Quantitative nucleic acid sequence based amplification (NASBA) to monitor malaria drug resistance (NFA.6009)

Malaria is one of the leading infectious diseases in the world, with 300-500 million clinical cases and 1-3 million deaths annually. Economic growth reduction due to malaria can be 1.3% per year, which contributes significantly to the ongoing poverty in endemic countries. Plasmodium falciparum is the most dangerous malaria parasite species for humans and responsible for the large number of casualties, which are mainly young children in Africa. Both asexual and sexual forms of the parasite circulate in the bloodstream of infected persons. Clinical disease is caused by proliferation of asexual forms while sexual stages develop in mosquitoes after ingestion of a blood meal (appendix 1: life cycle). Infected mosquitoes transmit the parasite in the population each time when a new bloodmeal is taken.
Antimalarial drugs are primarily directed against asexual parasite stages but some drugs also affect the development of sexual forms. Resistance against the limited portfolio of anti-malarial drugs have resulted in increases of malaria morbidity and mortality with major impact on public health, social and economic development (Nature 2002, 415:670-2). Monitoring of drug efficacy is a hallmark for malaria control and should be fast, sensitive, quantitative and parasite-stage specific. Measurement of both asexual and sexual forms will provide information on drug efficacy for clinical asexual forms and spread of (resistant) sexual forms over the population. At present monitoring of drug efficacy is performed by microscopy, which is slow, laborious and due to its limited sensitivity requires a patient follow-up of 28 days. Sexual stages are rarely included in the surveys. More sensitive molecular techniques based on DNA or RNA amplification experience problems with either quantification, or possibilities of stage-specific detection and are not applied routinely.
In this project, a quantitative nucleic acid sequence based amplification method (QT-NASBA) will be developed and tested for quantitative detection of parasites which can be used for efficient monitoring of drug resistance. A novel multiplex assay will be made, which consists of two components:

1. QT-NASBA based on P. falciparum 18S rRNA detection will be adapted to new detection methodology to allow combination into a multiplex assay. With the current detection method, this 18S QT-NASBA has shown to be a thousand times more sensitive than microscopy in quantification of malaria parasites.
2. Pfs25 mRNA QT-NASBA will be developed to quantify mature sexual transmission stages, based on sexual stage-specific expression of mRNA coding for the protein Pfs25. Detection of transmission stages is of major importance to monitor the spread of disease over a population, including the spread of drug resistance. Both assays will be adapted to molecular beacon detection, enabling high throughput of samples, decreasing risk of contamination and allowing combination of the two assays into one multiplex assay.

Utilisation
Drug resistance against most safe and affordable antimalarial drugs is spreading globally at an alarming rate, threatening to cause a public health disaster. Drug resistance surveys measure clinical response and persistence/recrudescence of parasitaemia after treatment. For these studies, microscopy has limited sensitivity and is laborious, especially when transmission stages need to be quantified. Molecular techniques based on amplification of DNA cannot be used for specific detection of various developmental stages and RNA amplification techniques other than NASBA experience problems since they require complete removal of DNA from the samples.
The multiplex QT-NASBA avoids all these limitations and allows for simultaneous monitoring of the effect of drug treatment on asexual disease-inducing stages and sexual transmission stages. Due to the high sensitivity, parasitaemia can be detected at an earlier stage, thus reducing follow-up times needed for reliable monitoring of drug resistance. Therefore, multiplex QT-NASBA is a powerful tool to optimise drug treatment policies, facilitating careful use of medicine such that the spread malaria and drug resistance will be minimised and the effective life span of safe and affordable antimalarial drugs will be prolonged.
The high accuracy, sensitivity and quick reactions times that allow for high throughput of samples, make multiplex QT-NASBA extremely suitable for large-scale epidemiological investigations such as monitoring of drug resistance and evaluation of control measures. Especially in endemic areas where resistances are quickly increasing, an effective monitoring technique for early warning will enable optimal use of available medicine and decreases the need to switch to more expensive drugs. The ongoing development of drug resistance under changing drug treatment policies causes a need for continuous application of multiplex QT-NASBA in national drug monitoring programmes.
The use of multiplex QT-NASBA for monitoring of malaria drug resistance is encouraged by bioMrieux and several research institutes. Additional to monitoring drug resistance, the assay can be used for evaluation of control measures and for epidemiological surveys. The malaria multiplex QT-NASBA will serve as a model for use in other parasitic diseases. It is expected that multiplex QT-NASBA and its individual components will be available for research purposes within the first half of the project. A complete protocol for monitoring of malaria drug resistance using multiplex QT-NASBA, will be available after evaluation, at the end of the 3-year research period. Potential users will be (inter)national institutes involved in malaria control, research institutions and hospitals.

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Er zijn vier bedrijven en één andere universiteit bij dit project betrokken.