Human Monoclonal Antibodies against Clostridium difficile Toxins A and B Inhibit Inflammatory and Histologic Responses to the Toxins in Human Colon and Peripheral Blood Monocytes

Clostridium difficile infection (CDI) is a common and debilitating nosocomial infection with high morbidity and mortality. C. difficile mediates diarrhea and colitis by releasing two toxins, toxin A and toxin B. Since both toxins stimulate proinflammatory signaling pathways in human colonocytes and both are involved in the pathophysiology of CDI, neutralization of toxin A and B activities may represent an important therapeutic approach against CDI. Recent studies indicated that human monoclonal antibodies (MAbs) against toxins A and B reduce their cytotoxic and secretory activities and prevent CDI in hamsters. Moreover, anti-toxin A and anti-toxin B MAbs together with antibiotics also effectively reduced recurrent CDI in humans. However, whether these MAbs neutralize toxin A- and toxin B-associated immune responses in human colonic mucosa or human peripheral blood monocyte cells (PBMCs) has never been examined. We used fresh human colonic biopsy specimens and peripheral blood monocytes to evaluate the effects of these antibodies against toxin A- and B-associated cytokine release, proinflammatory signaling, and histologic damage. Incubation of anti-toxin A (MK3415) or anti-toxin B (MK6072) MAbs with human PBMCs significantly inhibited toxin A- and toxin B-mediated tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) expression. MK3415 and MK6072 also diminished toxin A- and toxin B-mediated NF-κB p65 phosphorylation in human monocytes, respectively, and significantly reduced toxin A- and B-induced TNF-α and IL-1β expression as well as histologic damage in human colonic explants. Our results underline the effectiveness of MK3415 and MK6072 in blocking C. difficile toxin A- and toxin B-mediated inflammatory responses and histologic damage.     

Outcomes in patients tested for Clostridium difficile toxins

Clostridium difficile testing is shifting from toxin detection to C. difficile detection. Yet, up to 60% of patients with C. difficile by culture test negative for toxins and it is unclear whether they are infected or carriers. We reviewed medical records for 7046 inpatients with a C. difficile toxin test from 2005 to 2009 to determine the duration of diarrhea and rate of complications and mortality among toxin-positive (toxin+) and toxin− patients. Overall, toxin− patients had less severe diarrhea, fewer diarrhea days, and lower mortality (P < 0.001, all comparisons) than toxin+ patients. One toxin− patient (n = 1/6121; 0.02%) was diagnosed with pseudomembranous colitis, but there were no complications such as megacolon or colectomy for fulminant CDI among toxin− patients. These data suggest that C. difficile–attributable complications are rare among patients testing negative for C. difficile toxins. More studies are needed to evaluate the clinical significance of C. difficile detection in toxin− patients.     

Impact of toxigenic Clostridium difficile polymerase chain reaction testing on the clinical microbiology laboratory and inpatient epidemiology

Conversion from Clostridium difficile toxin A/B EIA to tcdB polymerase chain reaction for diagnosis of C. difficile infection (CDI) resulted in significant decreases in laboratory testing volume and largely unchanged C. difficile toxin detection rates. Decreases in healthcare-associated CDI rates (P ≤ 0.05) reflected a clinical practice benefit of this conversion.     

Usefulness of a newly developed high-speed polymerase chain reaction analysis system for the diagnosis of Clostridioides difficile infection

IntroductionThe incidence of Clostridioides difficile infection (CDI) has been continuously increasing and thereby became an important issue worldwide. Appropriate diagnosis, management, and infection control are required for patients with CDI. Enzyme immunoassay (EIA) is a widely used standard diagnostic tool for C. difficile-specific glutamate dehydrogenase (GDH) and C. difficile toxins (toxins A and B). However, the sensitivity of EIA in detecting C. difficile toxins has been reported to be relatively low, resulting in CDI underdiagnosis. Therefore, nucleic acid amplification tests (NAAT) are recently developed for higher sensitivity/specificity test.MethodsIn this study, a total of 279 stool samples submitted for CDI diagnosis were examined using an independently developed new high-speed polymerase chain reaction (PCR) device (PathOC RightGene, Metaboscreen). In parallel, results were compared with those of definitive diagnosis and conventional diagnostic methods (EIA, real-time PCR) to assess the inspection accuracy.ResultsPathOC RightGene showed high sensitivity (96.7%) and specificity (96.7%). Regarding the measurement time, C. difficile-specific and C. difficile toxin genes were simultaneously detected in approximately 25 min for one sample (including the preprocessing and measurement time).ConclusionPathOC RightGene has been found to show both excellent sensitivity and rapidity and thus can be used for the reliable and early diagnosis, which are needed for the appropriate management of CDI.     

Rare toxin A-negative and toxin B-positive strain of Clostridioides difficile from Japan lacking a complete tcdA gene

IntroductionClostridioides difficile (C. difficile) produces three kinds of toxins: toxin A (enterotoxin), toxin B (cytotoxin), and C. difficile transferase (CDT), a binary toxin. Some strains show positivity only for toxin B. These strains reportedly possess a gene for toxin A, tcdA. However, toxin A production is inhibited due to a mutated stop codon and/or deletion within the tcdA gene. Here for the first case in Japan, we describe toxin genomes and proteins of a strain possessing only toxin B and lacking a complete tcdA gene, along with clinical manifestations.MethodsC. difficile was isolated from the bloody stool of a 60-year-old female patient treated with meropenem. Although a rapid detection kit of toxins (C. DIFF QUIK CHEK COMPLETE®, TechLab, Blacksburg, VA, USA) showed positivity, Western blotting detected no toxins. Therefore, we explored the strain's toxin genes and their sequences to determine whether the strain possessed a toxin.ResultsPolymerase chain reaction did not identify toxin genes. Whole-genome sequencing analysis showed that a gene for toxin A, tcdA, was completely deleted in the strain. Moreover, 701 mutations and some deletions/insertions were identified on the tcdB gene.ConclusionsWe isolated a rare strain of C. difficile producing only toxin B and lacking a complete tcdA gene herein Japan. The possibility of a false negative needs to be considered with a genetic method for a diagnose of C. difficile infection.     

Fidaxomicin Inhibits Clostridium difficile Toxin A-Mediated Enteritis in the Mouse Ileum

Clostridium difficile infection (CDI) is a common, debilitating infection with high morbidity and mortality. C. difficile causes diarrhea and intestinal inflammation by releasing two toxins, toxin A and toxin B. The macrolide antibiotic fidaxomicin was recently shown to be effective in treating CDI, and its beneficial effect was associated with fewer recurrent infections in CDI patients. Since other macrolides possess anti-inflammatory properties, we examined the possibility that fidaxomicin alters C. difficile toxin A-induced ileal inflammation in mice. The ileal loops of anesthetized mice were injected with fidaxomicin (5, 10, or 20 μM), and after 30 min, the loops were injected with purified C. difficile toxin A or phosphate-buffered saline alone. Four hours after toxin A administration, ileal tissues were processed for histological evaluation (epithelial cell damage, neutrophil infiltration, congestion, and edema) and cytokine measurements. C. difficile toxin A caused histologic damage, evidenced by increased mean histologic score and ileal interleukin-1β (IL-1β) protein and mRNA expression. Treatment with fidaxomicin (20 μM) or its primary metabolite, OP-1118 (120 μM), significantly inhibited toxin A-mediated histologic damage and reduced the mean histology score and ileal IL-1β protein and mRNA expression. Both fidaxomicin and OP-1118 reduced toxin A-induced cell rounding in human colonic CCD-18Co fibroblasts. Treatment of ileal loops with vancomycin (20 μM) and metronidazole (20 μM) did not alter toxin A-induced histologic damage and IL-1β protein expression. In addition to its well known antibacterial effects against C. difficile, fidaxomicin may possess anti-inflammatory activity directed against the intestinal effects of C. difficile toxins.     

Reevaluation of the Premier Clostridium difficile toxin A and B immunoassay with comparison to glutamate dehydrogenase common antigen testing evaluating Bartels cytotoxin and Prodesse ProGastro Cd polymerase chain reaction as confirmatory procedures

Enzyme immunoassays are currently the most common tests used in the clinical laboratory for the detection of Clostridium difficile toxins; however, significant problems with their performance have recently been described. We prospectively reevaluated the Meridian Premier C. difficile toxin A/B assay with direct comparison to a 2-step algorithm that screened for C. difficile common antigen and compared cytotoxin and real-time polymerase chain reaction (PCR) as confirmatory procedures. The Premier assay lacked sufficient sensitivity, missing 25% of true-positive samples. PCR was the most sensitive method and the only procedure that allowed same day testing and reporting.     

A simple 3-step algorithm for improved laboratory detection of Clostridium difficile toxin without the need for tissue culture cytotoxicity neutralization assays

This report describes a 3-step algorithm for the detection of Clostridium difficile using an enzyme immunoassay for glutamate dehydrogenase (GDH) antigen, a lateral flow assay for C. difficile toxin, and anaerobic broth culture. This method was found to detect more toxin-containing stool samples than the use of a combination GDH antigen plus toxin A/B testing alone.     

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.     

A retrospective study of the epidemiology of Clostridium difficile infection at a University Hospital in Japan: genotypic features of the isolates and clinical characteristics of the patients

Clostridium difficile is a major cause of antibiotic-associated diarrhea and frequently results in healthcare-associated infections. The epidemiology of C. difficile infection (CDI), including the prevalent polymerase chain reaction (PCR) ribotypes and the clinical characteristics of the patients, is not well known in Japan, compared to the situation in the United States and Europe. We performed PCR ribotyping of C. difficile isolates from 71 consecutive patients with CDI at a University Hospital over a 3-year period and investigated the clinical features of those patients. CDI was diagnosed when a patient with diarrhea or colitis was found to have toxin B-positive C. difficile with no other enteropathogenic microorganisms. Toxin A-positive, toxin B-positive, binary toxin-positive (A⁺B⁺CDT⁺) strains; toxin A-positive, toxin B-positive, binary toxin-negative (A⁺B⁺CDT^?) strains; and toxin A-negative, toxin B-positive, binary toxin-negative (A^?B⁺CDT^?) strains were isolated from 4, 58, and 9 patients, respectively, indicating that infections with binary toxin-positive strains were uncommon (5.6%). PCR ribotyping of the isolates demonstrated that among the 71 strains, 20 different PCR ribotypes were identified and that types smz, yok, and hr were predominant (19, 14, and 13 isolates, respectively), all of which were A⁺B⁺CDT^?. No specific time periods or wards were found to be associated with the three types; PCR ribotyping analysis clearly showed that the three types spread almost evenly in all wards for the 3 years studied. Comparative analysis of the clinical characteristics of patients harboring the three C. difficile types indicated that the duration of CDI was longer in the yok group than in the hr group. PCR ribotyping, which is easy to perform, appears to give us useful information to trace CDI cases in clinical settings. Further, the analysis of a large number of CDI cases may allow evaluation of the possible relationship between specific C. difficile types and the clinical features of patients.     

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.     

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.     

Molecular epidemiologic study of Clostridium difficile infections in university hospitals: Results of a nationwide study in Japan

We conducted a nationwide molecular epidemiological study of Clostridium difficile infection (CDI) in Japan investigated the correlation between the presence of binary toxin genes and CDI severity. This is the first report on molecular epidemiological analyses for CDI in multiple university hospitals in Japan, to our knowledge. We examined 124,484 hospitalized patients in 25 national and public university hospitals in Japan between December 2013 and March 2014, investigating antimicrobial susceptibilities and toxin-related genes for C. difficile isolates from stools. Epidemiological genetic typing was performed by PCR-ribotyping and repetitive sequence-based (rep)-PCR to examine the genetic similarities. The results detected toxin A-positive, toxin B-positive, binary toxin-negative (A⁺B⁺CDT^?) detected from 135 isolates (80.8%) and toxin A-negative, toxin B-positive, binary toxin-negative (A^??B⁺CDT^?) in 23 (13.8%). Toxin A-positive, toxin B-positive, and binary toxin-positive (A⁺B⁺CDT⁺) were seen in 9 isolates (5.4%). Vancomycin (n = 81, 37.7%) or metronidazole (n = 88, 40.9%) therapies were undertaken in analyzed cases. Ribotypes detected from isolates were 017/subgroup 1, 070, 078, 126, 176, 449, 475/subgroup 1, 499, 451, 566 and newtypes. Rep-PCR classified 167 isolates into 28 cluster groups including 2–15 isolates. In addition, 2 pairs of strains isolated from different institutions belonged to the same clusters. Seven out of 9 (77.8%) of the patients with binary toxin producing strains had “mild to moderate” outcome in evaluated symptoms. In conclusion, we found that binary toxin did not show regional specificity and had no relevance to severity of CDI.     

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.     

Actoxumab + bezlotoxumab combination: what promise for Clostridium difficile treatment?

Introduction: Clostridium difficile infection (CDI) is the most common healthcare-associated infection worldwide. As standard CDI antibiotic therapies can result in unacceptably high recurrence rates, novel therapeutic strategies for CDI are necessary. A recently emerged immunological therapy is a monoclonal antibody against C. difficile toxin B.

Areas covered: In this review, the authors summarize the available pharmacological, preclinical, and clinical data for the CDI treatment based on anti-toxin A (actoxumab) and anti-toxin B (bezlotoxumab) human monoclonal antibodies (HuMabs), and discuss about the potentiality of a therapy that includes HuMab combined administration for CDI.

Expert opinion: Although only bezlotoxumab is indicated to reduce recurrence of CDI, experimental studies using a combination of HuMabs actoxumab and bezlotoxumab have shown that bolstering the host immune response against both the C. difficile toxins may be effective in primary and secondary CDI prevention. Besides neutralizing both the key virulence factors, combination of two HuMabs could potentially offer an advantage for a yet to emerge C. difficile strain, which is a steady threat for patients at high risk of CDI. However, as actoxumab development was halted, passive immunotherapy with actoxumab/bezlotoxumab is actually impracticable. Future research will be needed to assess HuMab combination as a therapeutic strategy in clinical and microbiological cure of CDI.     

Broad Coverage of Genetically Diverse Strains of Clostridium difficile by Actoxumab and Bezlotoxumab Predicted by In Vitro Neutralization and Epitope Modeling

Clostridium difficile infections (CDIs) are the leading cause of hospital-acquired infectious diarrhea and primarily involve two exotoxins, TcdA and TcdB. Actoxumab and bezlotoxumab are human monoclonal antibodies that neutralize the cytotoxic/cytopathic effects of TcdA and TcdB, respectively. In a phase II clinical study, the actoxumab-bezlotoxumab combination reduced the rate of CDI recurrence in patients who were also treated with standard-of-care antibiotics. However, it is not known whether the antibody combination will be effective against a broad range of C. difficile strains. As a first step toward addressing this, we tested the ability of actoxumab and bezlotoxumab to neutralize the activities of toxins from a number of clinically relevant and geographically diverse strains of C. difficile. Neutralization potencies, as measured in a cell growth/survival assay with purified toxins from various C. difficile strains, correlated well with antibody/toxin binding affinities. Actoxumab and bezlotoxumab neutralized toxins from culture supernatants of all clinical isolates tested, including multiple isolates of the BI/NAP1/027 and BK/NAP7/078 strains, at antibody concentrations well below plasma levels observed in humans. We compared the bezlotoxumab epitopes in the TcdB receptor binding domain across known TcdB sequences and found that key substitutions within the bezlotoxumab epitopes correlated with the relative differences in potencies of bezlotoxumab against TcdB of some strains, including ribotypes 027 and 078. Combined with in vitro neutralization data, epitope modeling will enhance our ability to predict the coverage of new and emerging strains by actoxumab-bezlotoxumab in the clinic.     

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.     

Real-time cellular analysis for quantitative detection of functional Clostridium difficile toxin in stool

Rapid and accurate diagnosis and monitoring of Clostridium difficile infection (CDI) is critical for patient care and infection control. We will briefly review current laboratory techniques for the diagnosis of CDI and identify aspects needing improvement. We will also introduce a real-time cellular analysis (RTCA) assay developed for the diagnosis and monitoring of CDI using electronic impedance to assess the cell status. The RTCA assay uses impedance measurement to detect minute physiological changes in cells cultured on gold microelectrodes embedded in glass substrates in the bottom of microtiter wells. This assay has been adapted for quantitative detection of C. difficile functional toxin directly from stool specimens. Compared to conventional techniques and molecular assays, the RTCA assay provides a valuable tool for the diagnosis of CDI as well as for the assessment of clinical severity and for monitoring therapeutic efficacies.     

Effects of Tigecycline and Vancomycin Administration on Established Clostridium difficile Infection

The glycylcycline antibiotic tigecycline was approved in 2005 for the treatment of complicated skin and soft tissue infections and complicated intra-abdominal infections. Tigecycline is broadly active against both Gram-negative and Gram-positive microorganisms, including Clostridium difficile. Tigecycline has a low MIC against C. difficile in vitro and thus may represent an alternate treatment for C. difficile infection (CDI). To assess the use of tigecycline for treatment of established CDI, 5- to 8-week-old C57BL/6 mice were colonized with C. difficile strain 630. After C. difficile colonization was established, mice (n = 10 per group) were treated with either a 5-day course of tigecycline (6.25 mg/kg every 12 h subcutaneously) or a 5-day course of vancomycin (0.4 mg/ml in drinking water) and compared to infected, untreated control mice. Mice were evaluated for clinical signs of CDI throughout treatment and at 1 week posttreatment to assess potential for disease development. Immediately following a treatment course, C. difficile was not detectable in the feces of vancomycin-treated mice but remained detectable in feces from tigecycline-treated and untreated control mice. Toxin activity and histopathological inflammation and edema were observed in the ceca and colons of untreated mice; tigecycline- and vancomycin-treated mice did not show such changes directly after treatment. One week after the conclusion of either antibiotic treatment, C. difficile load, toxin activity, and histopathology scores increased in the cecum and colon, indicating that C. difficile-associated disease occurred. In vitro growth studies confirmed that subinhibitory concentrations of tigecycline were able to suppress toxin activity and spore formation of C. difficile, whereas vancomycin did not. Taken together, these data show how tigecycline is able to alter C. difficile pathogenesis in a mouse model of CDI.