Laboratory Tests for the Diagnosis of Clostridium difficile

Abstract

Clostridium (reclassified as “ Clostridioides ”) difficile is an anaerobic, gram-positive bacterium that causes significant disease through elaboration of two potent toxins in patients whose normal gut microbiota has been altered through antimicrobial or chemotherapeutic agents (dysbiosis). The optimum method of laboratory diagnosis is still somewhat controversial. Recent practice guidelines published by professional societies recommend a two-step approach beginning with a test for glutamate dehydrogenase (GDH), followed by a toxin test and/or a nucleic acid test. Alternatively, in institutions where established clinical algorithms guide testing, a nucleic acid test alone is acceptable. Nucleic acid tests are the methods of choice in approximately 50% of laboratories in the United States. These tests are considered as the most sensitive methods for detection of C. difficile in stool and are the least specific. Because of the lower specificity with nucleic acid tests, some clinicians believe that toxin enzyme immunoassays are better predictors of disease, despite their known poor performance in certain patient populations. This review will discuss the advantages and disadvantages of the currently available test methods for the diagnosis of C. difficile with a brief mention of some novel assays that are currently in clinical trials.

As described in several manuscripts in this volume, Clostridium (new name Clostridioides ) ^a difficile , hereafter referred to as C. difficile , remains a common and virulent cause of healthcare-associated infections and some cases of community associated and traveler's diarrhea in vulnerable patient populations. The last several decades have seen emergence of more virulent strains and historically high-incident levels of disease. ^{1 2} Therefore, accurate clinical and laboratory diagnosis is essential for prompt patient management and effective infection control measures. Recent practice guidelines emphasize that the targeted populations for C. difficile testing are patients with unexplained and new-onset ≥3 unformed stools in 24 hours. ^{3 4} However, the optimum laboratory based approach is still a matter of debate and controversy. ⁵ A variety of methods exist for testing patients at risk for C. difficile . These can be broadly categorized into two groups: (1) those tests that detect the organism itself (or a component of the organism), such as anaerobic bacterial culture, glutamate dehydrogenase (GDH), the common antigen present in both toxigenic and nontoxigenic strains, and nucleic acid tests that detect the genes that encode toxins A and B; and (2) tests that detect free toxin, such as cell culture cytotoxicity neutralization assays (CCCNA) and enzyme immunoassays (EIAs). Bacterial culture with toxin testing of recovered isolates (toxigenic culture [TC]) and CCCNA are considered reference methods and the standards against which other methods are compared. ^{3 4 6} Each of these methods is described below and their performance characteristics are listed in Table 1 .

Table 1. Summary of performance characteristics of various test methods for C. difficile detection .

Methods/assays	Performance characteristics
	Sensitivity (%, range)	Specificity (%, range)
Toxigenic culture	Reference method	Reference method
Cell culture cytotoxicity neutralization assay a	33–86	97–100
Glutamate dehydrogenase b
Microwell EIA	88–95	94–98
Lateral flow membrane	60–100	76–100
ELFA/CLIA	87–99	91–97
Toxin A/B c
Microwell EIA	41–86	91–99
Lateral flow membrane	29–79	89–100
ELFA/CLIA	41–88	89–100
Nucleic acid tests d	62–100	89–100

Abbreviations: CLIA, chemiluminescent immunoassay; EIA, enzyme immunoassay; ELFA, enzyme linked fluorescent assay.

^a Compiled from various studies. ^{32 38 63 64 65 66}

^b Compiled from various studies. ^{16 30 31 32 33 67 68 69 70}

^c Compiled from various studies. ^{16 32 33 39 41 64 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81}

^d Compiled from various studies. ^{6 16 32 33 34 36 37 38 39 40 63 65 66 70 71 76 78 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103}

Toxigenic Bacterial Culture

C. difficile in an anaerobic, spore-forming, gram-positive bacterium, many strains of which produce toxin. Culture requires inoculation of stool to anaerobic media and after prolonged incubation, identifying suspicious colonies, and then performing toxin testing on the identified C. difficile organisms. The entire process may require up to 7 days and therefore this method is too labor-intensive and slow to be used in a clinical laboratory as a primary diagnostic approach. However, it is a very sensitive method for detection of C. difficile and TC is quite useful for recovering isolates for outbreak investigations, for surveying for drug resistance, and as a standard reference method for evaluation of other diagnostic tests. ⁶ There is no agreed upon standard for culture. Traditional methods apply preliminary treatment with “heat shock” or “alcohol shock” to minimize contaminating stool organisms, followed by inoculation to selective cycloserine–cefoxitin–fructose agar or a variant of this medium. ^{6 7 8} More recent studies have shown that inoculating an enrichment broth that contains taurocholate, antibiotics, carbohydrates, and lysozyme (e.g., cycloserine mannitol broth with taurocholate and lysozyme [CCMB-TAL, Anaerobe Systems, Morgan Hill, CA]) enhances the recovery of C. difficile without the need for heat or alcohol pretreatment. ^{9 10} In addition, the use of chromogenic agar medium, with or without enrichment techniques, has also been shown to produce rapid and sensitive results. ^{11 12} A detailed description of the various ways to culture C. difficile is beyond the scope of this review and the interested reader is referred to other publications for a more in-depth discussion of TC methods. ^{6 12}

Cell Culture Cytotoxicity Neutralization Assays

The development of a CCCNA by Bartlett et al and Chang et al using initially primary human amnion cell cultures, facilitated the discovery that cytotoxins elaborated by C. difficile were responsible for pseudomembranous colitis, and this became the first diagnostic test for detection of C. difficile in stool. ^{13 14} This test method requires multiple steps that include placing an aliquot of stool in a buffer solution, centrifuging it, and filtering the supernatant. The filtrate is diluted and then inoculated to a cell culture monolayer. A variety of different cell types can be used and human fibroblasts are considered the most sensitive. ^{14 15} After incubation at 37°C for 24 hours, the plates are assessed for cytopathic effect (CPE) that is characterized by rounding and spiculation of the cells. CPE must be neutralized by C. difficile or Clostridium sordellii antitoxin to prove that CPE is not related to nonspecific substances in stool. Although CCCNA is considered as a reference method, and is a sensitive method for the detection of toxin B, clinical sensitivity is <90% ( Table 1 ). This is likely due to a variety of preanalytical factors and the difficulty with test performance and interpretation. For these reasons and because few clinical laboratories have the capacity to support cell cultures, this method has decreased in popularity. However, despite these limitations, some believe that detection of cytotoxin in patient stool samples by CCCNA (or other toxin test) is a more accurate reflection of active disease than other methods. ^{5 16} In the prospective study by Reigadas et al, the authors noted that patients who were cytotoxin test positive by CCCNA had more severe colitis and complicated disease compared with patients positive by TC alone. ¹⁷ The authors did also observe that a significant proportion of symptomatic patients (31.9%) would have been missed if only cytotoxicity testing was used for treatment decisions, emphasizing the lower clinical sensitivity of this method. ¹⁷

Toxin Enzyme Immunoassays

The primary virulence factors in pathogenesis of C. difficile infection are toxins A and B. ^{18 19} CCCNA has been considered as a reference method for detecting C. difficile toxin. However, as discussed above, CCCNA is labor intensive, time consuming, requires technical expertise and maintenance of tissue culture facilities. Therefore, rapid enzyme immunoassays (EIAs) to detect toxin A and/or toxin B were the mainstay of C. difficile diagnostic tests for many years. Toxin EIAs are available in multiple formats including solid phase microwell, rapid immunoassays in chromatographic cassettes, immunocard, lateral flow membrane, and automated systems using an enzyme-linked fluorescent assay (ELFA) or a chemiluminescent immunoassay (CLIA). Although several C. difficile toxin EIAs are commercially available, they are all limited in sensitivities and variability within the assays (sensitivities range 44–99% when compared with CCCNA, and 29–88% when compared with TC [ Table 2 ]). The meta-analysis of the studies published from 2009 until June 2014 to evaluate commercial toxin EIA assays compared with a reference method showed pooled sensitivities of 83 and 57% when compared with CCCNA and TC, respectively. ³ Given inadequate performance, professional societies do not recommend toxin EIAs as stand-alone tests for the diagnosis of C. difficile . ^{3 4} Lack of sensitive toxin EIAs, as well as recent studies, demonstrating potential correlation between stool toxin levels and disease severity ^{16 20} led to development of the ultrasensitive digital enzyme-linked immunosorbent assays (ELISAs) for toxins A and B with quantification of toxins in stools. ²¹ Digital ELISA uses single-molecule array (SIMOA) technology to isolate and detect single proteins on paramagnetic beads in femtoliter-sized wells and enables more sensitive immunoassays than are possible using conventional EIAs. ²² In the validation study of the ultrasensitive digital ELISAs by Song et al toxin-B assay had 100% sensitivity compared with CCCNA and 97% specificity compared with TC. Correlation between mean toxin levels and C. difficile -attributable severity outcomes did not reach statistical significance. ²¹ In a more recent publication using SIMOA technology, similar to that evaluated by Song et al using the Quanterix analyzer (Quanterix Corporation, Lexington, MA), the SIMOA assays detected toxins in 24% more samples than the C. difficile toxin A-/B-II assay (Techlab, Blacksburg, VA). ²³ Another “ultrasensitive” assay for detecting toxin is also in development. ^{24 25} The Singulex Clarity C. diff Toxins A/B Assay (Singulex, Inc. Alameda, CA) uses single molecule counting technology to detect both toxins A and B in stool. ^{24 25} Paramagnetic microparticles, precoated with toxin-A and toxin-B antibodies (capture antibodies), and fluorescently labeled toxin-specific antibodies (detection antibodies) form immune complexes with toxins present in the stool sample. After some wash steps, elution buffer is added to cleave the immune complexes from the paramagnetic beads releasing the fluorescently bound detection antibodies. These particles are detected by the clarity system reader which is a confocal fluorescence microscope with a photodiode detector. An algorithm counts detected fluorescence “events” comparing them to a standard curve. Total assay time is 40 minutes. ^{24 25} This assay is not yet FDA (Food and Drug Administration)-approved and like the digital ELISA described above, there is potential to quantify the amount of toxin in the stool sample. In preliminary studies, this assay detected over 95% of EIA positive or CCCNA positive samples and an additional 23 to 27% toxin-positive samples missed by conventional toxin tests. ^{24 25} Although toxin detection alone may not be sufficient for diagnosis of C. difficile infection because toxins can be detected in asymptomatic carriers and can remain detected in patients with C. difficile after clinical cure, ^{26 27} digital ELISA, and single-molecule counting technology have the potential to improve clinical diagnostic accuracy by not detecting colonized C. difficile with no toxin or minimal toxin production.

Table 2. Summary of toxin EIA for detection of C. difficile .

	Comparison to CCCNA		Comparison to TC
Methods/assays	Sensitivity (%, range)	Specificity (%, range)	Sensitivity (%, range)	Specificity (%, range)
Microwell EIA
Premier toxin A + B a	58–99	94–100	40–86	91–100
TechLab Toxin A/B II b	72–91	87–100	58–85	96–99
Ridascreen Toxin A/B c	57–67	95–97	52–60	96–98
Remel ProSpecT d	90–91	93–97	82	93
Lateral flow membrane EIA
ImmunoCard toxins A/B e	85–96	97–99	41–69	93–99
Tox A/B Quik Chek f	61–84	99	40–74	94–100
Quick Chek Complete Tox A/B g	50–73	100	29–79	89–100
Xpect h	44–83	99–100	48–69	95–99
ELFA/CLIA
VIDAS CDAB i	53–98	99–100	44–80	95–100
Liaison C. difficile toxins A and B j	88	95	69–88	95–100

Abbreviations: CCCNA, cell culture cytotoxicity neutralization assays; CLIA, chemiluminescent immunoassay; EIA, enzyme immunoassay; ELFA, enzyme linked fluorescent assay; TC, toxigenic culture.

^a Meridian Bioscience, Inc.. Cincinnati, OH, compiled from various studies. ^{16 41 64 71 78 81 82 83 104 105}

^b TechLab, Blacksburg, VA, compiled from various studies. ^{16 64 75 81 104 106 92}

^c R-Biopharm AG Darmstadt, Germany, compiled from various studies. ^{64 88 108}

^d Remel, Lenexa, KS, compiled from two studies. ^{64 104}

^e Meridian Bioscience, Inc. Cincinnati, OH, compiled from various studies. ^{40 64 70 79 89 104}

^f TechLab, Blacksburg, VA, compiled from various studies. ^{64 68 80 90 107}

^g TechLab, Blacksburg, VA, compiled from various studies. ^{39 66 68 69 70 77 82}

^h Remel, Lenexa, KS, compiled from three studies. ^{64 79 91}

ⁱ bioMerieux, Durham, NC, compiled from various studies. ^{64 69 73 74 76 77 78 89}

^j DiaSorin, Saluggia, Italy, compiled from two studies. ^{72 75}

Glutamate Dehydrogenase Testing and Multistep Algorithms

C. difficile GDH is a metabolic enzyme present in high levels in all strains of C. difficile , both toxigenic and nontoxigenic isolates. GDH assays are available as microwell EIA, lateral flow EIA, and automated systems with ELFA or CLIA. GDH assays are simple to perform with minimal hands-on time, and less expensive compared with nucleic acid tests (NATs). Good sensitivity (>90%) and high-negative predictive value of GDH were demonstrated in systematic reviews and meta-analysis, ^{28 29} making the assay a useful first screening step in multistep algorithms combining a toxin EIA and/or NAT with GDH. In general, GDH negative specimens can be reported as negative and GDH positive/EIA positive specimens can be reported as positive (two-step algorithms). For GDH positive/EIA negative specimens, the third testing (NAT or TC) can be performed to rule out C. difficile with higher confidence (three-step algorithms). ^{3 4} Of note, a few studies reported sensitivities below 90% for GDH assays ^{30 31 32 33} which may be explained by variable sensitivities associated with ribotype. In a multicenter clinical trial conducted to assess the performance of a NAT (Cepheid Xpert C. difficile ) in North America, it was found that GDH was less sensitive than the NAT in detecting non-027 ribotype strains, and suggested that the sensitivity of GDH may vary according to ribotype. ³⁴ However, another study evaluating effect of strain type on detection of toxigenic C. difficile showed no difference in detection according to ribotype, although overall GDH was significantly less sensitive compared with nucleic acid amplification tests (NAATs). ³⁵ Considering the mixed literature on the performance of GDH assays, laboratories that implement it, should verify that the GDH assay has acceptable test performance for their patient population.

Nucleic Acid Detection Methods

Although polymerase chain reaction (PCR) based molecular tests were developed as early as the 1990s, ^{36 37} the first real-time PCR method for detection of C difficile in fecal samples (the BD-GeneOhm Cdiff assay, BD Diagnostics, Sparks, MD) was not FDA-approved until 2008. ³⁸ Currently there are 15 FDA-cleared molecular platforms available in the United States that detect either toxin A or toxin B. Table 3 lists the available assays in the United States and summarizes the test methodology and performance characteristics of each assay where available. The broad ranges in performance are likely related to the patient population that is studied and the type of test and analytical sensitivity of the comparative method used to assess the NAT.

Table 3. Currently available FDA cleared molecular assays in the United States ^a .

Assay	Manufacturer	Gene targets	Method	TAT (h)	Performancecharacteristics		References
					Sensitivity	Specificity
Prodesse ProGastro Cd	Hologic	tcdB	qPCR	4	77–100	94–99	38 93
Xpert	Cepheid	tcdB/tcdC	qPCR	1	90–100	93–99	69 78 85 94 100
Illumigene	Meridian	tcdA	LAMP	1	82–100	94–100	39 66 69 71 85 95
Simplexa	Focus Diagnostics	tcdB	qPCR	1	87–98	99–100	94 95
BDMAX	BD Diagnostics	tcdB	qPCR	2	86–98	89–100	81 83 85 90 92 94 100
Verigene	Nanosphere/Luminex	tcdB	PCR; nanoparticle hybridization	2	91–95	93–99	32 85
Aries C difficile Complete Kit	Luminex	tcdA/tcdB	qPCR	2.3	93	97	86
AmpliVue	Quidel	tcdA	Helicase dependent amplification	1.5	92–96	99–100	87 95
Lyra	Quidel	tcdA/tcdB	qPCR	2–3	82–89	97–99	96
Solana	Quidel	tcdA	Helicase dependent amplification	0.5	93	99	97
Cobas	Roche	tcdB	qPCR	0.5–1.5 b	93	99	98
ICEPlex	Primera DX	tcdB	qPCR	4	90	97	99
IMDx C difficile for Abbott m2000	IMDx	tcdA tcdB tcdBv	qPCR	3	62–84	94–99	91 100
Artus C difficile QS-RGQ	Qiagen	tcdA tcdB	qPCR	3.75 for 24 samples	100	90–100	101 102
GenePOC	GenePOC	tcdB	qPCR	1.5	81	97	103

Abbreviations: FDA, Food and Drug Administration; PCR, polymerase chain reaction; TAT, turnaround time.

^a As of August 2018. Comparative methods vary. qPCR, real-time PCR, LAMP, loop mediated amplification; tcdA , toxin A gene; tcdB , toxin B gene; tcdBv , toxin B variant genes; tcdC , toxin-C gene; cdt , binary toxin gene.

^bDepends on the platform used cobas 4800 (1.5 hours) or Liat instrument (20–30 minutes).

In general, NATs have analytical sensitivities that are 10 to 100 times higher than cytotoxin assays, ^{39 40} and they are substantially more expensive than EIAs when just the price of the actual tests are considered. ⁵ Some laboratories have successfully lowered the costs of these assays by including the NAT in a three step algorithm. ^{41 42 43} In these cases, the stool is first tested with a combination test (e.g., C diff CHEK Complete, TechLab, Inc., Blacksburg, VA) that detects both GDH and toxin. If the GDH and toxin tests are concordantly positive or negative, results can be reported and testing is completed. However, if the GDH is positive and the toxin test is negative, those specimens progress to a NAT. Bartsch et al compared the economic and health benefits of different approaches to diagnose C. difficile infections as follows: toxin A/B EIA alone, GDH antigen/toxin A, NAT testing, and a GDH antigen/toxin A/B combination test with NAT confirmation of indeterminate results (e.g., GDH positive or toxin negative). ⁴⁴ The authors found that GDH/toxin AB plus NAT testing led to the timeliest treatment and was the least costly. ⁴⁴ The NAT alone approach also led to timely treatment. ⁴⁴ A more recent study likewise found that when the laboratory switched from EIA to PCR testing, the decrease in the number of patients treated and the duration of treatment for a negative result, offset the costs of the implementation of a more expensive test. ⁴⁵ These findings corroborate the results of earlier studies. ^{46 47 48}

The trend among laboratories to adopt NATs as primary methods of testing for C. difficile diagnosis has recently been met with a ground swell of “antimolecular” sentiment, following the publication of two large prospective studies that demonstrated that positive toxin tests correlate better with patient outcomes. ^{5 16 49} These studies were performed among large general patient populations and some reports have challenged that the findings may not be applicable to all patient populations, especially immunocompromised hosts. ^{50 51 52} In the latter, albeit smaller studies, no differences in disease severity nor mortality were observed among patients who were NAT positive, toxin negative. ^{50 51 52}

Since NATs are more sensitive and some laboratories have noted a negative impact on their reporting of C. difficile rates to the National Healthcare Safety Network, it is advisable to improve test utilization by carefully selecting symptomatic patients at risk for C. difficile disease. Studies have shown that close to 50% of patients are tested inappropriately, either because they did not have diarrhea or they were on a laxative at the time of testing. ^{53 54}

Several different strategies have been used to improve the pretest probability of NAT testing. Some have looked at assessing the correlation of the PCR threshold cycle with free toxin. In general, the studies are mixed with a few demonstrating good predictive values ^{55 56} for presence of toxin and others showing no or suboptimal correlation. ^{57 58} Another strategy has been to look at the value of screening patients with biomarkers, such as fecal calprotectin, lactoferrin, and cytokines prior to or concomitant with NAT testing. ^{4 57 59 60} Both fecal lactoferrin and calprotectin are nonspecific markers of intestinal inflammation, and while studies demonstrate that they may be significantly elevated in patients with C. difficile , there is too much overlap with other inflammatory conditions to recommend their routine use in screening patients for C. difficile disease. However, it does appear to be the case that the absence of fecal lactoferrin or fecal calprotectin is inconsistent with an inflammatory cause for the diarrhea. Cytokine analysis in fecal specimens is an area of active research and may be an area to pursue in future studies with respect to assessing severity of infection, potentially to resolve whether a positive NAT is associated with disease or colonization, and perhaps to monitor a patient's response to treatment. The reference by Crobach et al provides a recent review of this topic. ⁶⁰

Regardless of the testing method or algorithm chosen, laboratories should have policies in place to ensure testing only on patients at risk for C. difficile infection. There should be clear specimen acceptance and rejection criteria. Specimens should be loose or take the shape of the container (Bristol stool chart 5 or 6), otherwise they should be rejected. Many laboratories have developed best practice alerts through the institutions' laboratory information system or electronic medical records preventing testing of patients on laxatives, repeat testing within a 7 day period and “test of cure” on positive patients. ^{61 62} The goal is to reduce detection of colonized patients and enhance diagnosis of patients likely to have actual disease.

Conclusion

Currently, there is no FDA-approved single rapid test that can be reliably used to diagnose C. difficile disease. Toxin EIAs provide good clinical specificity in symptomatic patients, but they are not sensitive enough to rule out C. difficile infection, and presence of toxins does not correlate with true C. difficile infection if asymptomatic carries or cured patients are tested. While NATs offer the excellent sensitivity to detect toxigenic C. difficile , they can detect patients colonized with C. difficile with no toxin production and may lead to overdiagnosis and overtreatment of C. difficile infection. Regardless of the testing method employed, improvement in test utilization is critically important for identification of true C. difficile cases. Institutional agreement on appropriate patient selection for C. difficile testing is needed to prevent negative consequences from detection of colonized C. difficile , such as falsely high C. difficile rates in hospitals and increased colonization and infection with multidrug resistant organisms in patients who received unnecessary antibiotic treatment for C. difficile . Multistep algorithms combining two or three assays can increase diagnostic accuracy of C. difficile infection and are recommended, especially when there are no institutional criteria for patient stool submission. The best performing diagnostic algorithm may differ in each institution, depending on test volume, patient population, laboratory work flow, and cost. Ultrasensitive immunoassays for toxins A and B have potential for stand-alone diagnostic testing for C. difficile pending further validation and accumulation of data.