Evaluation of three enzyme immunoassays and a nucleic acid amplifi cation test for the diagnosis of Clostridium diffi cile-associated diarrhea at a university hospital in Brazil

Introduction: Despite the known importance of Clostridium diffi cile as a nosocomial pathogen, few studies regarding Clostridium diffi cile infection (CDI) in Brazil have been conducted. To date, the diagnostic tests that are available on the Brazilian market for the diagnosis of CDI have not been evaluated. The aim of this study was to compare the performances of four commercial methods for the diagnosis of CDI in patients from a university hospital in Brazil. Methods: Three enzyme immunoassays (EIAs) and one nucleic acid amplifi cation test (NAAT) were evaluated against a cytotoxicity assay (CTA) and toxigenic culture (TC). Stool samples from 92 patients with suspected CDI were used in this study. Results: Twenty-fi ve (27.2%) of 92 samples were positive according to the CTA, and 23 (25%) were positive according to the TC. All EIAs and the NAAT test demonstrated sensitivities between 59 and 68% and specifi cities greater than 91%. Conclusions: All four methods exhibited low sensitivities for the diagnosis of CDI, which could lead to a large number of false-negative results, an increased risk of cross-infection to other patients, and overtreatment with empirical antibiotics.

Clostridium diffi cile was fi rst isolated in 1935, but this microorganism was only recognized as a human pathogen in the late 1970s.Currently, Clostridium diffi cile infection (CDI) is recognized as the main cause of nosocomial diarrhea.In the last few years, the emergence of highly virulent strains of C. diffi cile in several countries 1 and cases in outpatients with no history of antibiotic therapy have been reported, demonstrating the need for further studies related to the diagnosis and control of this pathogen 2 .
For many years, the detection of A/B toxins in feces via a cell cytotoxicity assay (CTA) was considered the gold standard method for the diagnosis of CDI 2 .More recently, studies have shown that toxigenic culture (TC) is more sensitive; therefore, it has been used as the new gold standard method 3 .However, both techniques are time consuming, laborious, and require trained personnel.Thus, commercial enzyme immunoassays (EIA) are currently the most widely used techniques for the diagnosis of CDI 3 .
Other potential options that have been widely cited include commercial real time polymerase chain reaction (PCR) kits and nucleic acid amplifi cation tests (NAATs).Studies investigating these assays found that they exhibit high sensitivity, suggesting that they could be useful for screening patients with CDI 2,4 .Currently, the main limitation of these kits is their high cost, which is typically up to ten times more expensive than EIAs 5 .
Despite the known importance of C. diffi cile as a nosocomial pathogen, few studies have been conducted regarding CDI in Brazil.To date, the diagnostic tests that are available on the Brazilian market for the diagnosis of CDI have not been evaluated.Therefore, the aim of this study was to compare the performance of three commercial EIAs and one NAAT for the diagnosis of diarrhea attributed to C. diffi cile versus cytotoxicity and toxigenic culture assays.

METHODS
Between December 2011 and June 2013, 92 stool samples were collected from patients at the University Hospital of the Federal University of Minas Gerais.All samples were obtained from inpatients with suspected C. diffi cile-associated diarrhea.Specimens were collected in sterile containers, and aliquots Cytotoxicity assays were performed using Vero (African green monkey kidney) cells (ATCC CCL 81) 6 .Briefl y, fecal samples were diluted 1:4 in phosphate-buffered saline (pH 7.0) and centrifuged at 3,000 x g for 5min at 4°C.The resulting supernatants were fi ltered through a 0.22-µm pore size fi lter and subject to 2fold dilutions until a dilution of 1:1024 was achieved.Serial dilutions and parallel samples with Clostridium sordellii antitoxin were added onto Vero cell monolayers.The cells were examined after 24 h of incubation at 37°C in a 5% CO 2 incubator.A specimen was considered positive by the CTA if at least 90% of cells were rounded and if the effects were neutralized by an antitoxin at the same dilution in a parallel sample.
For toxigenic culture, equal volumes of stool samples and 96% ethanol (v/v) were mixed.After incubation for 30min at room temperature, 50µl aliquots were inoculated on plates containing cycloserine-cefoxitin fructose agar supplemented with 7% horse blood and 0.1% sodium taurocholate 6 .After anaerobic incubation at 37°C for 96h, all colonies with suggestive morphologies were subjected to a previously described multiplex-PCR protocol involving a housekeeping gene (tpi), toxins A (tcdA) and B (tcdB), and a binary toxin gene (cdtB) 7 .All strains positive for tcdA or/and tcdB were considered toxigenic.In addition, all toxigenic isolates from the PCR were tested by CTA for in vitro toxin production as previously described 8 .
The following three commercial EIAs for A/B toxin detection were tested: C. diffi cile Tox A/B II (Techlab Inc., USA), Remel ProSpecT C. diffi cile Toxin A/B (Oxoid, UK), and Ridascreen Clostridium diffi cile toxins A/B (R-Biopharm, Germany).In addition, one commercial NAAT kit (Simplexa™ C. diffi cile Universal Direct Kit, Focus Diagnostics, USA) that directly detects the tcdB gene in stool samples was also tested.All EIAs and the NAAT were performed according to the manufacturers' recommendations.The sensitivity, specifi city, positive predictive value (PPV), negative predictive value (NPV), and 95% confidence interval were calculated for each EIA and NAAT versus CTA and TC.Additionally, the kappa coeffi cient 9 was calculated to compare the CTA and TC (Stata 12, College Station, Texas, USA).

DISCUSSION
Approximately ten years ago, it was believed that the CTA demonstrated sensitivity and specifi city values greater than 99% and was thus considered the primary technique to diagnose CDI 10,11 .However, when compared with toxigenic culture, the sensitivities of CTA protocols range between 60 and 86% with specifi cities greater than 90% 5 .Corroborating these fi ndings, the CTA protocol used in the present study demonstrated a sensitivity of 73.9% (Table 1).It is also important to note that no standard protocol exists for CTAs.In this study, Vero cells were used; this cell line is considered to be the most sensitive to A/B toxins, and a similar CTA protocol was used successfully in previous studies 6,8,[12][13][14][15] .
In the TC assay, 23 (25%) patients were positive for toxigenic strains.Of these patients, 15 (65.2%) were A + B + CDT -, six (26.1%) were A + B + CDT + , and two (8.7%) were A -B + CDT -.All of the C diffi cile isolates to be toxigenic by PCR produced toxins A and B in Conversely, the TC protocol had a sensitivity that was lower (73.9%)than the values commonly reported in previous studies, which have recorded sensitivities close to 100% 2,16 .No standard method for TC assays with C. diffi cile is available, making it diffi cult to compare results from other studies.A wide variety of media and differences in isolation protocols, such as the use of shock alcohol and variations in incubation time, are common 5,10 .In this study, we opted for a simple isolation protocol, which would be more applicable for diagnosis when compared with previous methods 17 .It is well known that some strains of C. diffi cile may not grow due to susceptibility to either one or both antibiotics used in the medium 18 .Recently, Malik et al. 19 demonstrated that antibiotics used in selective media are responsible for the addition of stress in the recovery of C. diffi cile spores, which might reduce isolation rates.In addition, the use of CCFA, even with supplemental taurocholate, can result in a variable sensitivity for the recovery of C. diffi cile spores compared with protocols that use pre-enrichment broth before plating on selective agar 19 .All of these factors potentially contributed to the sensitivity exhibited in the TC assay in this study.It should be noted that, even with a TC protocol with sensitivity slightly lower than commonly reported, the concordance (kappa) between TC and CTA was 0.71.This value could be considered refl ective of substantial agreement 9 and is similar to that reported by Keessen et al. 13 It also important to note that asymptomatic colonization by toxigenic or non-toxigenic C. diffi cile is considered rare in healthy adults.However, asymptomatic colonization is estimated to occur in approximately 9 to 20% in some groups, such as residents of long-term care facilities 2,5,20 .The detection of the tcdA and tcdB genes by PCR may decrease the occurrence of false-positive results by TC after patients with non-toxigenic strains have been identifi ed as not positive; in the present study, four (4.3%) patients who were suspected of having nosocomial diarrhea caused by C. diffi cile were negative for A/B toxins and carried non-toxigenic strains.Conversely, eight (8.7%) patients were positive for TC but negative for A/B toxins by CTA and the three EIAs tested.In this case, a false-positive result should be considered a possibility.According to Peterson et al. 20 , the false-positive rate for TC is approximately 10%.Therefore, comparisons of tests for the diagnosis CDI should be performed in parallel with CTA and TC as the gold standard methods.
Due to its ease of use, low cost and the fast turnaround for results, EIA remains the most commonly used test for the diagnosis of CDI in humans and animals worldwide 10,12,20 .However, the three EIAs tested in this study demonstrated low sensitivities ranging from 61 to 68%.This result is similar to previous studies with EIA kits and rapid tests, which reported sensitivities between 50 and 77% and specifi cities of approximately 90% 5,21,22 .
The NAAT tested in this study also exhibited a low sensitivity (less than 62%).This result contrasts with previous studies that have used other NAAT tests, which reported specifi cities and sensitivities greater than 90% 5,[21][22][23] .Compared with other kits on the market, the main advantage of the NAAT tested in this study is the lack of a protocol for deoxyribonucleic acid (DNA) extraction from stool samples, which could save time and labor.Briefl y, in this test, the contents of a clinical specimen swab are immersed in a lysis solution, followed by heating and centrifugation.The resulting supernatant is directly used as a DNA template in a PCR reaction.Some authors have stated that false-negative results in the NAAT are primarily caused by the presence of gene copy numbers below the detection limits of the test [20][21][22][23][24] .Considering the number of false negative results obtained in the present work, the potential use of a more accurate extraction protocol should be considered for better performance of diagnostic tests; this modifi cation might increase the number of gene copies and also reduce the presence of inhibitors in the sample reactions.
One sample exhibited an undetermined result in the NAAT.An additional unfrozen aliquot of this sample was retested, but an undetermined result was obtained.Thus, this sample was not considered when calculating the sensitivity, specifi city, and positive and negative predictive values for the NAAT test.This stool sample was also positive in TC (A -B + CDT -) but negative for A/B toxins in the CTA and in all three EIAs tested.Undetermined results are mainly caused by the presence of inhibitors in the stool sample 5 .It appears that this is not a common event, given that it occurred in only one sample out of 81 (1.2%) stool samples tested in the NAAT.This rate is similar to previous reports with other NAATs 5,21 .
Several alternatives have been proposed to improve the diagnosis of CDI.In contrast to our results, the vast majority of commercial NAATs demonstrated high sensitivities in previous studies, but their high costs prevent their wide use 2,5,21 .At institutions where EIAs are still used for the diagnosis of CDI, some authors have suggested the submission of more than one sample from the same patient, but this practice remains controversial given that some studies have demonstrated that multiple samples do not signifi cantly increase the positive predictive value of the test and may even increase the rate of false positives 5,25,26 .Some authors have also suggested that an

Silva ROS et al. -Evaluation of EIAs and NAAT for Clostridium diffi cile infection RESULTS TABLE 1 -Evaluation of three immunoassays and RT-PCR compared with a cell cytotoxicity assay and toxigenic culture assay as the gold standards for the diagnosis of Clostridium diffi cile infection in patients at a university hospital in Brazil.
held at -20°C until all tests were performed.All procedures were previously approved by the Research Ethics Committee of the School of Medicine of Federal University of Minas Gerais (CAAE -0710.0.203.0000.11).