Development of a lateral flow recombinase polymerase amplification assay for rapid and visual detection of Cryptococcus neoformans/C. gattii in cerebral spinal fluid

Abstract

BackgroundFor definitive diagnosis of cryptococcal meningitis, Cryptococcus neoformans and/or C. gattii must be identified within cerebral spinal fluid from the patients. The traditional methods for detecting Cryptococcus spp. such as India ink staining and culture are not ideal. Although sensitive and specific enough, detection of cryptococcal antigen polysaccharide has a high dose hook effect. Therefore, the aim of this study was to introduce a new rapid and simple detection method of Cryptococcus neoformans and C. gattii in cerebral spinal fluid.MethodsThe lateral flow strips combined with recombinase polymerase amplification (LF-RPA) assay was constructed to detect the specific DNA sequences of C. neoformans and C. gattii. The detection limit was evaluated using serial dilutions of C. neoformans and C. gattii genomic DNA. The specificity was assessed by excessive amount of other pathogens genomic DNA. The optimal detection time and amplification temperature were also analyzed. The diagnostic parameters were first calculated using 114 clinical specimens and then compared with that of other diagnostic method. A brief analysis and comparison of different DNA extraction methods was discussed, too.ResultsThe LF-RPA assay could detect 0.64 pg of genomic DNA of C. neoformans per reaction within 10 min and was highly specific for Cryptococcus spp.. The system could work well at a wide range of temperature from 25 to 45 °C. The overall sensitivity and specificity were 95.2 and 95.8% respectively. As amplification template for LF-RPA assay, both cell lysates and genomic DNA produce similar experimental results.ConclusionsThe LF-RPA system described here is shown to be a sensitive and specific method for the visible, rapid, and accurate detection of Cryptococcus spp. in cerebral spinal fluid and might be useful for clinical preliminary screening of cryptococcal meningitis.

Serology anno 2021—fungal infections: from invasive to chronic

“…Simultaneous detection of anti-mannan antibodies (AMA) improved sensitivity and specificity (83% and 86%) [45], respectively. Several test kits for the detection of mannan antigen (MA) of C. albicans and AMA show variable performance [46][47][48], see [55][56][57][58], which avoid serum pretreatment and support urinary cryptococcal antigen testing, which is useful for screening [59,60]. The LFA is a rapid procedure, there are various test kits on the market, see Table 5;…”

Point-of-Care Strategies for Detection of Waterborne Pathogens

“…Lateral flow devices are one of the most commercialized POC testing methods, attributable to low-cost, easy interpretation, high specificity, and portability for detection and quantification of different analytes in complex mixtures. In the modern era, lateral flow assay-based rapid tests have been developed and widely accepted by regulatory authorities for screening of diseases [104], chemicals [105], pathogens [106], water pollutants [107], and toxins [108]. Lateral flow assays offer one-step detection, without the involvement of washing steps, in a simple manner, with relatively short development time, low-cost production, and low sample volume requirements.…”

Bushmeat Species Identification: Recombinase Polymerase Amplification (RPA) Combined with Lateral Flow (LF) Strip for Identification of Formosan Reeves’ Muntjac (Muntiacus reevesi micrurus)

“…The reaction can be completed in 5 to 20 min and visualized by either gel electrophoresis, nucleic acid dye, or a lateral flow (LF) strip [19,20]. RPA is also resistant to common PCR inhibitors, and the reaction can be performed with heated, lysed, or crude DNA extract [21][22][23][24][25]. Recently, diagnostic techniques using RPA have successfully been developed for various pathogens, including viruses [23,25], bacteria [26,27], parasites [21,24], and fungi [28].…”