The frequency with which blood films were positive at given parasite densities measured by PCR were analysed. The poisson distribution was used to calculate the theoretical likelihood of diagnosis. Further in vitro studies used serial dilutions to prepare thick films from malaria cultures at known parasitaemia.
Even in expert hands, thick blood films were considerably less sensitive than might have been expected from the parasite numbers measured by quantitative PCR. In vitro work showed that thick films prepared from malaria cultures at known parasitaemia consistently underestimated parasite densities.
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Microscopy of thick blood films is the usual diagnostic test for Plasmodium falciparum malaria. Density is usually assessed by thick films, either by counting parasites per microscope field, or by counting parasites per hundred white blood cells [1]. Thick films contain several layers of red cells, whereas thin films contain a single layer of spread red cells. Thus, for a fixed number of microscope fields, thick films allow the microscopist to examine a larger number of red cells for the presence of parasites, and low parasitaemias can be more readily identified by thick film. Thin films are preferred to examine the morphology of parasites and determine species. Non-immune individuals may be unwell when one parasite or less is present in an entire thick film, requiring laborious, repeated examinations to make a diagnosis.
The sensitivity of thick blood films was studied using data obtained during these trials, compared this with quantitative PCR data, and further investigated these findings with in vitro studies.
Volunteers gave informed consent. Procedures were reviewed by OXREC (Oxford Research Ethics Committee), the local ethics committee, and were in accordance with the declaration of Helsinki (revised 1983). Twice daily blood samples were taken from day 6 until day 14, then daily until day 21. At least 100 high powered fields of a thick blood film were viewed and quantitative PCR performed on each sample. Volunteers were treated when a single parasite was seen by blood film, after the appearance of the parasite was confirmed by a second microscopist. Neither managing clinicians nor microscopists were aware of PCR data during the trial.
Giemsa staining was used for the first two sporozoite challenge studies, and Field's stain in coplin jars for the later two studies. The thick film was air dried in both methods. For giemsa staining, the film was stood in 5% Giemsa for 30 minutes, then washed gently in tap water and air dried. Field's stain was applied by dipping the slide into Field's stain A for 3 seconds, then into tap water for 3 seconds (with gentle agitation), into Field's stain B for a further 3 seconds and then washing gently in tap water to remove excess stain. The slide was then air dried for at least 30 minutes. The lead microscopist held a post in the London School for Hygiene and Tropical Medicine Clinical Parasitology Laboratory, the UK national reference laboratory, and others at the Medical Research Council, the Gambia. The lead microscopist examined slides produced by serial dilutions, blind to source. The average thick film uses 10 μl of blood spread over one thousand high powered fields, so the 100 high powered fields routinely examined during views 1 μl of blood [7].
This surprising finding suggested that a significant number of parasites were not visualized on a thick film despite being theoretically present in the original blood sample used to make the film. Results did not vary according to staining protocol (Giemsa or Field stain) or by microscipist. A similar density threshold for reliable diagnosis of malaria by thick film examination is reported elsewhere [8].
At each serial dilution (x axis), parasite densities seen by PCR (open circles) and parasite densities seen by thick blood film examination (filled circles) are both plotted on the y axis. The PCR readings are the result of a single experiment, the thick film readings are the results of two experiments. The solid line (least squares regression line for PCR results against densities known from serial dilution) is given by y = 0.98x + 0.07 (95% CI -0.12 to 0.27). The dotted line (regression line for thick film densities against serial dilution, ignoring the outlier) is given by y = 0.78x - 1.5 (95% CI -0.61 to -2.4) (y = 0.81x - 1.26, 95% CI - 0.07 to -2.5 including the outlier). Densities measured by thick film are therefore approximately 1 log lower than those calculated by serial dilution, whereas PCR readings match the serial dilution more closely.
Thin film and thick film parasite density estimates have been compared in previous studies. Although thick films are more sensitive than thin films, they significantly underestimated the parasite density in some studies [7, 9, 10], but not in others [11]. These previous studies lacked accuracy, since at high parasitaemias thick films are difficult to count accurately, and thin films cannot be counted accurately at low parasitaemias. In the study presented here, this difficulty was avoided by using serial dilution to provide known concentrations of parasites, and the accuracy of serial dilution was confirmed by quantitative real time PCR. PCR counts gene copy number, and this might have led to an over-estimate of parasite numbers when counting multi-nucleated schizonts. However, only mononucleated parasites are identified in peripheral blood at the low parasitaemias seen in this study, and in vitro cultures were synchronous.
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In this chapter it is proved that with thick-film technology, planar voltam-metric sensors can be produced. A working thick-film glucose sensor realized by the authors and a commercial blood glucose test strip based on a ferrocene biosensor manufactured by MediSense is discussed. A new range of planar voltammetric sensors has been developed by the authors using screen-printing technology. A new range of planar voltammetric sensors has been developed by the authors using screen-printing technology. The world-wide interest in publications indicates that thick-film technology applied to the development of planar voltammetric sensors is a worthy alternative to high-cost ic technology and labour-intensive manual production methods. Packaging of thick-film sensors is easy if the sensors are large. Due to the larger dimensions, a higher current is measured at the electrodes, so leakage currents are not that important. 2ff7e9595c