# Emerging Diagnostics in _Clostridioides difficile_ Infection
## 3 Sampling the Community (Ribotyping and Mass Spectrometry)
Whether using ML or a CAM, developing and training these tools necessitates exposure to many different ribotypes to accurately and specifically classify strains of clinical and epidemiological significance. Thus, it may be argued that machine learning paired with MALDI-TOF MS could be a powerful tool for epidemiological surveillance centers involved in tracking CDI worldwide, akin to WGS. Like WGS, better performance will require continuous updating based on epidemiological data to include new unknown circulating ribotypes. NAAT and CRISPR-Cas12a (Toxin Identification)  
NAAT provides the most useful diagnostic information [76], offering several advantages, including excellent sensitivity, low complexity, simplified reporting, and a reduced need for repeat testing. To date, the Food and Drug Administration (FDA) have approved 23 nucleic tests ([[https://www.fda.gov/medical-devices/in-vitro-diagnostics/nucleic-acid-based-tests#microbial](https://www.fda.gov/medical-devices/in-vitro-diagnostics/nucleic-acid-based-tests#microbial)]([https://www.fda.gov/medical-devices/in-vitro-diagnostics/nucleic-acid-based-tests#microbial](https://www.fda.gov/medical-devices/in-vitro-diagnostics/nucleic-acid-based-tests#microbial)), accessed on 10 June 2024). Five utilize either isothermal helicase-dependent (HDA) or loop-mediated isothermal amplification (LAMP), while the remaining assays predominantly use real-time or multiplex PCR (mPCR). The majority of NAATs target the genes for tcdB, tcdA, and/or the binary toxin in diarrhead stool specimens and have exhibited good sensitivity (80-100%) and specificity (87-99%) [77] values. Of note is the recent work by [PERSON] et al. in which a new single-tube multiplex real-time PCR method for simultaneously detecting tcdA, tcdB, and cdtB targets in 74 fecal samples was reported [78]. The method demonstrated an intraspecies specificity of 100%, a consistency value of 98.4%, and similar sensitivities compared with RT-PCR. Moreover, the technique identified toxicgenic C. _difficile_ in 54 fecal samples, exhibiting sensitivities and specificities of 96.49% and 94.12%, respectively. PCR can only detect toxin-encoding genes rather than directly detecting the toxins in stool, which may result in the overdiagnosis of CDI, potentially ruling out other causes of diarrhea [79]. Several PCR panels/platforms have sought to address this concern, such as Luminex xTAG GPP (Luminex Molecular Diagnostics Inc., Toronto, ON, Canada) and FilmArray(tm) Gastrointestinal Panel (BioFire Diagnostics, Salt Lake City, UT, USA), which can detect multiple microbial pathogens (seven bacteria, two viruses, and two parasites, and fourteen bacteria, five viruses, and four parasites), including C. _difficile_, frequently encountered in stool samples. Regarding the former platform, the sensitivity ranges from 90% to 100% (depending on the target organism), with an overall sensitivity of 94.5% and a specificity of 99.1% being reported [80]. A multicenter trial using the BioFire panel identified 1180 potential pathogens in 1556 specimens tested, demonstrating 100% sensitivity for 12/22 targets and 94.5% for an additional 7/22 targets [81]. More recently, a direct comparison between the two showed that samples with low-concentration targets were more often detected by FilmArray than with Luminex xTAG GPP, and xTAG GPP was more likely to be affected by amplification inhibitors [82]. While employing multiple targets offers benefits, MPCR is still vulnerable to proteinous interferements, which can delay or elevate the C\({}_{\text{T}}\) (threshold cycle) and lead to inaccurately low template estimates [83]. The benefits of an established or fixed C\({}_{\text{T}}\) value were demonstrated in a large study by [PERSON] et al. using the GeneXpert assay with C\({}_{\text{T}}\) values correlating with toxin positivity. In addition, the researchers found that a lower CT correlated with a higher failure rate with first-line therapy and higher mortality in patients [84].