Clinical Characteristics of Patients Who Test Positive for Clostridium difficile by Repeat PCR

Abstract

The high sensitivity of PCR assays for diagnosing Clostridium difficile infection (CDI) has greatly reduced the need for repeat testing after a negative result. Nevertheless, a small subset of patients do test positive within 7 days of a negative test. The aim of this study was to evaluate the clinical characteristics of these patients to determine when repeat testing may be appropriate. The results of all Xpert C. difficile PCR (Cepheid, Sunnyvale CA) tests performed in the clinical microbiology laboratory at New York-Presbyterian Hospital, Columbia University Medical Center (NYPH/CUMC) from 1 May 2011 through 6 September 2013, were reviewed. A retrospective case-control study was performed, comparing patients who tested positive within 7 days of a negative test result to a random selection of 50 controls who tested negative within 7 days of a negative test result. During the study period, a total of 14,875 tests were performed, of which 1,066 were repeat tests (7.2%). Eleven of these repeat tests results were positive (1.0%). The only risk factor independently associated with repeat testing positive was history of a prior CDI (odds ratio [OR], 19.6 [95% confidence interval {CI}, 4.0 to 19.5], P < 0.001). We found that patients who test positive for C. difficile by PCR within 7 days of a negative test are more likely to have a history of CDI than are patients who test negative with repeat PCR. This finding may be due to the high rate of disease relapse or the increased likelihood of empirical therapy leading to false-negative results in these patients.

INTRODUCTION

The incidence of Clostridium difficile infection (CDI) has been rising over the last 2 decades, leading to significant morbidity and mortality, particularly in elderly and hospitalized patients (1). PCR assays for the diagnosis of CDI have gained in popularity in clinical microbiology laboratories in recent years. These PCR tests are rapid, simple to perform, and offer a high degree of sensitivity and specificity for detecting toxigenic C. difficile strains, usually by detection of the tcdB gene that encodes toxin B production. The high sensitivities of these assays have greatly reduced the need for repeat testing after a negative result, especially within the first 7 days of a negative result. Multiple studies have demonstrated that repeat testing within 7 days yields positive results in only about 1 to 3% of cases (2–5). However, clinical microbiology laboratories continue to receive test requests for patients with a recent negative test result, presumably in part due to the perceived poor sensitivity of previously used enzyme immunoassays (EIAs). These unnecessary PCR tests lead to increased costs and inefficient utilization of resources.

Our laboratory began using PCR testing for C. difficile on 1 May 2011, and due to the low yield of repeat testing, instituted a policy on 30 October 2013 to reject stool specimens received within 7 days of a negative PCR result unless permission was granted by the laboratory director. Despite the low probability of a positive result within 7 days of a negative result, we have nevertheless observed a small number of such cases since we began using PCR in our laboratory. The aim of the present study was therefore to evaluate the clinical characteristics of the patients who test positive within 7 days of a negative test in order to identify a potential subset of patients for whom repeat testing may in fact be appropriate.

MATERIALS AND METHODS

The results of all Xpert C. difficile PCR (Cepheid, Sunnyvale CA) tests performed in the clinical microbiology laboratory at the Columbia University Medical Center from 1 May 2011 through 6 September 2013, were reviewed. A retrospective case-control study was performed comparing patients who tested positive within 7 days of a negative result to a random selection of controls who tested negative within 7 days of a negative result. The testing intervals were defined as the number of days or hours between the order of consecutive tests in the electronic health information system, rounded to the nearest day or hour. The electronic medical records were then reviewed for each of these patients, and data were gathered on patient age, gender, antibiotic use within the past 30 days, any history of CDI, use of empirical CDI therapy within 1 day of stool collection, number of previous positive tests, hospitalization, diarrhea, fever, and leukocytosis at the time of physician order of the second PCR test. Leukocytosis was defined as a white blood cell count of >11,000/μl, and fever was defined as a temperature of ≥100.4°F (38°C). A random selection of 60 controls was initially chosen, but 10 patients were excluded because information about one or more of the clinical characteristics listed above could not be ascertained from the electronic medical record. Univariate analyses using Fisher's exact test were performed to find risk factors associated with repeat positive testing, using a P value of 0.05 for statistical significance. Student's t test was used to compare the mean ages between the two groups using a P value of 0.05 for statistical significance.

This study was approved by the Columbia University institutional review board.

RESULTS

From 1 May 2011 to 6 September 2013, a total of 14,875 Xpert C. difficile PCR tests were performed in the laboratory, of which 1,066 (7.2%) were repeat tests performed within 7 days of a negative test result. Of these 1,066 repeat tests, a total of 11 tests from 11 patients were positive (1.0%). The interval between the initial negative and subsequent positive result ranged from 3 h to 6 days and 6 h, with a median interval of 3 days and 1 h. The 11 case patients had a mean age of 56.1 years, and the 50 control patients had a mean age of 58.7 years (P = 0.58). The univariate analyses of the patient demographics and clinical data are summarized in Table 1. Of the variables analyzed, a history of prior CDI was the only statistically significant risk factor associated with testing positive within 7 days of a negative test result (odds ratio, 19.6 [95% confidence interval {CI}, 4.0 to 94.7], P = 0.0001). Empirical therapy was more common among those who tested positive, but this finding was not statistically significant (P = 0.13). A subanalysis showed that empirical therapy was significantly associated with a history of CDI (P = 0.0007), and a stratified analysis revealed empirical therapy to be an effect modifier, although the sample size was too small to accurately calculate the stratified odds ratio. The clinical characteristics of the 11 case patients are summarized in Table 2.

TABLE 1.

Demographic and clinical features of cases and controls

Demographic or clinical factor	No. (%) for:		P value using Fisher's exact test
	Cases (n = 11)	Controls (n = 50)
Male gender	6 (55)	28 (56)	1.00
History of C. difficile infectiona	8 (73)	6 (12)	0.0001
Leukocytosis	9 (82)	31 (62)	0.30
Diarrhea	11 (100)	39 (78)	0.19
Fever	4 (36)	12 (24)	0.46
Hospitalization	11 (100)	48 (96)	1.00
Antibiotic use within last 30 days	11 (100)	45 (90)	0.57
Empirical therapy	5 (45)	11 (22)	0.13

^aThe only significant risk factor influencing a positive test result within 7 days of a negative test result.

TABLE 2.

Clinical characteristics of patients testing positive for C. difficile on repeat PCR

Patient	Age (yr)	Sexa	Diarrhea	Leukocytosis	Fever	History of CDIb	No. of previous positive tests	Time since most recent positive test	Interval between negative and positive testsc	Empirical therapy
1	74	M	Yes	Yes	No	Yes	1	16 days	5 h	Yes
2	86	F	Yes	Yes	No	Yes	1	21 days	11 h	Yes
3	45	F	Yes	Yes	No	Yes	3	5 mo	5 days	Yes
4	67	F	Yes	No	No	Yes	1	7 days	2 days	Yes
5	52	M	Yes	Yes	Yes	Yes	1	35 days	2 days	No
6	35	M	Yes	Yes	Yes	Yes	2	21 days	5 days	No
7	65	M	Yes	Yes	No	Yes	1	25 days	6 days	No
8	51	F	Yes	Yes	Yes	Yesd	NAd	NA	3 days	No
9	64	F	Yes	Yes	Yes	No	NA	NA	5 days	Yes
10	21	M	Yes	Yes	No	No	NA	NA	6 days	No
11	57	M	Yes	No	No	No	NA	NA	3 h	No

^aM, male; F, female. NA = Not Applicable.

^bCDI, C. difficile infection.

^cRounded to the nearest day if >1 day, rounded to the nearest hour if <1 day.

^dPatient 8 had a reported history of CDI but had no prior laboratory results at our institution. NA, not applicable.

DISCUSSION

Given the high sensitivity of C. difficile PCR assays, repeat testing after a negative result, especially within the first 7 days of that result, usually has little diagnostic value. Only 1.0% of such repeat tests in the present study led to a positive result, a rate similar to that observed in previous studies (2–5). There was no single uniform reason for ordering a second test; some patients had recurrent or worsening diarrhea, others had persistent watery stools with leukocytosis, and yet others simply required two consecutive negative tests for admission to a rehabilitation center. Khanna et al. (4) showed that patients who tested positive for C. difficile by PCR after the initial negative test result had similar ages, gender, and location (inpatient versus outpatient) as those of the patients who tested negative on the second test; to our knowledge, the present study is the first to compare these two groups on other clinically relevant risk factors, such as history of CDI, empirical CDI therapy, and clinical signs and symptoms of CDI. The only variable that was independently associated with a positive result within 7 days of a negative result was history of a prior CDI, with 8 of the 11 (73%) case patients having a documented history of CDI compared to just 6 of the 50 control patients (12%).

An examination of the electronic medical records of the 11 case patients revealed that these patients generally fell into one of two categories: those who developed new or recurrent infections during the period between the negative and positive test results, and those who likely falsely tested negative on the first test. Up to 25% of patients with a history of CDI will experience relapse of the initial infecting strain or reinfection with a new strain (6), placing them at higher risk for CDI during hospitalization compared to patients without a history of CDI. The more recent the initial infection, the more likely that recurrence is associated with relapse rather than reinfection, as demonstrated by a molecular typing study by Kamboj (7), in which 88% of the episodes that occurred within 8 weeks were due to relapse, whereas only 65% that occurred after 8 weeks were due to relapse. In the present study, 6 of the 8 case patients with a history of CDI had a positive test within the past 5 weeks (Table 2), suggesting that the majority of these cases were due to relapse.

With regard to false negatives, two patients tested positive just hours after the initial negative test, which is strongly suggestive of false-negative results, especially since both of these patients had clinical evidence of C. difficile-associated diarrhea. Prior studies have shown the Cepheid Xpert C. difficile PCR assay to have a sensitivity of 96 to 97% (8–11), so a significant number of false negatives should be expected in a laboratory that performs thousands of tests annually; the overall prevalence of CDI for inpatients at New York-Presbyterian Hospital, Columbia University Medical Center (NYPH/CUMC) was 1.6% in the year 2013. Furthermore, many patients with suspected CDI receive empirical antimicrobial therapy prior to stool sample collection, which can potentially lead to an increased rate of false-negative results. In one prospective study of patients with CDIs, the cumulative percentages of patients who converted from a positive to a negative result after 1, 2, and 3 days of therapy were 14%, 35%, and 45%, respectively (12). In the present study, 5 of the 11 case patients had received empirical treatment prior to stool collection, suggesting that the initial PCR results for some of these patients may have been false negatives. Patients with a history of CDI may be more likely to receive empirical therapy, since there is stronger suspicion of infection in these patients. Our data show that patients who had history of CDI were indeed more likely to receive empirical therapy (P < 0.001).

One of the major limitations of this study is the small sample size of the patients included. Only 11 patients met the study criteria after >3 years since the implementation of the Xpert C. difficile PCR assay in our laboratory. A more thorough evaluation of patient risk factors would require larger multicenter studies. A second important limitation is the retrospective nature of this study and the possibility that other unmeasured risk factors may confound or modify the results.

Due to the low yield of repeat PCR testing after a negative result, many hospitals, including our own, have implemented health information system alerts when a repeat test is ordered. One recent study demonstrated that the implementation of such an alert reduced repeat testing within 7 days from 14.5% to 1.3% (13). If widely implemented, such interventions might meaningfully address the overuse of repeat C. difficile PCR testing, leading to cost savings and more efficient resource utilization.

Nevertheless, physicians and clinical microbiology laboratories should be aware that there are occasional circumstances for which repeat testing may be warranted. Our data demonstrate that patients who test positive with repeat PCR have significantly increased odds of having a prior CDI, likely due to disease relapse during hospitalization or false-negative results on the initial test due to empirical therapy. Further prospective studies are needed to determine the overall likelihood of a repeat positive test result in patients who have a history of CDI in order to determine if this risk factor should be considered an exception to the rule against repeat testing within 7 days.