Can a toxin gene NAAT be used to predict toxin EIA and the severity of Clostridium difficile infection?

C. difficile is an anaerobic, spore forming Gram positive bacillus found in diverse environments that is able to colonise and proliferate in the human gastrointestinal tract, especially following changes in the indigenous colonic microbiota after antibiotic use [1, 2]. C. difficile is one of the most common causes of healthcare associated infection and is responsible for 15 to 25% of all cases of antibiotic associated diarrhoea [2‐4]. The infection causes an estimated 3000 deaths every year in the UK and 15,000–20,000 deaths in the USA, with associated case-fatality rates of 6–17% [4, 5]. It is associated with gut overgrowth of C. difficile and the production of toxins A, B, or both, which cause a range of effects, including gut mucosal damage, colitis, and pseudomembranous colitis [2, 4]. The primary mediators of inflammation in C. difficile infection (CDI) are large clostridial toxins, toxin A and toxin B. The toxins trigger a complex cascade of host cellular responses to cause diarrhoea, inflammation and tissue necrosis — the major symptoms of CDI [2, 4]. It must be noted a small number of healthy people naturally carry C. difficile in their large intestine and do not have ill effects from the infection [1].

Since C. difficile diarrhoea cannot be reliably distinguished from other causes of healthcare associated diarrhoea on clinical grounds alone, laboratory confirmation is essential [4, 6‐10]. However, the diagnosis of CDI remains a contentious issue [4, 8, 9, 11]. For decades, toxin tests were favoured over culture for diagnosis of CDI because toxins mediate disease, detection was faster and this method provided evidence of toxin production in vivo that typically correlated better with clinical disease [3, 10, 12, 13]. Molecular tests such as nucleic acid amplification tests (NAAT) target toxin genes but are similar to culture in detecting C. difficile bacteria regardless of toxin production, making it unclear whether positive NAAT results reflect clinical disease [4, 12, 14]. A disadvantage for NAATs is that they detect toxin genes alone and not toxin production which is thought to be associated with CDI [15]. The uncertain clinical significance of positive NAAT results is problematic in inpatient healthcare facilities where C. difficile colonization is 5 to 10 times more common than CDI and non-infectious causes of diarrhoea [4, 12, 15]. A large multi-centre study in the UK led to a change in the Department of Health diagnostic guidance, after showing that an algorithmic approach was optimal for laboratory diagnosis [4]. Two stage algorithms have been widely adopted, and consist of; an initial screen using glutamate dehydrogenase (GDH) enzyme immunoassay (EIA) or toxin gene NAAT to detect the presence of C. difficile, followed by a second step which detects faecal toxin using either EIA or a cell cytotoxin assay [4, 8, 11, 13, 16, 17]. The cell cytotoxin assay is used to detect the presence of C. difficile toxin by its effect on human tissue cells grown in culture. This test is considered to be the ‘gold standard’ for diagnosing CDI, however it requires technical expertise and takes 24 to 48 h to produce a result [10].

In the UK, screen positive/toxin negative patients are usually regarded as being colonized with C. difficile rather than infected, based on a large multicentre prospective study showing that only patients with detectable faecal toxin had adverse outcomes [2‐4]. However, outside the UK, such patients typically identified with NAATs as a screening test alone, are often regarded as having CDI [3, 18]. The role of C. difficile toxin gene NAAT for CDI diagnosis has largely been polarised between Europe and the US [3, 19]. Data from the National Healthcare Safety Network indicated in 2014 ~44% of US hospitals use NAAT for CDI diagnosis, whilst in Europe that level was much lower at 5% [3, 19]. Over diagnosis of CDI in hospitals using standalone NAAT can lead to unnecessary treatment causing clinicians to miss the real underlying diagnosis [3]. The outcome of patients who are NAAT positive/ toxin EIA negative has been shown to be indistinguishable from that of NAAT negative and toxin EIA negative patients (in terms of diarrhoea and CDI related complications) [3, 15]. Additionally, increased mortality is associated with the presence of faecal toxin but not with NAAT positive/ toxin negative sample results [15]. While qualitative results of NAAT have poor positive predictive value for CDI, could there be an alternative role for NAATs in CDI diagnosis? [12, 15] Here we aimed to determine if a low NAAT cycle threshold result can predict severity of CDI and/or mortality using data from our institution over the past 5 years.

In this study, regression analysis demonstrated that low CT values were associated with toxin EIA positivity. With a CT value ≤26 we saw ≥72% toxin EIA positivity; applying a logistic regression model we demonstrated an association between low CT values and toxin EIA positivity. Conversely a CT value of ≤26 would miss 28% of patients that were EIA positive. It is not surprising that a low CT value is linked with toxin EIA positivity as previous studies have suggested expression of toxin is associated with the severity of CDI, and a correlation between faecal toxin levels and C. difficile counts has been reported in mice [12]. Dionne et al., previously identified that amplification CT values correlate to bacterial load [29]. They demonstrated toxin positive samples had higher bacterial loads than toxin negative samples and there was a significant inverse correlation between PCR CT values and bacterial loads [29]. In the present study, low CT values inversely correlate to toxin assay positivity, one could imply that CT values of toxin B genes act as a marker of the amount of toxin produced [12]. However, investigations seeking to link quantitative toxin production with C. difficile virulence have not been conclusive [12]. Further work is warranted to explore this.

In the UK, two stage algorithms for CDI have been adopted [4, 10]. Often a GDH test for screening is employed followed by a toxin EIA [4, 10]. Eyre et al., (2017) sequenced all GDH positive patients and compared the probability of faecal toxin positive and toxin negative patients being likely sources of transmission, that is, having C. difficile genetically linked to a subsequent isolate in another patient [30]. They concluded faecal toxin negative patients were similarly infectious to faecal toxin positive patients; faecal toxin status did not affect transmission rates [30]. They concluded strategies to identify and institute infection control measures around patients with potentially toxigenic C. difficile without detected faecal toxin are likely to reduce CDI incidence [30]. NAAT could be used as a screening test as it is rapid and highly sensitive to aid detection of such cases from an infection control perspective [31]. In addition with concerns about the lower sensitivity of toxin EIA testing increased usage of NAAT in hospitals worldwide has been seen [12]. The results in our study suggest a NAAT CT value of ≤26 corresponds to toxin EIA positivity. In our setting, a GDH test followed by NAAT using the CT value as marker of EIA positivity would seem to be sufficient to pick up the majority of EIA positive cases. NAAT detects the DNA of the toxin gene of C. difficile rather than the presence of toxin in stool samples, however in our study a low CT value shows a strong association with toxin EIA positivity. Our finding is in line with the observations of Jazmati et al., (2016) that free toxin detection is associated with low CT-values [32]. As in our study Jazmati et al., (2016) showed CT-values were significantly lower in specimens that were positive in toxigenic culture (26.2 ± 4.5, n = 100 versus 30.5 ± 5.0, n = 65; p < 0.001) [32]. Senchyna et al., (2017) also found low CT values predicted free toxin results; a CT value of <26.35 could sensitively predict 96.0% of toxin EIA / PCR stool samples with a negative predictive value of 97.1% and with a specificity of 78.0% using both EIA toxin and cell cytotoxin assay [33]. In our study it is important to note that the toxin EIA has a sensitivity of 94.7% which could affect the CT value of ≤26 corresponding to toxin EIA positivity. One main disadvantage of NAATs is that they do not detect the presence of biologically active toxin in stool specimens [15]. Toxins expressed by C. difficile are the main virulence factor, and some feel that the presence of toxin in stool is a positive correlation of disease [4, 12, 15]. The significance of detecting C. difficile in the absence of the toxins, such as in the patient who tests positive by NAAT but negative by toxin EIA, is unclear [15].

The majority of large studies on C. difficile diagnostics show toxin EIA is the best predictor of outcome, with NAAT tests providing no additional information about disease severity [4, 9, 11, 13]. However, in our study 72% (n = 99/138) patients with a CT value between 18 and 21 had severe/recurrent CDI based on PHE guidelines for management of CDI [20]. In the mild cases, with a CT value between 18 and 21, we often saw treatment failure with our first line therapy of metronidazole requiring escalation of treatment. In contrast, 92% (n = 74/80) of the patients with a high CT value had mild CDI and responded to treatment. In our setting, the CT value could be used to guide treatment of patients with CDI. It must be noted we have only looked at a small number of patients with either the lowest or highest CT values. Reigadas et al., (2016) also showed patients with severe disease had significantly lower CT-values (toxin B CT value of <23.5) compared with patients in the other groups [12]. In our study, we also looked at the mortality within a month of CDI. It must be noted we have looked at all-cause mortality and not mortality specifically attributed to CDI. However, a significant association between low CT value and mortality was observed. Rao et al. (2015) found that there was no correlation between amplification CT values and severe CDI or all-cause mortality, however in our study; the mean toxin B amplification CT value was 27.1, which was much lower than that reported by Rao et al. (34.3) [34]. There were limitations in the study by Rao et al., (2015) which included sampling amounts and specimen transport. [34]

It is often difficult to distinguish between healthcare associated diarrhoea and CDI [1]. In CDI, the spectrum of clinical disease ranges from mild diarrhoea to toxic megacolon, colonic perforation and death [1]. Research suggests that both strain characteristics and the host’s immune response influence CDI severity, recurrence risk and mortality [1]. Jazmati et al., Reigadas et al., and Senchyna et al., all showed a correlation between low NAAT CT values and severity and/or patient outcome [12, 32, 33]. To our knowledge, this is the largest study reporting a significant association between CT values of a NAAT C. difficile test vs toxin EIA positivity and mortality. There is growing evidence that NAAT CT values could be used to predict CDI severity, recurrence risk and mortality.