined with mosquito infections in the laboratory and confirmed with transmission studies in the field [chapter 7]. The infectivity of (sub)microscopic gametocytes to Anopheles gambiae was studied in the field in Kenyan children after antimalarial drug treatment [chapter 8]. The results presented in this thesis are summarised and discussed in chapter ...