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Background  For 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.    Methods  The 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.    Results  The 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.    Conclusions  The 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.

bmc infectious diseases

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