Prevalence of diagnostically-discrepant Clostridioides difficile clinical specimens: insights from longitudinal surveillance

Abstract

Background: Clostridioides difficile Infection (CDI) is a healthcare-associated diarrheal disease prevalent worldwide. A common diagnostic algorithm relies on a two-step protocol that employs stool enzyme immunoassays (EIAs) to detect the pathogen, and its toxins, respectively. Active CDI is deemed less likely when the Toxin EIA result is negative, even if the pathogen-specific EIA is positive for C. difficile. We recently reported, however, that low-toxin-producing C. difficile strains recovered from Toxin-negative ('discrepant') clinical stool specimens can be fully pathogenic, and cause lethality in a rodent CDI model. To document frequency of discrepant CDI specimens, and evaluate C. difficile strain diversity, we performed longitudinal surveillance at a Southern Arizona tertiary-care hospital.

Methods: Diarrheic stool specimens from patients with clinical suspicion of CDI were obtained over an eight-year period (2015-2022) from all inpatient and outpatient Units of a > 600-bed Medical Center in Southern Arizona. Clinical laboratory EIA testing identified C. difficile-containing specimens, and classified them as Toxin-positive or Toxin-negative. C. difficile isolates recovered from the stool specimens were DNA fingerprinted using an international phylogenetic lineage assignment system ("ribotyping"). For select isolates, toxin abundance in stationary phase supernatants of pure cultures was quantified via EIA.

Results: Of 8,910 diarrheic specimens that underwent diagnostic testing, 1733 (19.4%) harbored C. difficile. Our major findings were that: (1) C. difficile prevalence and phylogenetic diversity was stable over the 8-year period; (2) toxigenic C. difficile was recovered from 69% of clinically Tox-neg ('discrepant') specimens; (3) the six most prevalent USA ribotypes were recovered in significant proportions (>60%) from Tox-neg specimens; and (4) toxin-producing C. difficile recovered from discrepant specimens produced less toxin than strains of the same ribotype isolated from non-discrepant specimens.

Conclusion: Our study highlights the dominance of Toxin EIA-negative CDI specimens in a clinical setting and the high frequency of known virulent ribotypes in these specimens. Therefore, a careful reevaluation of the clinical relevance of diagnostically-discrepant specimens particularly in the context of missed CDI diagnoses and C. difficile persistence, is warranted.

Keywords: Clostridioides difficile; discrepant; longitudinal; prevalence; ribotyping; surveillance.

Figure 1

Study design and diagnostic results. From 2015–2022, 8,910 samples were diagnostically assayed for C. difficile utilizing a GDH-EIA and TcdA/B-EIA. 692 specimens were positive for both EIA (GDH+/Toxin+) and 1,041 specimens were GDH+ but Toxin–. C. difficile was isolated from 636 of 1,041 GDH+/Toxin– specimens and ribotyped. 170 specimens harbored non-toxigenic C. difficile, and 439 specimens harbored toxigenic C. difficile. These 439 specimens are considered discrepant, highlighted in red. Of the 692 GDH+/Toxin+ specimens, C. difficile isolates were isolated from 472 and ribotyped. 429 specimens (90.9%) harbored toxigenic C. difficile, and 15 specimens (3.2%) harbored non-toxigenic C. difficile.

Figure 2

Clostridioides difficile ribotype distribution during an 8-year prospective surveillance (2015–2022). Tree map of the most prevalent ribotypes, in color, where the area of the box corresponds to the n-value. RT027 and RT106 were consistently the most common ribotypes per year. The non-toxigenic ribotypes RT010 and RT009 were the fourth and ninth most common ribotypes isolated from patient specimens.

Figure 3

Annual distribution of most common C. difficile ribotypes is largely invariant. Pie charts showing the prevalence of the six most common ribotypes (RT027, RT106, RT002, RT014, RT056, and RT076) in totality, per year. While the number of cases varied by year, the relative frequencies did not vary. RT027 was consistently the most common ribotype isolated each year and RT106 was the second-most common ribotype, with the exception of 2020.

Figure 4

Toxin EIA results do not predict C. difficile toxigenic potential. Comparisons of toxin EIA results and strain toxigenicity as inferred by ribotype. (A) Toxin EIA-negative (peach) specimens were more numerous than toxin EIA-positive (green) specimens (615 vs. 449, respectively). (B) Annual distribution of specimens by toxin EIA result. (C) Of the toxin EIA-positive specimens, 434/449 (96.7%) harbored toxigenic C. difficile with 15/449 (3.3%) harboring non-toxigenic ribotypes. For the toxin EIA-negative specimens, 171/615 (27.8%) harbored non-toxigenic C. difficile but 444/615 (72.2%) specimens harbored toxigenic C. difficile. There were more non-toxigenic strains in toxin EIA-negative specimens but there were similar numbers of toxigenic C. difficile isolated from both groups (n.s. p > 0.4; Mann–Whitney).

Figure 5

Toxigenic C. difficile ribotypes are isolated from toxin EIA-negative specimens. (A) The percentage of toxin EIA-negative strains, compared to the total number of strains, of the six most prevalent toxigenic ribotypes. Percentage values noted at top of bar. RT027 was significantly less prevalent in toxin EIA-negative specimens compared to the other five ribotypes (^***p < 0.001; ANOVA). However, 26% of RT027 isolates, and > 60% of other ribotypes, were isolated from GDH+/Toxin– specimens. (B) Annual frequency of the 6 most common ribotypes, isolated from GDH+/Toxin– specimens. Goodness-of-fit (R²) of a linear regression reported for each ribotype.

Figure 6

Clostridioides difficile from discrepant and non-discrepant specimens produce the major intoxicants TcdA/B. Five strains from toxin EIA-positive and toxin EIA-negative specimens each, belonging to four ribotypes (RT027, RT106, RT002, RT014), were assayed for toxin production. Bars represent average toxin produced (OD_450nm) per mg of protein. Irrespective of ribotype, all strains produced C. difficile toxin A/B; however, strains isolated from discrepant specimens produced less toxin compared to strains from non-discrepant, toxin EIA-positive specimens. (^*p < 0.05; t-test).