Multiplex detection of meningitis pathogens by a vertical flow paper microarray and signal enhancement suitable for low-resource settings: Proof of concept

• The vertical flow microarray (VFM) can detect multiple pathogens simultaneously. • The primers (sodC, hpd, lytA, and invA) amplified their targets with specificity. • The limit of detection was 10 2 initial copies of DNA for all target bacteria. • Clinical samples were tested on VFMs with the FilmArray ME panel as the reference. Meningitis is a medical emergency, and it is crucial to diagnose it accurately and promptly in order to manage patients effectively. It would, therefore, be essential to introduce and have fast, accurate, and user-friendly methods to determine the cause of these infections. This study aimed to demonstrate a potentially cost-effective new approach for detecting meningitis using a paper-based vertical flow microarray, which could be useful in settings with limited resources. We describe a multiplex paper microarray for detecting Neisseria meningitidis, Haemophilus influenzae, Streptococcus pneumoniae , and Salmonella spp. by the passive vertical flow of PCR-amplified clinical samples. A multibiotinylated amplicon was obtained as a product of PCR in the presence of both a biotinylated primer and biotin-11-dUTP. An enhancement step based on an enzyme-free gold enhancement protocol was also used to facilitate visual detection. This study showed that the vertical flow microarray (previously evaluated for one pathogen) can discriminately detect the amplification results down to the 10 2 copies of DNA limit for four meningitis pathogens in a multiplexed set-up. The study further demonstrated the ability of this device and setup to detect three of the four pathogens from clinical biosamples. This study demonstrated the capacity of a vertical flow microarray device to detect amplification products for four prevalent meningitis pathogens in a multiplex format. The vertical flow microarray demonstrated consistent visualization of the expected gene amplification results; however, indicating limitations in the pre- and amplification steps. This study highlights the potential of this multiplexing method for diagnosing meningitis and other syndromic diseases caused by various pathogens, especially in resource-limited areas.