Clostridium difficile: association with thrombocytosis and leukocytosis.

Background: Apart from leukocytosis, few laboratory markers suggestive of Clostridium difficile infections have been described.

Methods: We retrospectively analyzed the association between thrombocytosis, leukocytosis and C difficile infections at the Atlanta Veterans Affairs Medical Center.

Results: Of 162 patients with C difficile infection, 36 (22%) had thrombocytosis, and 97 (60%) had leukocytosis. C difficile toxin A ELISA was performed in 46/695 (6.6%) patients with thrombocytosis and was positive in 18 (39.1%). Leukocytosis was present in 16/18 (89%) of patients with positive C difficile toxin A ELISA and thrombocytosis, but also in 21/28 (75%) of patients with negative C difficile toxin A ELISA and thrombocytosis. Among patients with marked leukocytosis, C difficile toxin A was more frequently detected in those with concomitant thrombocytosis (P = 0.07).

Conclusions: The presence of thrombocytosis may be helpful to improve the pretest probability for C difficile infections.

Key Words: Clostridium difficile, thrombocytosis, leukocytosis

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Infection with Clostridium difficile is the leading cause of antibiotic-associated and pseudomembranous diarrhea affecting up to 25% of patients receiving [beta]-lactam antibiotics. (1) Its clinical presentation varies from mild diarrhea that subsides spontaneously with withdrawal of the offending agents to life-threatening colitis with septic shock. Recent reports suggest a changing epidemiology with a rising incidence, more aggressive clinical courses, and a higher mortality of up to 25%. (2) Standard diagnostic tests, which detect C difficile toxins in stool by enzyme immunoassays, are both sensitive and specific. However, clinical scenarios that are suggestive of C difficile colitis and should lead to diagnostic evaluation are less well defined. Leukocytosis has been reported both in patients with C difficile colitis and in patients harboring C difficile in the absence of diarrhea. (3-6) Anecdotally, we observed an association between thrombocytosis and the presence of C difficile in several patients regardless of the presence of leukocytosis. We are not aware of reports of this association in the medical literature. Thrombocytosis may serve as an additional surrogate marker for the presence of C difficile.

The aim of this study was to evaluate the relationship between the presence of C difficile toxin A, thrombocytosis and leukocytosis. We compared the prevalence of thrombocytosis and leukocytosis in patients with positive C difficile toxin A ELISA, and the value of thrombocytosis and leukocytosis for the prediction of C difficile in hospitalized patients.

Materials and Methods

The computerized medical records for inpatients of the Atlanta Veterans Affairs Medical Center (VAMC) were reviewed. All hospitalized patients with a positive fecal ELISA for C difficile toxin A between January 1, 2002 and July 28, 2003 were assessed for an elevated thrombocyte count (>400,000/[mm.sup.3]) and for leukocytosis (WBC > 10,000/[mm.sup.3]) at the time of the toxin A assay. If no cell count was performed on the day of detection of C difficile toxin A, the closest cell counts within four days before or after toxin detection were used. Medical records were reviewed for all patients hospitalized between January 1, and June 30, 2002 with documented thrombocytosis (thrombocyte count >450,000/[mm.sup.3]) for the concurrent presence of C difficile toxin A and for leukocytosis. Finally, medical records of all patients with a white blood cell count (WBC) of >30,000/[mm.sup.3] hospitalized during the same interval were reviewed for the performance of a C difficile toxin A ELISA and for the presence of concurrent thrombocytosis (thrombocyte count >450,000/[mm.sup.3]). While a single patient could have had thrombocytosis several times during the hospital stay, only one episode per patient was evaluated for each analysis.

The [chi square] test or Fisher exact test was performed, as appropriate, for the comparison between the presence of leukocytosis and thrombocytosis among patients with C difficile. The correlation between leukocyte and thrombocyte count for patients with C difficile was determined using linear regression (SAS 8.2, SAS Institute Inc., Cary, NC). All P-values were 2-sided and considered statistically significant if [less than or equal to]0.05.

The study was approved by the Institutional Review Boards of Emory University and of the Atlanta VAMC.

Results

A total of 162 hospitalized patients were identified between January 1, 2002 and July 28, 2003 with a positive C difficile toxin A ELISA. Thrombocyte counts >400,000/[mm.sup.3] were present in 36 (22.2%; mean: 320,000/[mm.sup.3]) and leukocytosis in 97 (59.9%; mean: 15,000/[mm.sup.3]) patients, respectively. The Pearson correlation coefficient was 0.3516 (P < 0.0001) for the correlation between leukocytosis and thrombocytosis among patients with C difficile toxin A. Of all 695 hospitalized patients with thrombocytosis, a C difficile toxin A ELISA was performed in 46 (6.6%), which was positive in 18 (39.1%) (Fig.). Of patients with positive C difficile toxin A ELISA and thrombocytosis, 16/18 (88.9%) had leukocytosis. However, the prevalence of leukocytosis among patients with thrombocytosis and a negative C difficile toxin A ELISA (21/28; 75%) was not statistically different (P = 0.448). Among 82 patients with marked leukocytosis (WBC >30,000/[mm.sup.3]), tests for C difficile were performed in 20 (24.3%). Of those, 12 (60%) were positive, 7 (35%) negative and 1 (5%) indeterminate. Thrombocytosis was present in 21/82 (25.6%). There was a trend toward more frequent performance of C difficile toxin A ELISA among patients with thrombocytosis (8/21; 38.1%) compared with those without thrombocytosis (12/61; 19.7%) (OR = 2.51; P = 0.09; 95% CI: 0.75-8.49). C difficile toxin A was more frequently detected in patients with thrombocytosis, although not statistically significant (6/21 versus 6/61; OR = 3.67; P = 0.07; 95% CI: 0.83-15.71). If the patient with the indeterminate C difficile toxin A ELISA (thrombocyte count: 372,000/[mm.sup.3]) were considered positive, the respective OR for presence of C difficile toxin A for patients with thrombocytosis compared with patients without thrombocytosis would be 3.09 (P = 0.085; 95% CI: 0.73-12.45). Specificity of thrombocytosis for the presence of C difficile was 6/8 (75%) in this group.

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Discussion

Our study suggests an association between C difficile infection and thrombocytosis. Although thrombocytosis had lower sensitivity for the presence of C difficile than leukocytosis (22.2% versus 59.9%) it may have better specificity, particularly in patients with marked leukocytosis. In concordance with previous reports, (3-6) C difficile was considered in the differential diagnosis of 24% of patients with leukemoid reactions (WBC >30,000/[mm.sup.3]), regardless of the presence of diarrhea. C difficile toxin A was confirmed in 60% of those. The presence of thrombocytosis was predictive for C difficile infection in patients with leukocytosis, although our study was underpowered to reach statistical significance. Interestingly, C difficile toxin A was detected in 39.1% of patients with thrombocytosis who had at least one stool sample submitted for testing. This is considerably higher than the 10 to 20% positivity rate per submitted stool sample among patients with antibiotic-associated diarrhea in most other studies. (1,7) The association between C difficile and thrombocytosis is not surprising since reactive thrombocytosis is a well-recognized, nonspecific phenomenon in several inflammatory conditions, both infectious and noninfectious. More specifically, C difficile toxins A and B activate human monocytes to release cytokines such as interleukin-1 (IL-1), IL-6 and IL-8, (8,9) which have been shown to be responsible for reactive thrombocytosis. (10-12)

Our study has several limitations. Only patients, in whom attempts for detection of C difficile were made based on clinical suspicion, were assessed. We cannot exclude potential misclassification bias since there may have been patients who were treated empirically for C difficile without attempted laboratory diagnosis. However, due to the wide availability of the test and the ease of ordering and performing the assay, we consider this a relatively rare event. In addition, there may have been patients with subclinical infection who were never tested for C difficile, another potential source of misclassification bias. We are therefore unable to make any inferences on the correlation between asymptomatic carriage of C difficile and thrombocytosis. The analysis was performed retrospectively in a single urban hospital with a predominantly male population, thereby limiting generalizability. However, the mean leukocyte count in our study (15,000/[mm.sup.3]) was very similar to those reported previously, (3) suggesting comparability of our study results. We did not assess for confounding factors such as underlying hematologic disorders among the patients with leukocytosis and thrombocytosis. Our decision not to control for hematologic disorders could have affected the results in several ways. Firstly, hematologic diseases may lead to thrombocytopenia, leukocytopenia or suppressed abilities to mount reactive thrombocytosis or leukocytosis in the presence of C difficile. Therefore, the correlation between unexplained thrombocytosis and C difficile may have been underestimated. However, patients with thrombocytosis or leukocytosis due to hematologic disorders were not excluded from the analysis. This could lead to an overestimation of the correlation since these patients might be at increased risk for C difficile due to frequent antibiotic or antineoplastic therapy. We did not attempt to assess the exact temporal relationship between onset of thrombocytosis, infection with C difficile and institution of therapy. Hence we cannot exclude that thrombocytosis was an effect of successful therapy rather than infection itself. However, this seems unlikely, since we primarily assessed blood counts at the time of stool sample collection when patients typically had not received therapy yet or had therapy for only a very brief period of time.

At the time of this study, our laboratory only performed toxin A assays. It is unclear whether infection with toxin A-[B.sup.+] C difficile strains might lack the association with thrombocytosis. Apart from toxin B being several times more potent than toxin A, the patterns of activating human monocytes are very similar. (8,9) Thus we would expect an association between thrombocytosis and C difficile to be at least as strong as the one observed, if we had used a test to detect both toxin A and toxin B.

Conclusion

In summary, we observed an association between thrombocytosis and C difficile. Thrombocytosis may help better identify a clinical scenario suggestive of C difficile. Further studies will be necessary to determine whether thrombocytosis may be the only marker of C difficile infection in some patients and whether asymptomatic carriage is associated with thrombocytosis.

References

1. Bartlett JG. Clinical practice. Antibiotic-associated diarrhea. N Engl J Med 2002;346:334-339.

2. Pepin J, Valiquette L, Alary ME, et al. Clostridium difficile-associated diarrhea in a region of Quebec from 1991 to 2003: a changing pattern of disease severity. CMAJ 2004;171:466-472.

3. Bulusu M, Narayan S, Shetler K, et al. Leukocytosis as a harbinger and surrogate marker of Clostridium difficile infection in hospitalized patients with diarrhea. Am J Gastroenterol 2000;95:3137-3141.

4. Wahanita A, Goldsmith EA, Musher D. Conditions associated with leukocytosis in a tertiary care hospital, with particular attention to the role of infection caused by clostridium difficile. Clin Infect Dis 2002;34:1585-1592.

5. Wahanita A, Goldsmith EA, Marino BJ, et al. Clostridium difficile infection in patients with unexplained leukocytosis. Am J Med 2003;115:543-546.

6. De Toledo FG, Symes SN. Leukemoid reaction due to Clostridium difficile infection in acquired immunodeficiency syndrome: two case reports and a review of the literature. South Med J 2004;97:388-392.

7. Katz DA, Lynch ME, Littenberg B. Clinical prediction rules to optimize cytotoxin testing for Clostridium difficile in hospitalized patients with diarrhea. Am J Med 1996;100:487-495.

8. Flegel WA, Muller F, Daubener W, et al. Cytokine response by human monocytes to Clostridium difficile toxin A and toxin B. Infect Immun 1991;59:3659-3666.

9. Linevsky JK, Pothoulakis C, Keates S, et al. IL-8 release and neutrophil activation by Clostridium difficile toxin-exposed human monocytes. Am J Physiol 1997;273:G1333-1340.

10. Hamaguchi H, Takano N, Saito K, et al. Interaction of monocytes and T cells in the regulation of normal human megakaryocytopoiesis in vitro: role of IL-1 and IL-2. Br J Haematol 1990;76:12-20.

11. Hsu HC, Tsai WH, Jiang ML, et al. Circulating levels of thrombopoietic and inflammatory cytokines in patients with clonal and reactive thrombocytosis. J Lab Clin Med 1999;134:392-397.

12. Kaser A, Brandacher G, Steurer W, et al. Interleukin-6 stimulates thrombopoiesis through thrombopoietin: role in inflammatory thrombocytosis. Blood 2001;98:2720-2725.

Werner C. Albrich, MD, MS, and David Rimland, MD

From the Division of Infectious Diseases, Veterans Affairs Medical Center, School of Medicine, Emory University, Atlanta, GA.

Reprint requests to Dr. Werner C. Albrich, Respiratory and Meningeal Pathogens Research Unit, Chris Hani Baragwanath Hospital, Old Nurses Home, 1st Floor West Wing, Bertsham, Gauteng 2013, South Africa. Email: [e-mail]

Werner C. Albrich is now with the Respiratory and Meningeal Pathogens Research Unit, Johannesburg, South Africa.

Presented in part: 15th Annual Meeting of Society for Healthcare Epidemiology of America, Los Angeles, CA, April 2005 (abstract 57).

Accepted August 1, 2006.

RELATED ARTICLE: Key Points

* Twenty-two percent of patients with Clostridium difficile infection had thrombocytosis and 60% had leukocytosis.

* C difficile toxin A ELISA was performed in 6.6% of patients with thrombocytosis and was positive in 39.1% of those.

* Patients with thrombocytosis and a positive C difficile toxin A ELISA had leukocytosis 89% of the time, compared to 75% when the C difficile toxin A ELISA was negative.

* Among patients with marked leukocytosis, C difficile toxin A was more frequently detected in those with concomitant thrombocytosis.