Development and Application of Rapid Clinical Visualization Molecular Diagnostic Technology for Cryptococcus neoformans/C. gattii Based on Recombinase Polymerase Amplification Combined With a Lateral Flow Strip.

Wei Liang,Yingzhi Lu,Lipin Liu,Wenjun Zhu,Xuzhu Gao,Lei Wang,Huimin Chen,Na Li,Fang Wang,Ping Zhou,Kun Wang,Mengdi Zhao,Qing Zhu,Yujiao Chen,Yan Wang,Weiguo Zhao,Xia Liu

doi:10.3389/fcimb.2021.803798

Abstract

Cryptococcus neoformans (C. neoformans)/C. gattii can easily invade the human central nervous system and cause cryptococcal meningitis (CM). The clinical fatality rate of these fungi is extremely high and causes more than 180,000 deaths worldwide every year. At present, the common clinical identification methods of these fungi are traditional culture methods and Indian ink staining. In addition, enzyme-linked immunosorbent assay (ELISAs), polymerase chain reaction (PCR), real-time quantitative PCR detecting system (qPCR), mass spectrometry, and metagenomic next-generation sequencing (mNGS) have also been applied to detect these fungus. Due to the rapid progress of meningitis caused by C. neoformans/C. gattii infection, there is a desperate need for fast, sensitive, and on-site detection methods to meet the clinical diagnosis. Recombinase polymerase amplification (RPA) is a promising isothermal amplification technique that can compensate for the shortcomings of the above techniques, featuring short reaction time, high specificity, and high sensitivity, thus meeting the demand for in-field detection of C.neoformans/C. gattii. In our study, RPA- lateral flow strip (LFS) was used to amplify the capsule-associated gene, CAP64, of C. neoformans/C. gattii, and the primer-probe design was optimized by introducing base mismatches to obtain a specific and sensitive primer-probe combination for clinical testing, and specificity of the detection system was determined for 26 common clinical pathogens. This system was developed to obtain results in 20 min at an isothermal temperature of 37°C with a lower limit of detection as low as 10 CFU/μL or 1 fg/μL. A total of 487 clinical samples collected from multicenter multiplexes were tested to evaluate the detection performance of the RPA-LFS system, which revealed that the system could specifically detect C. neoformans/C. gattii, meeting the need for rapid, specific, and sensitive detection.

Highlights

Cryptococcus neoformans and Cryptococcus gattii are the two main pathogenic cryptococci that cause human infections (Torda et al, 2001)
We propose to establish a rapid detection technique for C. neoformans/C. gattii using recombinase polymerase amplification (RPA)-Lateral flow strip (LFS)
PCR amplification and gel electrophoresis showed that all five primer pairs could effectively amplify the target gene CAP64 (Figure 2A) and RPA-LFS results showed that only F2/ R2/P met the detection requirements (Figure 2B)

Summary

Introduction

Cryptococcus neoformans and Cryptococcus gattii are the two main pathogenic cryptococci that cause human infections (Torda et al, 2001). CM is fungal meningitis caused by C. neoformans and C. gattii infection. C. neoformans is the main pathogen of cryptococcosis and mainly affects immunocompromised individuals (Rodrigues et al, 1999; Voelz and May 2010), showing a preference for CNS infection (Rodrigues et al, 2007; Bielska and May 2016; May et al, 2016). C. gattii can establish Cryptococcus infections and infect the lungs, exhibiting a high pathogenicity that is difficult to treat. C. neoformans is a more common causative agent of cryptococcal infections in China and elsewhere compared to C. gattii (Bromilow and Corcoran, 2007; Chen et al, 2008). The clinical presentation is chronic or subacute onset, with progressive worsening of symptoms that include swelling of the brain and headache, which may be accompanied by fever, nausea, vomiting, and irritability (George et al, 2017; Rajasingham et al, 2017)

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Conclusion