Abstract
Background
Hospitalized patients with diarrhea who have a negative Clostridoides difficile (C. difficile) test are not routinely evaluated for alternative causes of infectious diarrhea. This study assessed for potential infectious causes of diarrhea in hospitalized patients with an order for repeat C. difficile toxin enzyme immunoassay (tEIA) testing after an initial tEIA test was negative.
Methods
For patients age ≥18 years who had a second C. difficile tEIA test ordered within 96 h after a negative tEIA test, remnant fecal specimens from the first (negative) tEIA test were evaluated using the BioFire FilmArray Gastrointestinal Panel PCR, C. difficile toxigenic culture, and culture on a blood agar plate (BAP) to identify other potential causes of infectious diarrhea. Growth of organisms on the BAP was also used to assess potential disruptions in the gastrointestinal microbiota.
Results
Among 84 remnant specimens, toxigenic C. difficile was identified in 9 (11%) by culture or PCR, while potential alternative causes of infectious diarrhea, including norovirus, rotavirus, enteropathogenic Escherichia coli, and Salmonella, were identified in 11 specimens (13%) by PCR. For the majority of patients, no infectious cause of diarrhea was identified, but 84% exhibited disrupted gastrointestinal microbiota, which may contribute to diarrhea.
Conclusions
When a hospitalized patient has a negative C. difficile tEIA test but continues to have diarrhea, alternative infectious and noninfectious causes of diarrhea should be considered. If the patient has clinical signs and symptoms suggestive of infection or risk factors for gastrointestinal infection, laboratory testing for other etiologic agents may be appropriate.
Keywords: diarrhea, Clostridoides difficile, infectious diarrhea
Introduction
IMPACT STATEMENT.
The findings of this study indicate that, for hospitalized patients with diarrhea who have a negative test for Clostridoides difficile, alternative causes of diarrhea, including the potential for other infectious causes, should be considered. Identification of non-Clostridoides difficile causes of infectious diarrhea would impact infection prevention, patient care, and treatment options.
Clostridoides difficile (C. difficile) is the most common infectious cause of healthcare-associated diarrhea in the United States (1). Although other microbes can cause diarrhea in hospitalized patients (2), due to the low likelihood of acquiring an enteric or food-borne pathogen in the hospital, alternative infectious causes of diarrhea are often not considered when C. difficile is suspected. This remains true even after a negative initial C. difficile test, with studies indicating that it is common practice to repeat C. difficile testing multiple times before considering it to be a true negative, rather than considering other causes of diarrhea (1). In addition, a 3-day rule is a common policy in microbiology laboratories, limiting the use of diagnostic tests on feces for infectious pathogens other than C. difficile in patients who have been hospitalized for >3 days (3). Our objective was to assess for alternative infectious causes of diarrhea in hospitalized patients for whom clinicians had ordered repeat C. difficile toxin enzyme immunoassay (tEIA) testing after an initial tEIA test was negative. This is of clinical importance to understand potential alternative causes of diarrhea in patients with an initial negative test for C. difficile. Identification of non-C. difficile causes of infectious diarrhea may impact patient care and treatment options, as well as infection control and prevention.
Methods
This study was conducted at a 1260-bed academic medical center as part of a larger quality improvement project (4, 5). The study protocol was reviewed by the Washington University Human Research Protection Office, which determined that it did not constitute human subjects research. Between November 27, 2014 and August, 20, 2015, we identified hospitalized patients age ≥ 18 years, who had a second C. difficile tEIA test (C. difficile Tox A/B II EIA, TechLab) ordered within 96 h after a negative tEIA test.
Among the patients who received repeat C. difficile tEIA testing, those with remnant fecal specimens associated with the first (negative) tEIA test were identified. Following collection in a sterile cup, fecal specimens were stored at 4 °C for up to 4 days, and then aliquots were made and stored at −80°C until further analysis. Specimens were evaluated using the BioFire FilmArray Gastrointestinal (GI) Panel PCR (BioFire Diagnostics) according to manufacturer instructions. Stool specimens were processed for culture of C. difficile, as previously described (6). Briefly, a pea-sized amount of stool was placed in a microcentrifuge tube and incubated at 80 °C for 10 min. In an anaerobic chamber, the specimen was added to a tube containing cycloserine cefoxitin mannitol broth with taurocholate and lysozyme and incubated anaerobically at 35 °C for up to 7 days. After 7 days or when growth was detected (i.e., turbidity or color change in the broth), the sample was pelleted and streaked for isolation on blood agar plates (BAP). The BAP were incubated anaerobically at 35 °C for 48 to 72 h. Any colony suggestive of C. difficile was identified using VITEK matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS; bioMerieux). Toxigenic C. difficile was differentiated from nontoxigenic C. difficile using a multiplex PCR with oligonucleotide primers that amplify the tcdA and tcdB genes (5, 7).
To qualitatively assess normal or disrupted enteric microbiota, specimens were plated for microbiologic culture on to a BAP and incubated aerobically at 35 °C for 48 h, as is routine practice for fecal specimens in the clinical laboratory setting (8). The enteric microbiota was considered normal if any enteric Gram-negative bacilli were isolated. The enteric microbiota was considered disrupted if there was a pure culture of yeast or only mixed Gram-positive organisms isolated. Any mucoid colonies suggestive of Klebsiella spp. were identified using VITEK MALDI-TOF MS (bioMerieux). Any colonies suggestive of Staphylococcus aureus (S. aureus) were also confirmed using MALDI-TOF MS.
Results
A total of 2728 C. difficile tEIA tests were performed during the study period, and 2549 (93%) were negative. Among patients with a negative tEIA test, 134 (5%) had a repeat tEIA test ordered within 96 h. Six of these patients (4%) had a positive tEIA test on the repeat sample. The majority of patients who had repeat tEIA testing (115, 86%) had been hospitalized >7 days, and 92 (69%) were hospitalized for >14 days. Thirty-three (25%) had received metronidazole for empiric treatment of C. difficile infection (CDI) in the 48 h before fecal samples were collected for testing, and 20 (15%) had received oral vancomycin.
Of the patients who had repeat tEIA testing, 84 (63%) had a remnant fecal specimen from the first (negative) tEIA test that was sufficient for study testing (Table 1). Toxigenic C. difficile was identified in 9 specimens (11%) while nontoxigenic C. difficile was identified in 4 (5%). Five patients had C. difficile identified by both culture and the BioFire FilmArray GI Panel PCR; 2 were identified only by culture, and 2 were identified only by BioFire FilmArray GI Panel PCR. Among the 9 patients for whom study testing identified toxigenic C. difficile, 3 (33%) were diagnosed with CDI during their hospital admission. These were the 3 patients who had positive repeat C. difficile tEIA testing. Only 2 of these 3 patients reported diarrhea during their admission, which resolved after treatment with metronidazole.
Table 1.
Results of the additional testing of 84 initial stool samples from patients who had an order for a repeat tEIA test for C. difficile after an initial tEIA test was negative.
| Test/organism | Total by any test, n (%) | C. difficile toxigenic culture, n (%) | BioFire GI Panel PCR, n (%) |
|---|---|---|---|
| (n = 84) | n = 84 | n = 84 | |
| No organism identified | 61 (72.6) | 73 (86.9) | 67 (79.8) |
| Toxigenic C. difficile (toxin A and toxin B) | 9 (10.7) | 7 (8.3) | 7 (8.3)a |
| Non-toxigenic C. difficile | 4 (4.8) | 4 (4.8) | NA |
| Other infectious organisms by BioFire GI Panel PCR | 11 (13.1) | NAb | 11 (13.1) |
| Norovirus | 5 (6.0) | NA | 5 (6.0) |
| Enteropathogenic E. coli | 2 (2.4) | NA | 2 (2.4) |
| Enteroaggregative E. coli | 1 (1.2) | NA | 1 (1.2) |
| Campylobacter | 1 (1.2) | NA | 1 (1.2) |
| Rotavirus | 1 (1.2) | NA | 1 (1.2)a |
| Salmonella spp. | 1 (1.2) | NA | 1 (1.2) |
| Mixed gram positive microbiota on BAP | 55 (65) | NA | NA |
| Growth suggestive of normal GI microbiota on BAP | 13 (15) | NA | NA |
| No growth of aerobic microorganisms on BAP | 8 (10) | NA | NA |
| Predominant organism and/or putative pathogen detected on BAPs | |||
| Staphylococcus aureus | 3 (3.6) | NA | NA |
| Yeast species | 2 (2.4) | NA | NA |
| Enterococci | 2 (2.4) | NA | NA |
| Klebsiella oxytoca and yeast species | 1 (1.2) | NA | NA |
One patient had a Biofire GI panel that was positive for toxigenic C. difficile and rotavirus.
NA, not applicable.
Among the 6 patients who were not diagnosed with CDI at the time of index or repeat tEIA testing but had a positive result for the study toxigenic C. difficile culture, clinical outcomes varied. Patient 1 had resolution of diarrhea without CDI treatment. Patient 2 had diarrhea, which resolved after treatment with loperamide, without CDI treatment. Patient 3 had multiple subsequent hospital admissions for abdominal pain and diarrhea over the next several months with unremarkable endoscopic findings, negative tEIA testing, and no change after treatment with metronidazole. This patient was later diagnosed with chronic pancreatitis related to alcohol use. Patient 4 was diagnosed with septic shock secondary to a polymicrobial bloodstream and urinary tract infection related to a malpositioned nephrostomy tube. This patient developed diarrhea that was thought to be a side effect of antibiotic use and that resolved with loperamide. Patient 5 had a history of Churg-Strauss syndrome and presented with shortness of breath, without documented diarrhea. This patient was found to have a necrotizing pneumonia, myocardial infarction, septic shock, and subsequently developed an ileus and mild colitis. The cause of the ileus and mild colitis was unspecified by clinicians. Patient 6 was diagnosed with neutropenic typhlitis in the setting of cancer chemotherapy for diffuse large B-cell lymphoma. The patient improved after treatment with cefepime and metronidazole and, on outpatient follow-up, was noted to have no diarrhea or abdominal pain. However, 1 month later, this patient was readmitted with a line-related bloodstream infection and progressive leukemia and was diagnosed with CDI during this subsequent admission.
For 11 of the 84 specimens (13%), the BioFire FilmArray GI Panel PCR identified a microorganism potentially associated with diarrhea other than C. difficile. Norovirus was identified in 5 (6%), enteropathogenic Escherichia coli (E. coli) in 2 (2%), and enteroaggregative E. coli, Campylobacter, Salmonella, and rotavirus were each identified in 1 (1%). Among the patients who had stool specimens that were found to be positive for organisms other than C. difficile during study testing, only 3 had been in the hospital for more than 7 days at the time that their stool specimen was collected. This included 2 patients with specimens positive for norovirus, who had been admitted 22 and 56 days, respectively, prior to collection of their index stool specimen and the patient with Campylobacter, who had been admitted 9 days prior to collection of their index stool specimen. Among the 3 patients with study specimens that were positive for enteropathogenic or enteroaggregative E. coli, 2 had been admitted for 2 days at the time of index stool specimen collection and 1 had been admitted for 5 days.
The patient with Salmonella detected by BioFire FilmArray GI Panel PCR had a history of recurrent salmonella enteritis and was clinically diagnosed with Salmonella through a standard of care stool culture. The patient (age 66) with a stool specimen that was positive for rotavirus was also positive for toxigenic C. difficile, and study stool antigen testing for rotavirus (Remel Xpect) was positive. The patient had presented with vomiting and diarrhea and was diagnosed with a nonspecific gastroenteritis, which resolved without CDI treatment. None of the other 9 patients who had organisms other than C. difficile detected by BioFire FilmArray GI Panel PCR had a diagnostic test ordered for the aforementioned organisms within 30 days of C. difficile tEIA testing. For these patients, the cause of their symptoms remained undetermined or was attributed to antibiotics by their treating clinicians.
A total of 84 samples underwent microbiologic culture for assessment of normal gastrointestinal microbiota (Table 1). Only 13 (16%) exhibited normal enteric microbiota. The rest (84%) exhibited microbiologic growth suggestive of disruption in normal gastrointestinal flora. The majority of specimens (55, 65%) exhibited Gram-positive microbiota, and 8 (10%) exhibited no growth of aerobic bacteria. S. aureus was identified in 3 samples and Klebsiella oxytoca (K. oxytoca) in 1; these organisms have been postulated to cause a C. difficile-like diarrhea.
Of the 3 patients with S. aureus identified as the predominant organism on BAPs, 1 had a recent history of bloodstream infection due to S. aureus. The other 2 patients did not have a clinical or surveillance culture positive for S. aureus within 30 days before or after the date of the positive BAP. One patient had a pelvic abscess and sacral osteomyelitis due to Streptococcus anginosus and mixed microbes, and 1 had influenza B. The patient with K. oxytoca as the predominant organism on BAP was diagnosed with a liver abscess and small bowel perforation, with peritoneal fluid cultures positive for Candida albicans and E. coli; no clinical cultures were positive for K. oxytoca.
Discussion
In this study focusing on patients for whom clinicians had heightened suspicion of CDI, additional testing of the original (tEIA negative) stool specimens identified toxigenic C. difficile in 9 (11%) of patients, only 3 of whom were identified by repeat tEIA testing. A potential alternative cause of infectious diarrhea was identified in 11 patients (13%). Additionally, our results indicate that 84% of the patients in this study exhibited a shift in normal gastrointestinal flora, which may contribute to diarrhea. When a hospitalized patient has a negative C. difficile tEIA test but continues to have diarrhea, alternative infectious and noninfectious causes of diarrhea are considered. If the patient has clinical signs and symptoms suggestive of infection or risk factors for GI infection, laboratory testing for other etiologic agents may be appropriate.
For hospitalized patients with diarrhea, not having a specific diagnosis can hinder appropriate management and interfere with infection control (9). Diagnostics should be utilized with the clinical picture in mind, as multiplex PCR testing may increase diagnostic yield but can also detect organisms of uncertain clinical importance (10, 11). It is important to use a syndromic-based approach to the diagnosis of infectious diarrhea, specifically through a thorough interview and physical exam and then ordering a diagnostic test directed toward the organisms of concern.
Other studies that have used multiplex PCR testing to examine stool specimens from hospitalized patients with diarrhea have identified a range of pathogens, including norovirus and E. coli (12, 13). Nevertheless, the low positivity rate of the BioFire Film Array GI Panel PCR compounded with the potential for false positives has called into question its use in hospitalized patients with diarrhea (14). A study by Hitchcock et al. reported a BioFire Film Array positivity rate of 4.8% (20/414) in patients hospitalized for >72 h, with no testing restrictions based on symptoms; however, the rate dropped to 3.0% once false positives and chronic viral shedders were excluded (14). Our study was smaller and found a positivity rate of 13%, not excluding potential viral shedders, when testing was restricted to patients suspected of CDI undergoing a repeat tEIA test. In another study that also reexamined stool specimens from hospitalized patients who had tested negative for C. difficile, at least 1 unsuspected agent of infectious gastroenteritis was identified in 22% of specimens (15). As in our study, the majority of patient specimens were negative for infectious pathogens.
For the majority of the patients included in our study, no infectious causes of diarrhea were identified, although the majority exhibited culture-based evidence of GI microbiota disruption. Current guidelines for the diagnosis and management of patients with infectious diarrhea recommend that in situations where a pathogen has not been identified, stool samples be reevaluated if there is evidence of systemic symptoms in an attempt to identify an undetected pathogen (2). While it is possible that some of our patients may have had an unidentified infectious agent, negative testing points toward the diagnosis of other, noninfectious conditions associated with diarrhea (5, 9).
Strengths of this study include the real-world hospital population and the use of additional diagnostic testing to evaluate for alternative infectious causes of diarrhea. This study also had limitations, including retrospective testing of stored specimens, which could have negatively impacted the recovery of organisms, and a lack of patient clinical information that might aid in data interpretation. Overall, our sample size was small, and further study is warranted to determine the utility of adding additional infectious diseases testing for hospitalized patients with persistent diarrhea. The presence of norovirus in 6% of the stool specimens is of unclear significance but could indicate viral shedding or symptomatic disease. Although there is no specific therapeutic intervention for norovirus, detecting this virus it could have implications regarding infection prevention, contact precautions, and room cleaning.
Potential approaches to improve syndromic-based diagnostic testing for evaluation of non-CDI causes of infectious diarrhea include (a) if patients continue to have clinical signs and symptoms suggestive of infection, not rejecting testing for infectious agents on fecal specimens from patients who have been in the hospital for >3 days; (b) recommending expanded infectious diagnostic testing for hospitalized patients with worsening diarrhea without a clear noninfectious cause or significant clinical suspicion (3); and (c) utilizing the electronic health record to set alerts when patients have had recent tEIA testing, prompting the consideration of alternative causes of diarrhea (4).
In summary, although further study is necessary to guide policies surrounding the evaluation of infectious causes of diarrhea in hospitalized patients, the results of this study suggest that clinicians and laboratorians should have a heightened suspicion for alternative infectious and noninfectious causes of diarrhea in patients with an initial negative test for C. difficile.
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 4 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved.
Nonstandard Abbreviations: C. difficile; Clostridoides difficile, tEIA; C. difficile toxin enzyme immunoassay; GI, gastrointestinal; BAP, blood agar plate; CDI, C. difficile infection; S. aureus, Staphylococcus aureus; K. oxytoca, Klebsiella oxytoca; E. coli, Escherichia coli.
Authors’ Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest: Employment or Leadership: None declared. Consultant or Advisory Role: C.-A.D. Burnham, Cepheid. Stock Ownership: None declared. Honoraria: C.-A.D. Burnham, BioFire, Roche. Research Funding: J.H. Kwon, the Washington University Institute of Clinical and Translational Sciences and the National Center for Advancing Translational Sciences (NCATS) of the National Institutes of Health [UL1 TR000448, sub-award KL2 TR000450] and the National Institute of Allergy and Infectious Diseases [1K23 AI137321-01A1]. C.-A.D. Burnham, grants from Cepheid, bioMerieux, Luminex, BioFire to institution. Expert Testimony: None declared. Patents: None declared.
Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, preparation of manuscript, or final approval of manuscript.
This manuscript contains original material that is not already available elsewhere. Preliminary data from this study was presented in part at ID Week 2017 10/5/2017, San Diego, CA.
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