Prospective evaluation of the Meridian Illumigene™ loop-mediated amplification assay and the Gen Probe ProGastro™ Cd polymerase chain reaction assay for the direct detection of toxigenic Clostridium difficile from fecal samples

Clostridium difficile is the most common and important cause of toxigenic colitis in the health care setting. Laboratory diagnostics have included bacterial culture with further identification of toxigenic stains, or more commonly, direct detection of preformed toxin in stool samples using biological or immunochemistry assays. Recently, molecular amplification assays for the direct detection of toxin-encoding genes have become available commercially. We prospectively evaluated 2 FDA-cleared molecular amplification tests, the Illumigene C. difficile and the ProGastro Cd PCR assay, for the direct detection of toxigenic C. difficile from fecal samples. Of 446 samples tested, 418 produced matching amplification results, 88 positive and 330 negative, and 13 resolved with repeat testing. Toxigenic culture and direct cytotoxin testing were used to resolve the remaining 15 discordant samples. Overall, each assay performed well and correctly identified 97% of positive samples.     

Diagnostic utilities of a fully automated molecular test for toxigenic Clostridium difficile

Laboratory underdiagnosis of toxigenic Clostridium difficile can lead to inappropriate management of C. difficile infection (CDI). A fully automated molecular test (FAMT), BD MAX, and enzyme immunoassays for C. difficile glutamate dehydrogenase (GDH) and for toxin A/B antigen test were evaluated using clinical specimens. Laboratory analysis of 231 fecal specimens from patients suspected with CDI, indicated that the sensitivity (Sn), specificity (Sp), positive predictive value (PPV), and negative predictive value (NPV) of FAMT was 98.1%, 98.9%, 96.3%, and 99.4%, while that of toxin A/B antigen was 52.8%, 100.0%, 100.0%, and 87.7%, respectively, compared to toxigenic culture. Sn, Sp, PPV, and NPV of GDH test compared to toxigenic culture was 92.5%, 94.4%, 83.1%, and 97.7%, respectively. FAMT can support the accurate laboratory diagnosis of toxigenic C. difficile and be an effective tool for appropriate treatment of CDI.     

Multicenter evaluation of the Luminex® ARIES® HSV 1&2 Assay for the detection of herpes simplex virus types 1 and 2 in cutaneous and mucocutaneous lesion specimens

Introduction: The ARIES® HSV 1&2 Assay is a new FDA cleared real-time PCR test for detection and differentiation of HSV-1 and HSV-2 DNA from cutaneous and mucocutaneous lesions. The test is performed on the ARIES® System, an automated sample to answer real-time PCR instrument that provides a closed system and simple workflow for performing molecular testing.

Areas covered: This article reports the clinical performance of the ARIES® HSV 1&2 Assay assessed on 1963 prospectively collected specimens. Assay sensitivities were 91.1–95% (cutaneous) and 97–98.5% (mucocutaneous), and specificities were 88.8–94.2% (cutaneous) and 93.2–95.4% (mucocutaneous), as compared to the ELVIS® HSV test system.

Expert commentary: Detection of HSV DNA by PCR is rapid and more sensitive than traditional culture and immunoassay methods and is being widely adopted in many laboratory settings. Sample to answer molecular platforms like ARIES® will enable routine and non-molecular labs to perform sensitive and rapid molecular testing with ease.     

Portrait Toxigenic Clostridium difficile assay,an isothermal amplification assay detects toxigenic C. difficile in clinical stool specimens

The Portrait Toxigenic Clostridium difficile assay is a rapid, qualitative assay for the detection of the tcdB gene of C. difficile in stool specimens from patients suspected of C. difficile infections, and received 510(k) clearance by the US FDA in March 2012. The Portrait Toxigenic C. difficile assay combines novel blocked-primer-mediated helicase-dependent multiplex amplification (bpHDA) technology and chip-based detection in an automated sample-to-result format. The assay requires minimal sample preparation and results are available within 90 min. In a multicenter evaluation, the Portrait Toxigenic C. difficile assay had a sensitivity of 98.2% and specificity of 92.8% compared with toxigenic culture. A comparative study between the Portrait Toxigenic C. difficile assay and three FDA-cleared molecular assays for the detection of toxigenic C. difficile exhibited a high degree of agreement (93.8–97.5%). The Portrait Toxigenic C. difficile assay provides a simple, cost-effective method with broad applicability to panel-based approaches, potentially simplifying workflow.     

Using a Novel Lysin To Help Control Clostridium difficile Infections

As a consequence of excessive antibiotic therapies in hospitalized patients, Clostridium difficile, a Gram-positive anaerobic spore-forming intestinal pathogen, is the leading cause of hospital-acquired diarrhea and colitis. Drug treatments for these diseases are often complicated by antibiotic-resistant strains and a high frequency of treatment failures and relapse; therefore, novel nonantibiotic approaches may prove to be more effective. In this study, we recombinantly expressed a prophage lysin identified from a C. difficile strain, CD630, which we named PlyCD. PlyCD was found to have lytic activity against specific C. difficile strains. However, the recombinantly expressed catalytic domain of this protein, PlyCD_1–174, displayed significantly greater lytic activity (>4-log kill) and a broader lytic spectrum against C. difficile strains while still retaining a high degree of specificity toward C. difficile versus commensal clostridia and other bacterial species. Our data also indicated that noneffective doses of vancomycin and PlyCD_1–174 when combined in vitro could be significantly more bactericidal against C. difficile. In an ex vivo treatment model of mouse colon infection, we found that PlyCD_1–174 functioned in the presence of intestinal contents, significantly decreasing colonizing C. difficile compared to controls. Together, these data suggest that PlyCD_1–174 has potential as a novel therapeutic for clinical application against C. difficile infection, either alone or in combination with other preexisting treatments to improve their efficacy.     

Risk Factors for Acquisition and Loss of Clostridium difficile Colonization in Hospitalized Patients

Asymptomatic colonization may contribute to Clostridium difficile transmission. Few data identify which patients are at risk for colonization. We performed a prospective cohort study of C. difficile colonization and risk factors for C. difficile acquisition and loss in hospitalized patients. Patients admitted to medical or surgical wards at a tertiary care hospital were enrolled; interviews and chart review were performed to determine patient demographics, C. difficile infection (CDI) history, medications, and health care exposures. Stool samples/rectal swabs were collected at enrollment and discharge; stool samples from clinical laboratory tests were also included. Samples were cultured for C. difficile, and the isolates were tested for toxins A and B and ribotyped. Chi-square tests and univariate logistic regression were used for the analyses. Two hundred thirty-five patients were enrolled. Of the patients, 21% were colonized with C. difficile (toxigenic and nontoxigenic) at admission and 24% at discharge. Ribotype 027 accounted for 6% of the strains at admission and 12% at discharge. Of the patients colonized at admission, 78% were also colonized at discharge. Cephalosporin use was associated with C. difficile acquisition (47% of patients who acquired C. difficile versus 25% of patients who did not; P = 0.03). β-lactam–β-lactamase inhibitor combinations were associated with a loss of C. difficile colonization (36% of patients who lost C. difficile colonization versus 8% of patients colonized at both admission and discharge; P = 0.04), as was metronidazole (27% versus 3%; P = 0.03). Antibiotic use affects the epidemiology of asymptomatic C. difficile colonization, including acquisition and loss, and it requires additional study.     

Nontoxigenic Clostridium difficile Protects Hamsters against Challenge with Historic and Epidemic Strains of Toxigenic BI/NAP1/027 C. difficile

Nontoxigenic Clostridium difficile (NTCD) has been shown to prevent fatal C. difficile infection in the hamster model when hamsters are challenged with standard toxigenic C. difficile strains. The purpose of this study was to determine if NTCD can prevent C. difficile infection in the hamster model when hamsters are challenged with restriction endonuclease analysis group BI C. difficile strains. Groups of 10 hamsters were given oral clindamycin, followed on day 2 by 10⁶ CFU of spores of NTCD strain M3 or T7, and were challenged on day 5 with 100 CFU of spores of BI1 or BI6. To conserve animals, results for control hamsters challenged with BI1 or BI6 from the present study and controls from previous identical experiments were combined for statistical comparisons. NTCD strains M3 and T7 achieved 100% colonization and were 100% protective against challenge with BI1 (P ≤ 0.001). M3 colonized 9/10 hamsters and protected against BI6 challenge in the colonized hamsters (P = 0.0003). T7 colonized 10/10 hamsters, but following BI6 challenge, cocolonization occurred in 5 hamsters, 4 of which died, for protection of 6/10 animals (P = 0.02). NTCD colonization provides protection against challenge with toxigenic BI group strains. M3 is more effective than T7 in preventing C. difficile infection caused by the BI6 epidemic strain. Prevention of C. difficile infection caused by the epidemic BI6 strain may be more challenging than that of infections caused by historic BI1 and non-BI C. difficile strains.     

Direct Clostridioides difficile ribotyping from stool using capillary electrophoresis

Clostridioides difficile(C. difficile) genotyping is essential for surveillance of emerging strains, transmissions, and outbreak investigations, but culture is lengthy and may not be routinely performed, which necessitates culture-independent genotyping methods. We aimed to develop a direct from stool C. difficile PCR ribotyping algorithm using capillary electrophoresis. Ribotypes were generated directly from 66.8% of stools with 33.2% requiring broth enrichment. 16S and tcdB cycle thresholds (Ct) were significantly lower (P< 0.001) in directly ribotyped stools compared to enriched stools, and Ct correlated with direct ribotyping (area under the curve: 0.97 and 0.96, respectively). Direct and isolate ribotypes were 94.7% concordant. Mixed C. difficile ribotypes were presumptively identified in 14 (7.5%) samples with 12 (6.4%) mixtures confirmed. We have developed a rapid PCR ribotyping algorithm allowing for direct C. difficile genotyping from stool using capillary electrophoresis with occasional detection of mixed C. difficile populations in stool, which is a limitation of conventional isolate genotyping.     

Real-time polymerase chain reaction method for detection of toxigenic Clostridium difficile from stools and presumptive identification of NAP1 clone

This study describes the development of a cost-effective, multiplex real-time polymerase chain reaction (RTPCR) method for detection of toxigenic Clostridium difficile from stools and presumptive identification of the NAP-1 strain. The diagnostic value of the new method is for the detection of toxigenic C. difficile which has the following performance characteristics: 99.8% specificity, 95.1% sensitivity, 97.5% positive predictive value, and 99.5% negative predictive value. Examination of 24 specimens presumptively identified as NAP1 strain by RTPCR with Pulsed-field gel electrophoresis performed on C. difficile isolated from those specimens showed 100% agreement. This RTPCR showed equivalent test performance characteristics as the 2 commercially available assays which were evaluated. The estimated cost per test is CAD$9.50 and which is significantly less than the commercial assays. The average turnaround time from setup to detection is 3.5 h. The RTPCR method described here is a cost-effective and highly sensitive test which can be implemented in a clinical laboratory to assist clinicians in establishing the diagnosis of C. difficile infection and indirectly determine the presence of the hypervirulent epidemic binary toxin (BI)/NAP 1 strain for prompt infection control interventions.     

In Vitro and In Vivo Antibacterial Evaluation of Cadazolid,a New Antibiotic for Treatment of Clostridium difficile Infections

Clostridium difficile is a leading cause of health care-associated diarrhea with significant morbidity and mortality, and new options for the treatment of C. difficile-associated diarrhea (CDAD) are needed. Cadazolid is a new oxazolidinone-type antibiotic that is currently in clinical development for treatment of CDAD. Here, we report the in vitro and in vivo antibacterial evaluation of cadazolid against C. difficile. Cadazolid showed potent in vitro activity against C. difficile with a MIC range of 0.125 to 0.5 μg/ml, including strains resistant to linezolid and fluoroquinolones. In time-kill kinetics experiments, cadazolid showed a bactericidal effect against C. difficile isolates, with >99.9% killing in 24 h, and was more bactericidal than vancomycin. In contrast to metronidazole and vancomycin, cadazolid strongly inhibited de novo toxin A and B formation in stationary-phase cultures of toxigenic C. difficile. Cadazolid also inhibited C. difficile spore formation substantially at growth-inhibitory concentrations. In the hamster and mouse models for CDAD, cadazolid was active, conferring full protection from diarrhea and death with a potency similar to that of vancomycin. These findings support further investigations of cadazolid for the treatment of CDAD.     

Comparison of the Verigene Clostridium difficile, Simplexa C. difficile Universal Direct,BD MAX Cdiff,and Xpert C. difficile assays for the detection of toxigenic C. difficile

We compared the Verigene Clostridium difficile test (Nanosphere, Northbrook, IL, USA), the Simplexa C. difficile Universal Direct (Focus Diagnostics, Cypress, CA, USA), the BD MAX Cdiff (Becton Dickinson, Franklin Lakes, NJ, USA), and the Xpert C. difficile (Cepheid, Sunnyvale, CA, USA) assays for the detection of toxigenic C. difficile. One hundred and ninety deidentified, remnant diarrheal specimens were included in this study. After resolution of discordant results by toxigenic culture, the Xpert C. difficile assay displayed the highest sensitivity (100%), with a specificity of 98.8%. The sensitivity and specificity were 95.2% and 99.4% and 87% and 100% for the Verigene CDF and Simplexa Universal Direct assays, respectively. Finally, the BD MAX assay showed a sensitivity of 87% and a specificity of 98.8%. Despite differences in the overall performance of these assays, these results support the routine use of these platforms for the detection of toxigenic C. difficile in the clinical laboratory.     

Plasma cell-free metagenomic next generation sequencing in the clinical setting for the diagnosis of infectious diseases: a systematic review and meta-analysis

Plasma cell-free metagenomic next-generation sequencing (cf-mNGS) is a non-invasive method that may be able to identify thousands of pathogens through a hypothesis-free approach. There is a lack of consensus on how this test compares to conventional microbiologic testing. We conducted a systematic review and meta-analysis of published studies evaluating the accuracy of plasma cf-mNGS in hospitalized patients and present pooled estimates of the positive (PPA) and negative percent agreement (NPA) compared to a composite reference standard that included all conventional microbiological testing and clinical history as assessed by an adjudication panel or clinical treatment team. Five retrospective studies (n = 552) were included. The majority of the patients (56%?88%) were immunocompromised. The pooled PPA was 67% (95% CI, 54%?81%) and the pooled NPA was 70% (95% CI, 63%?77%). The pooled diagnostic performance characteristics suggest that cf-mNGS provides limited evidence for ruling in or out the presence of infection as commonly used.     

Comparison of a frozen human foreskin fibroblast cell assay to an enzyme immunoassay and toxigenic culture for the detection of toxigenic Clostridium difficile

This study set out to validate the Hs27 ReadyCell assay (RCCNA) as an alternative CCNA method compared against a commonly used commercial enzyme immunoassay (EIA) method and toxigenic culture (TC) reference standard. A total of 860 samples were identified from those submitted to the Health Protection Agency microbiology laboratories over a 30-week period. RCCNA performed much better than EIA when using TC as a gold standard, with sensitivities of 90.8% versus 78.6% and positive predictive value of 87.3% to 81.9%, respectively. The Hs27 Human Foreskin Fibroblast ReadyCells are an easy-to-use and a sensitive CCNA method for the detection of toxigenic Clostridium difficile directly from stool. A turnaround time of up to 48 h for a negative result and possible need for repeat testing make it an unsuitable method to be used in most clinical laboratory setting.     

Detection of toxigenic Clostridium difficile in diarrheal stools by rapid real-time polymerase chain reaction

The Cepheid Xpert polymerase chain reaction assay (Sunnyvale, CA) had a sensitivity of 100%, specificity of 96.7%, and positive and negative predictive values of 90.5% and 100%, respectively, compared with toxigenic culture for the laboratory diagnosis of Clostridium difficile in diarrheal stool samples. This test appears to be a significant improvement to poorly performing enzyme immunoassays.     

Kibdelomycin Is a Potent and Selective Agent against Toxigenic Clostridium difficile

Clostridium difficile is the causative agent of C. difficile-associated diarrhea (CDAD), with increased risk in elderly populations. Kibdelomycin, a novel natural-product inhibitor of type II topoisomerase enzymes, was evaluated for activity against C. difficile and gastrointestinal anaerobic organisms. Toxigenic C. difficile isolates (n = 168) from U.S. hospitals and anaerobic Gram-positive and Gram-negative organisms (n = 598) from Chicago-area hospitals were tested. Kibdelomycin showed potent activity against toxigenic C. difficile (MIC₉₀ = 0.25 μg/ml) and most Gram-positive aerobic organisms but had little activity against Bacteroides species (MIC₅₀ > 32 μg/ml; n = 270). Potent anti-C. difficile activity was also observed in the hamster model of C. difficile colitis. Dosing at 1.6 mg/kg (twice-daily oral dose) resulted in protection from a lethal infection and a 2-log reduction in C. difficile cecal counts. A 6.25-mg/kg twice-daily oral dose completely eliminated detectable C. difficile counts in cecal contents. A single 6.25-mg/kg oral dose showed that cecal contents were exposed to the drug at >2 μM (eightfold higher than the MIC), with no significant plasma exposure. These findings support further exploration of kibdelomycin for development of an anti-C. difficile agent.     

Subinhibitory Concentrations of LFF571 Reduce Toxin Production by Clostridium difficile

LFF571 is a novel semisynthetic thiopeptide antibacterial that is undergoing investigation for safety and efficacy in patients with moderate Clostridium difficile infections. LFF571 inhibits bacterial protein synthesis by interacting with elongation factor Tu (EF-Tu) and interrupting complex formation between EF-Tu and aminoacyl-tRNA. Given this mechanism of action, we hypothesized that concentrations of LFF571 below those necessary to inhibit bacterial growth would reduce steady-state toxin levels in C. difficile cultures. We investigated C. difficile growth and toxin A and B levels in the presence of LFF571, fidaxomicin, vancomycin, and metronidazole. LFF571 led to strain-dependent effects on toxin production, including decreased toxin levels after treatment with subinhibitory concentrations, and more rapid declines in toxin production than in inhibition of colony formation. Fidaxomicin, which is an RNA synthesis inhibitor, conferred a similar pattern to LFF571 with respect to toxin levels versus viable cell counts. The incubation of two toxigenic C. difficile strains with subinhibitory concentrations of vancomycin, a cell wall synthesis inhibitor, increased toxin levels in the supernatant over those of untreated cultures. A similar phenomenon was observed with one metronidazole-treated strain of C. difficile. These studies indicate that LFF571 and fidaxomicin generally result in decreased C. difficile toxin levels in culture supernatants, whereas treatment of some strains with vancomycin or metronidazole had the potential to increase toxin levels. Although the relevance of these findings remains to be studied in patients, reducing toxin levels with sub-growth-inhibitory concentrations of an antibiotic is hypothesized to be beneficial in alleviating symptoms.     

Prospective comparison of a commercial multiplex real-time polymerase chain reaction and an enzyme immunoassay with toxigenic culture in the diagnosis of Clostridium difficile–associated infections

Clostridium difficile infections (CDI) is a leading cause of nosocomial infections worldwide. The changes in the epidemiology of CDI during the past years, including the appearance of new epidemic strains of C. difficile that cause CDI episodes with increased severity, have led to the development of molecular methods with improved sensitivity and specificity. This study was designed to compare the performances of one antigen assay (Vidas, bioMérieux) and one molecular assay (GeneXpert, Cepheid). Fecal specimens from hospitalized patients (n = 230) suspected of having CDI were tested by both assays. Eleven specimens were positive and 202 were negative for both methods. After discrepant analysis by C. difficile toxigenic culture with broth enrichment and neutralization assay, the total numbers of stool specimens classified as positive and negative for toxigenic C. difficile were 23 (10%) and 206 (89.6%), respectively. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value for GeneXpert were 91.7%, 99%, 91.7%, and 99%, and for Vidas were 48%, 99%, 84.6%, and 94.5%, respectively. The sensitivity and PPV of polymerase chain reactoin GeneXpert assay far exceeded those of the EIA Vidas assay. The clinical characteristics of concordant and discrepant study patients were similar with the exception of the number of previous CDI episodes, which were higher in the concordant study patients; the clinical characteristics of both groups were similar. In conclusion, due to the appearance of more virulent strains of C. difficile during the last years that have produced dramatic changes in the epidemiology of C. difficile, we recommend that toxin enzyme immunoassays be replaced with rapid molecular-based tests for toxigenic C. difficile.     

Evaluation of a simultaneous detection kit for the glutamate dehydrogenase antigen and toxin A/B in feces for diagnosis of <Emphasis Type="Italic">Clostridium difficile</Emphasis> infection

Rapid detection kits for toxin A/B in feces are widely used as a diagnostic tool for Clostridium difficile infection (CDI). Their low sensitivity, however, has been considered a problem. In this study, we evaluated a new rapid diagnostic kit for simultaneous detection of the glutamate dehydrogenase (GDH) antigen and toxin A/B, C. DIFF QUIK CHEK COMPLETE. A total of 60 stool specimens from 60 patients with antibiotic-associated diarrhea were examined. Using C. difficile culture as the reference method, the GDH portion of this kit indicated a sensitivity, specificity, and negative predictive value of 100, 93.3, and 100%, respectively. The toxin A/B portion showed a sensitivity and specificity of 78.6 and 96.9%, respectively, compared to the culture results of toxin B-positive C. difficile (toxigenic culture). Of the 23 specimens that showed “dual positives” for GDH and toxin A/B, 22 were toxigenic culture positive, whereas C. difficile culture was negative in all the 28 specimens that showed “dual negatives” for GDH and toxin A/B. Of the nine “GDH-positive and toxin A/B-negative” specimens, six exhibited positive results by toxigenic culture. Results showing “dual positives” and “dual negatives” for GDH and toxin A/B can be reported as “true positive” and “true negative,” respectively, whereas additional testing for confirmation, such as toxigenic culture, is required for specimens with discrepant results. Diagnostic algorithms, utilizing the simultaneous detection kit for GDH and toxin A/B as an initial screening test, may be useful for accurate and efficient diagnosis of CDI as well as the control of healthcare-associated infections.     

Clostridium difficile Isolates with High Linezolid MICs Harbor the Multiresistance Gene cfr

We studied the molecular mechanisms of linezolid resistance in 9 isolates of toxigenic Clostridium difficile with high linezolid MICs. The activity of linezolid was determined against 891 clinical isolates of toxigenic C. difficile. The MIC₅₀ and MIC₉₀ of linezolid were 0.75 μg/ml and 1.5 μg/ml, respectively. Nine strains (1%) showed high linezolid MICs (6 μg/ml to 16 μg/ml) and also were resistant to clindamycin, erythromycin, and chloramphenicol. These strains were selected for molecular studies: sequencing of domain V of the 23 rRNA gene, detection of the cfr methyltransferase gene, and sequencing of the ribosomal protein genes rplC and rplD. Molecular relatedness between strains was assessed using PCR ribotyping and MLVA (multilocus variable-number tandem-repeat analysis) typing. The strains belonged to ribotypes 001 (2/9), 017 (6/9), and 078 (1/9). MLVA showed that strains of ribotype 001 and 017 belonged to the same clonal complex in each ribotype. We did not detect mutations in the 23S rRNA gene. The cfr gene was detected in 7 of 9 strains. Sequencing of cfr amplicons revealed a similarity of 100% to a fragment of transposon Tn6218 of C. difficile, which was annotated as a putative chloramphenicol/florfenicol resistance protein. We were unable to detect mechanisms of resistance to linezolid in the 2 strains belonging to ribotype 001. While the relevance of our results lies in the detection of the cfr gene as a possible mechanism of resistance to linezolid in C. difficile, our findings should be assessed by further investigations to characterize these possible cfr genes and their contribution to linezolid resistance.