RESUMEN
BACKGROUND: Thick blood films are routinely used to diagnose Plasmodium falciparum malaria. Here, they were used to diagnose volunteers exposed to experimental malaria challenge. METHODS: 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. RESULTS: 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. CONCLUSION: It appears large numbers of parasites are lost during staining. This limits their sensitivity, and leads to erroneous estimates of parasite density.
Asunto(s)
Sangre/parasitología , Malaria Falciparum/diagnóstico , Malaria Falciparum/parasitología , Plasmodium falciparum/aislamiento & purificación , Animales , Humanos , Malaria Falciparum/sangre , Microscopía , Sensibilidad y EspecificidadRESUMEN
The demand for an effective malaria vaccine is high, with millions of people being affected by the disease every year. A large variety of potential vaccines are under investigation worldwide, and when tested in clinical trials, researchers need to extract as much data as possible from every vaccinated and control volunteer. The use of quantitative real-time polymerase chain reaction (PCR), carried out in real-time during the clinical trials of vaccines designed to act against the liver stage of the parasite's life cycle, provides more information than the gold standard method of microscopy alone and increases both safety and accuracy. PCR can detect malaria parasites in the blood up to 5 days before experienced microscopists see parasites on blood films, with a sensitivity of 20 parasites/mL blood. This PCR method has so far been used to follow 137 vaccinee and control volunteers in Phase IIa trials in Oxford and on 220 volunteer samples during a Phase IIb field trial in The Gambia.
Asunto(s)
Vacunas contra la Malaria , Malaria/diagnóstico , Reacción en Cadena de la Polimerasa/métodos , Animales , Anopheles/parasitología , Secuencia de Bases , Ensayos Clínicos como Asunto , Cartilla de ADN , Humanos , Malaria/microbiología , Plasmodium/genética , ARN Ribosómico 18S/genética , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Inhibition of parasite growth is a major objective of blood-stage malaria vaccines. The in vitro assay of parasite growth inhibitory activity (GIA) is widely used as a surrogate marker for malaria vaccine efficacy in the down-selection of candidate blood-stage vaccines. Here we report the first study to examine the relationship between in vivo Plasmodium falciparum growth rates and in vitro GIA in humans experimentally infected with blood-stage malaria. METHODS: In this phase I/IIa open-label clinical trial five healthy malaria-naive volunteers were immunised with AMA1/C1-Alhydrogel+CPG 7909, and together with three unvaccinated controls were challenged by intravenous inoculation of P. falciparum infected erythrocytes. RESULTS: A significant correlation was observed between parasite multiplication rate in 48 hours (PMR) and both vaccine-induced growth-inhibitory activity (Pearson râ=â-0.93 [95% CI: -1.0, -0.27] Pâ=â0.02) and AMA1 antibody titres in the vaccine group (Pearson râ=â-0.93 [95% CI: -0.99, -0.25] Pâ=â0.02). However immunisation failed to reduce overall mean PMR in the vaccine group in comparison to the controls (vaccinee 16 fold [95% CI: 12, 22], control 17 fold [CI: 0, 65] Pâ=â0.70). Therefore no impact on pre-patent period was observed (vaccine group median 8.5 days [range 7.5-9], control group median 9 days [range 7-9]). CONCLUSIONS: Despite the first observation in human experimental malaria infection of a significant association between vaccine-induced in vitro growth inhibitory activity and in vivo parasite multiplication rate, this did not translate into any observable clinically relevant vaccine effect in this small group of volunteers. TRIAL REGISTRATION: ClinicalTrials.gov [NCT00984763].
Asunto(s)
Adyuvantes Inmunológicos , Vacunas contra la Malaria/inmunología , Malaria/prevención & control , Malaria/parasitología , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/inmunología , Vacunación/métodos , Adyuvantes Inmunológicos/efectos adversos , Adolescente , Adulto , Hidróxido de Aluminio/inmunología , Anticuerpos/inmunología , Antígenos de Protozoos/inmunología , Femenino , Humanos , Vacunas contra la Malaria/efectos adversos , Masculino , Proteínas de la Membrana/inmunología , Persona de Mediana Edad , Oligodesoxirribonucleótidos/inmunología , Proteínas Protozoarias/inmunología , Vacunación/efectos adversos , Adulto JovenRESUMEN
There is increasing interest in malaria vaccines targeting the asexual blood stage of Plasmodium falciparum. Without accepted immunologic correlates of clinical protection, challenge studies are useful for assessing the efficacy of candidate vaccines in vivo in healthy volunteers. We report a pilot study of a safe and robust challenge protocol using a blood-stage inoculum. We have applied well-validated trial endpoints and twice daily real-time quantitative polymerase chain reaction monitoring of parasitemia to blood-stage challenge, which enabled direct comparison with sporozoite challenge. We found that greater accuracy in quantification of blood-stage growth rates can be achieved with blood-stage challenge. This finding may provide greater power to detect partial efficacy of many blood-stage candidate vaccines. We discuss the potential utility of blood-stage challenge studies in accelerating malaria vaccine development.