Evaluation of Three Enzyme Immunoassays for Diagnosis of Clostridium difficile–Associated Diarrhea in Foals

Journal of Equine Veterinary Science 34 (2014) 1032–1035

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Short Communication

Evaluation of Three Enzyme Immunoassays for Diagnosis of Clostridium difficile–Associated Diarrhea in Foals Rodrigo Otávio Silveira Silva PhD a, *, Monique Silva Neves MSc a, Márcio Garcia Ribeiro PhD b, Maristela Silveira Palhares PhD a, Renata de Pino Albuquerque Maranhão PhD a, Francisco Carlos Faria Lobato PhD a a

Veterinary School, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil Department of Veterinary Hygiene and Public Health, School of Veterinary Medicine and Animal Sciences, Universidade Estadual PaulistadUNESP, Botucatu, Sao Paulo, Brazil b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 26 January 2014 Received in revised form 2 May 2014 Accepted 9 May 2014 Available online 12 June 2014

The aim of the present study was to compare the test performances of three commercial enzyme immunoassays (EIAs) against the toxigenic culture using the cytotoxicity assay as the gold standard. All EIAs showed >78% sensitivity, and the lowest specificity was 92.6%. These results suggest that EIAs could be useful for the diagnosis of Clostridium difficile infection in foals. Ó 2014 Elsevier Inc. All rights reserved.

Keywords: Colitis Enteritis Diarrhea Zoonosis Equine

1. Introduction Clostridium difficile is a spore-forming, anaerobic, grampositive bacillus responsible for most cases of antibioticassociated diarrhea in humans [1]. In veterinary medicine, this bacterium is the major cause of diarrhea and colitis in several domestic animals and some wildlife [2,3]. In foals, C. difficile causes diarrhea, ranging from a mild, self-limiting form to a peracute and fatal disease [4]. In addition, studies have also shown that strains isolated from humans suffering from C. difficile infection (CDI) exhibit high genetic relatedness with strains isolated from animals, suggesting the possibility of a zoonotic disease [5]. There is no “gold standard” for the diagnosis of CDI in foals. In contrast, for humans, the cytotoxicity assay (CTA) * Corresponding author at: Rodrigo Otávio Silveira Silva, PhD, Veterinary School, Universidade Federal de Minas Gerais, Antônio Carlos Avenue, 6627, Belo Horizonte, Minas Gerais, Brazil. E-mail address: [email protected] (R.O.S. Silva). 0737-0806/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jevs.2014.05.012

and, more recently, toxigenic culture (TC) are considered to be “gold-standard” diagnostic assays [1]. Both assays are labor intensive and time consuming. Thus, commercial enzyme immunoassays (EIAs) remain the most common method used for the diagnosis of CDI in humans [6,7]. However, only one study has evaluated the use of a single EIA for equine stool samples, thus the performance of most commercially available detection methods remains unknown [6]. Therefore, the aim of the present study was to compare the test performances of three commercial EIAs to the performances of the gold-standard CTA and TC for the diagnosis of C. difficile in foal stool samples. 2. Methods and Materials A total of 104 samples were obtained from foals aged between 1 day and 6 months; 68 of these samples were obtained from diarrheic foals submitted to the Veterinary Hospital of Universidade Federal de Minas Gerais (UFMG) for the routine diagnosis of diarrhea, and 36 were normal

R.O.S. Silva et al. / Journal of Equine Veterinary Science 34 (2014) 1032–1035

stool samples collected from clinically healthy foals. All specimens were collected at the time of clinical examination in sterile containers and were stored at
20 C until further analysis up to 7 days. The CTA for the detection of C. difficile A/B toxins was performed using Vero cells (Vero–ATCC CCL 81) as previously described [8]. Briefly, the fecal samples were diluted 1:4 in phosphate-buffered saline (pH 7.0) and centrifuged at 3,000  g for 5 minutes at 4 C. The resulting supernatant was filtered through a 0.22-mm pore size filter and diluted twofold until a dilution of 1:1,024 was achieved. The serial dilutions and parallel samples containing Clostridium sordellii antitoxins (National Institute for Biological Standards and Control, England) were applied onto the Vero cell monolayers. The cells were examined after 24 hours of incubation at 37 C in a 5% CO2 incubator. A specimen was considered positive if at least 90% of the cells were rounded, and the effect was neutralized with antitoxin at the same dilution in a parallel sample. For isolation, the stool samples were submitted to alcohol shock, and 50 mL of aliquots were inoculated onto plates containing cycloserine-cefoxitin fructose agar (TCCFA-Hi-media, Mumbai, India) supplemented with 7% horse blood and 0.1% sodium taurocholate (Sigma-Aldrich Co, St. Louis) [9]. After anaerobic incubation at 37 C for 72 hours, all colonies with morphology and Gram staining suggestive of C. difficile were subjected to a multiplex polymerase chain reaction for a housekeeping gene (tpi), toxins A (tcdA) and B (tcdB), and a binary toxin gene (cdtB) as previously described [10]. A toxigenic C. difficile strain (ATCC 9689) was used as a control for the TC method. Three commercial EIAs for A/B toxin detection were tested: C. difficile Tox A/B II (Techlab Inc), Remel ProSpecT C. difficile Toxin A/B (Oxoid, UK), and Ridascreen C. difficile toxins A/B (R-Biopharm, Germany). The EIAs were performed according to the manufacturer’s instructions. The sensitivity, specificity, positive predictive value (PPV), negative predictive value, and 95% confidence interval were calculated for each EIA against CTA and TC, and kappa coefficient was calculated to compare CTA and TC (STATA 12, College Station, TX). 3. Results The A/B toxins were detected by CTA in 10 foals (14.7%) from the diarrheic group. Clostridium difficile strains were recovered from 12 (11.5%) animals, of which nine strains were considered toxigenic and three were nontoxigenic (A
B
CDT
) by polymerase chain reaction. Three toxigenic strains were AþBþCDTþ, whereas the remaining six strains were AþBþCDT
. The clinical status and age of the animals that were positive for A/B toxins or/and TC are summarized in Table 1. The kappa concordance between TC and CTA was 0.71 (0.51–0.9 in a 95% confidence interval). All EIAs showed sensitivities of 77.8% and 100% compared with TC and CTA, respectively, whereas the specificity was >92.6% (Table 2). 4. Discussion The high sensitivity and specificity of the EIAs for the foal stool samples were consistent with the results of

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Table 1 Details of all foals positive for toxin detection and/or toxigenic culture. Foal Age

Clinical Status

Toxin Detectiona

Toxigenic Culture

20 d 2d 2 mo 5d 15 d 15 d 27 d 30 d 2 mo 2 mo 3 mo 5 mo 5 mo

Mild diarrhea Nondiarrheic Nondiarrheic Profuse diarrhea Profuse diarrhea Profuse diarrhea Profuse diarrhea Profuse diarrhea Profuse diarrhea Profuse diarrhea Profuse diarrhea Profuse diarrhea Profuse diarrhea




þ þ þ þ þ þ þ þ þ þ

Nontoxigenic (A
B
CDT
) Toxigenic (AþBþCDT
) Nontoxigenic (A
B
CDT
) Toxigenic (AþBþCDT
) Toxigenic (AþBþCDT
) Toxigenic (AþBþCDTþ) Toxigenic (AþBþCDT
) Negative Negative Negative Toxigenic (AþBþCDT
) Toxigenic (AþBþCDTþ) Toxigenic (AþBþCDTþ)

a Toxin detection by cytotoxicity assay and three commercial enzyme immunoassays.

Medina-Torrez et al [6], who examined a single EIA and reported a sensitivity and specificity of 84% and 96%, respectively. These results suggest that EIAs could be useful for diagnosis of C. difficile–associated diarrhea in foals. The TC showed a sensitivity of 70% compared with CTA; three samples were negative by TC but positive by CTA. These samples were also positive in the three EIAs tested, suggesting that the absence of isolation was associated with the TC protocol and did not result from a false-positive result by CTA. This result might have underestimated the sensitivity of the EIAs tested. Compared with CTA, all EIAs showed 100% sensitivity, but showed 77.8% sensitivity compared with TC. Notably, even with this potential underestimation, the resulting sensitivity of the tested EIAs could be considered high. Unfortunately, there is no standard method for the TC of C. difficile, making it difficult to compare the results of TC from similar studies. Various mediums have been reported, and differences in the isolation protocols, such as the use of alcohol shock, and differences in incubation time have also been proposed. Thus, the present study reported a simple isolation method, which would be faster and easier to perform compared with previous studies [6,11,12]. In this protocol, the samples were submitted to an alcohol shock and were directly plated onto TCCFA agar, followed by 72-hour incubation. Some C. difficile strains did not grow, reflecting a susceptibility to one or both of the antibiotics used on this medium [13]. Recently, Malik et al [14] also showed that the antibiotics used in the selective media are responsible for an additional stress to the recovery of C. difficile spores, potentially reducing the isolation rates. In addition, the use of TCCFA, even with the supplementation of taurocholate, could have a variable sensitivity compared with enrichment medium, such as TCCFB (cefoxitin-cycloserine broth, supplemented with taurocholate) [14,15]. These factors might have influenced the low sensitivity observed by TC for the foal samples. However, even with this slightly lower sensitivity, the concordance (kappa) between TC and CTA was 0.71, which could be considered a substantial agreement [16]. Anyway, the results of TC took at least 6 days to obtain, even with the simple protocol used in the present study. Considering that CDI in foals is commonly a clinical emergency [4], TC might not applicable for diagnosis.

(92.5–99.4) (92.3–99.4) (92.3–99.4) (35.4–84.8) (26.8–73.2) (26.8–73.2) (89.7–98.4) (85.6–96.4) (85.6–96.4)

NPV

(39.7–89.2)

70 d 63.6 50 50

PPV

(92.6–99.4) (94.2–99.8) (89.6–98.3) (89.6–98.3) (39.7–89.2) (72.1–100) (72.1–100) (72.1–100)

Acknowledgments

Abbreviations: CI, confidence interval of 95%; NPV, negative predictive value; PPV, positive predictive value.

d 96.8 100 100 100 d 77.8 90.9 71.4 71.4 d 97.9 98.9 95.7 95.7

Specificity Sensitivity

d 70 100 100 100 Cytotoxicity assay Toxigenic culture C. difficile Tox A/B II (Techlab) Remel ProSpecT C. difficile Toxin A/B (Oxoid) Ridascreen C. difficile toxins A/B (R-Biopharm)

Method

Cytotoxicity Assay % (95% CI)

(45.3–93.7) (62.3–98.4) (45.4–88.3) (45.4–88.3)

NPV PPV

(91.1–98.9) (96–100) (95.9–100) (95.9–100)

(45.3–93.7) (45.3–93.7) (45.3–93.7)

96.8 d 95.8 92.6 92.6

(91.1–98.9)

Specificity

77.8 d 77.8 77.8 77.8

(45.3–93.7)

Sensitivity

Toxigenic Culture % (95% CI)

Although the absence of A/B toxins in nondiarrheic foals has been previously reported [8,17,18], a recent survey showed that clinicians and pathologists commonly request the inclusion of normal stool samples from foals for the laboratory diagnosis of CDI [6]. Similar to previous studies reported for human samples, the inclusion of normal samples might lead to a decrease in the PPV [1,6,19]. A total of 36 nondiarrheic samples were included in the present study, and none of these samples were positive by CTA. However, four nondiarrheic samples showed false-positive results in the EIAs tested. Similar to a previous report by Medina-Torres et al [6], our findings suggest that nondiarrheic samples should not be tested when using an EIA for the diagnosis of CDI in foals. In the present study, a toxigenic strain was isolated from a nondiarrheic 2-day-old foal, negative for A/B toxins by CTA and all EIAs tested. The main hypothesis in this case is related to an asymptomatic carrier status. This possibility was already reported for equines, particularly for animals younger than 14-days-old [20]. The asymptomatic carrier status is a known event in human, although it is considered rare in healthy adults; however, asymptomatic carrier status could account for approximately 9%–20% in some groups, such as residents of long-term care facilities [1,21].

97.9 d 97.8 97.8 97.8

(92.6–99.4)

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Table 2 Comparison of the three commercial enzyme immunoassays to cytotoxicity assay and toxigenic culture as gold standards for the diagnosis of Clostridium difficile–associated diarrhea in foals (n ¼ 104).

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This work was supported by funds from FAPEMIG, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Conselho Nacional de Desenvolvimento Científico e Tecnológico, INCT-Pecuária, and PRPq-UFMG.

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