Clinical Evaluation of a Real-Time PCR Assay for Identification of Salmonella,Shigella, Campylobacter (Campylobacter jejuni and C. coli), and Shiga Toxin-Producing Escherichia coli Isolates in Stool Specimens

Enteric illness affects millions of individuals annually in the United States and results in >50,000 hospitalizations. The rapid and accurate identification of bacterial pathogens associated with gastroenteritis can aid acute patient management decisions, including the use of antibiotic therapy and infection control. This study compared the ProGastro SSCS multiplex real-time PCR assay (Gen-Probe Prodesse, San Diego, CA) to culture for the identification of Campylobacter spp. (Campylobacter jejuni and Campylobacter coli), Salmonella spp., and Shigella spp. and to broth enrichment followed by an FDA-cleared enzyme immunoassay (EIA) for the identification of Shiga toxin-producing Escherichia coli (STEC) isolates in stool specimens. Stool samples submitted in preservatives for routine culture and EIA were prospectively enrolled and tested at four clinical centers. Discrepancies between the ProGastro SSCS assay and culture or EIA were resolved using bidirectional sequencing. The overall prevalence of the pathogens as detected by culture was 5.6% (1.8% Campylobacter, 1.8% Salmonella, 1.3% Shigella, and 0.8% STEC). When results based on the ProGastro SSCS assay and bidirectional sequencing were applied, the overall prevalence increased to 8.3% (2.3% Campylobacter, 2.6% Salmonella, 1.8% Shigella, and 1.6% STEC). Following resolution of the discrepant results, the sensitivity of the ProGastro SSCS assay was 100% for all pathogens, and the specificities ranged from 99.4% to 100%. The sensitivity of culture compared to sequence-confirmed ProGastro SSCS results ranged from 52.9% to 76.9%, with the specificities ranging from 99.9% to 100%. Overall, these results suggest that the ProGastro SSCS assay is highly sensitive and specific in a clinical setting.     

Evaluation of a New Commercial TaqMan PCR Assay for Direct Detection of the Clostridium difficile Toxin B Gene in Clinical Stool Specimens

The ProGastro Cd assay (Prodesse, Inc., Waukesha, WI) is a new commercial TaqMan PCR assay that detects tcdB. The ProGastro Cd assay was compared to the Wampole Clostridium difficile toxin B test (TOX-B test; TechLab, Blacksburg, VA), a cell culture cytotoxicity neutralization assay (CCCNA), and to anaerobic toxigenic bacterial culture, as the “gold standard,” for 285 clinical stool specimens. Assays were independently performed according to manufacturers'' directions. A 1.0-ml sample was removed from the stool specimen, of which 20 μl was used for extraction on the NucliSENS easyMAG platform (bioMérieux, Inc., Durham, NC) for the Prodesse ProGastro Cd assay and 200 μl of the stool filtrate was used for the TOX-B CCCNA. Anaerobic toxigenic culture was done by heating an additional 1.0 ml of the stool sample to 80°C for 10 min before inoculation onto modified cycloserine, cefoxitin, and fructose agar with horse blood (Remel, Lenexa, KS) and into a prereduced chopped meat glucose broth (BBL, BD Diagnostics, Sparks, MD). The prevalence of toxin-producing strains of C. difficile was 15.7% (n = 44) as determined by anaerobic toxigenic culture. The sensitivity, specificity, and positive and negative predictive values of the Prodesse ProGastro Cd assay compared to the TOX-B test were 83.3%, 95.6%, 69.4%, and 98%, respectively. Compared to toxigenic culture, the sensitivity, specificity, and positive and negative predictive values of the Prodesse ProGastro Cd assay were 77.3%, 99.2%, 94.4%, and 95.9%, respectively, and those of the TOX-B test were 63.6%, 99.2%, 93.3%, and 93.6%, respectively. Although no statistical difference (Fisher''s exact test) was detected (P = 0.242) between the sensitivities of the Prodesse ProGastro Cd assay and a standard CCCNA compared to anaerobic culture for the detection of toxigenic C. difficile, the Prodesse ProGastro Cd assay did detect more toxigenic C. difficile isolates than the CCCNA.The severity of disease associated with Clostridium difficile infection (CDI) can vary from asymptomatic colonization or mild gastroenteritis to severe manifestations, such as colitis, pseudomembrane formation, and toxic megacolon (2, 3, 7). The increased recognition of CDI-associated morbidity and mortality with the apparent rise of hypervirulent C. difficile epidemic strains (BI/NAP1/027) necessitates a dependable diagnostic assay for the detection of toxigenic C. difficile as fast as possible (7, 8).A large number of diagnostic methods are available for the detection of toxigenic C. difficile in stool samples. Individual laboratories must balance the performance characteristics with test complexity, costs, and time to results. The cell culture cytotoxicity neutralization assay (CCCNA), once considered a “gold standard,” has been replaced in most laboratories by more rapid technologies. Rapid traditional methods for detection of toxins A and B, i.e., lateral flow devices and enzyme immunoassay methods, are quicker, less complex, and less expensive, but their performances differ with regard to sensitivity and specificity (1, 10, 12, 13, 15-17, 21). Although time-consuming, the most sensitive and specific method is anaerobic culture with selective media for C. difficile followed by testing of recovered isolates for cytotoxin production (1, 6, 12, 15-17, 21). A few real-time PCR assays have been evaluated in routine clinical laboratories for direct detection of toxin A and/or toxin B directly in stool, but only three PCR assays have been directly compared to anaerobic toxigenic culture (1, 4, 12, 15, 17-20). The only consensus on testing methods is that a rapid, sensitive, and specific assay that differentiates between toxigenic and nontoxigenic C. difficile isolates is the preferred tool to assist clinicians in their decision-making process.The real-time PCR assay manufactured by Prodesse, Inc. (Waukesha, WI), is performed on stool after extraction with the NucliSENS easyMAG platform (bioMérieux, Inc., Durham, NC). The PCR assay uses TaqMan chemistry and consists of proprietary primers specific to the toxin B gene (tcdB) as well as an internal control (IC), incorporated into every reaction, that is amplified on the Cepheid SmartCycler II (Sunnyvale, CA). The IC, added on initial processing of the stool sample, acts as a general process control and monitors for the presence of PCR inhibitors. In this study, we compared the ProGastro Cd assay (Prodesse, Inc., Waukesha, WI) to the Wampole C. difficile toxin B test (TOX-B test; TechLab, Blacksburg, VA) for a Prodesse (Waukesha, WI)-sponsored clinical trial. In addition, both assays were also compared to an anaerobic toxigenic culture method that was not a component of the sponsored clinical trial.(This research was presented in part at the 25th Annual Clinical Virology Symposium, Daytona Beach, FL, 19 to 22 April 2009.)     

Detection of non-jejuni and -coli Campylobacter Species from Stool Specimens with an Immunochromatographic Antigen Detection Assay

The STAT! Campy immunochromatographic assay for Campylobacter antigen was compared to culture for 500 clinical stool specimens. Antigen was detected in six culture-negative, PCR-positive specimens. C. upsaliensis, a pathogenic species that is traditionally difficult to recover in routine stool cultures, was detected in two of these culture-negative specimens. This study provides evidence that antigen testing may cross-react with at least one additional non-jejuni and -coli Campylobacter species that may be missed by routine culture for campylobacteriosis.     

Panfungal PCR Assay for Detection of Fungal Infection in Human Blood Specimens

A novel panfungal PCR assay which detects the small-subunit rRNA gene sequence of the two major fungal organism groups was used to test whole-blood specimens obtained from a series of blood or bone marrow transplant recipients. The 580-bp PCR product was identified after amplification by panfungal primers and hybridization to a 245-bp digoxigenin-labeled probe. The lower limit of detection of the assay was approximately four organisms per milliliter of blood. Multiple whole-blood specimens from five patients without fungal infection or colonization had negative PCR results. Specimens from 11 infected patients had positive PCR results. Blood from three patients with pulmonary aspergillosis had positive PCR results: one patient’s blood specimen obtained in the week prior to the diagnosis of infection by a positive bronchoalveolar lavage fluid culture result was positive by PCR, and blood specimens obtained from two patients 1 to 2 days after lung biopsy and which were sterile by culture were positive by PCR. The blood of four patients with candidemia, three patients with mixed fungal infections, and one patient with fusariosis also had positive PCR signals. The panfungal PCR assay can detect multiple fungal genera and may be used as an adjunct to conventional methods for the detection of fungal infection or for describing the natural history of fungal infection. Further studies are needed to define the sensitivity and specificity of this assay for the diagnosis of fungal infection prior to the existence of other clinical or laboratory indications of invasive fungal infection.     

A Powerful DNA Extraction Method and PCR for Detection of Microsporidia in Clinical Stool Specimens

The diagnosis of intestinal microsporidiosis has traditionally depended on direct visualization of the parasite in stool specimens or intestinal biopsy samples by light and/or electron microscopy. Limited information about the specificity and sensitivity of PCR for the detection microsporidia in clinical stool specimens is available. To establish a sensitive and specific method for the detection of microsporidia in clinical samples, we studied clinical stool specimens of 104 randomly selected human immunodeficiency virus-infected patients with diarrhea to compare light microscopy and PCR. Fluorochrome Uvitex 2B staining was used for light microscopy. To raise the sensitivity of PCR, we used a powerful and fast DNA extraction method including stool sedimentation, glass bead disruption, and proteinase K and chitinase digestion. PCR was performed with primer pairs V1-PMP2, V1-EB450, and V1-SI500, and the nature of the PCR products was confirmed by Southern blot hybridization. Microsporidiosis was diagnosed by light microscopy in eight patients. Ten patients tested positive for microsporidiosis by PCR. Enterocytozoon bieneusi was found in seven cases, and Encephalitozoon intestinalis was found in four cases. In one case a double infection with E. bieneusi and E. intestinalis was diagnosed by PCR, whereas light microscopy showed only E. bieneusi infection. PCR testing of stool specimens is useful for diagnosis and species differentiation of intestinal microsporidiosis in HIV patients.     

Multicenter Evaluation of Clinical Diagnostic Methods for Detection and Isolation of Campylobacter spp. from Stool

The use of culture-independent diagnostic tests (CIDTs), such as stool antigen tests, as standalone tests for the detection of Campylobacter in stool is increasing. We conducted a prospective, multicenter study to evaluate the performance of stool antigen CIDTs compared to culture and PCR for Campylobacter detection. Between July and October 2010, we tested 2,767 stool specimens from patients with gastrointestinal illness with the following methods: four types of Campylobacter selective media, four commercial stool antigen assays, and a commercial PCR assay. Illnesses from which specimens were positive by one or more culture media or at least one CIDT and PCR were designated “cases.” A total of 95 specimens (3.4%) met the case definition. The stool antigen CIDTs ranged from 79.6% to 87.6% in sensitivity, 95.9 to 99.5% in specificity, and 41.3 to 84.3% in positive predictive value. Culture alone detected 80/89 (89.9% sensitivity) Campylobacter jejuni/Campylobacter coli-positive cases. Of the 209 noncases that were positive by at least one CIDT, only one (0.48%) was positive by all four stool antigen tests, and 73% were positive by just one stool antigen test. The questionable relevance of unconfirmed positive stool antigen CIDT results was supported by the finding that noncases were less likely than cases to have gastrointestinal symptoms. Thus, while the tests were convenient to use, the sensitivity, specificity, and positive predictive value of Campylobacter stool antigen tests were highly variable. Given the relatively low incidence of Campylobacter disease and the generally poor diagnostic test characteristics, this study calls into question the use of commercially available stool antigen CIDTs as standalone tests for direct detection of Campylobacter in stool.     

Multicentric Evaluation of a New Real-Time PCR Assay for Quantification of Cryptosporidium spp. and Identification of Cryptosporidium parvum and Cryptosporidium hominis

Cryptosporidium is a protozoan parasite responsible for gastroenteritis, especially in immunocompromised patients. Laboratory diagnosis of cryptosporidiosis relies on microscopy, antigen detection, and nucleic acid detection and analysis. Among the numerous molecular targets available, the 18S rRNA gene displays the best sensitivity and sequence variations between species and can be used for molecular typing assays. This paper presents a new real-time PCR assay for the detection and quantification of all Cryptosporidium species associated with the identification of Cryptosporidium hominis and Cryptosporidium parvum. The sensitivity and specificity of this new PCR assay were assessed on a multicentric basis, using well-characterized Cryptosporidium-positive and -negative human stool samples, and the efficiencies of nine extraction methods were comparatively assessed using Cryptosporidium-seeded stool samples and phosphate-buffered saline samples. A comparison of extraction yields showed that the most efficient extraction method was the Boom technique in association with mechanical grinding, and column extraction showed higher binding capacity than extraction methods based on magnetic silica. Our PCR assay was able to quantify at least 300 oocysts per gram of stool. Satisfactory reproducibility between laboratories was observed. The two main species causing human disease, Cryptosporidium hominis and Cryptosporidium parvum, were identified using a duplex real-time PCR assay with specific TaqMan minor-groove-binding ligand (MGB) probes for the same amplicon. To conclude, this one-step quantitative PCR is well suited to the routine diagnosis of cryptosporidiosis since practical conditions, including DNA extraction, quantification using well-defined standards, and identification of the two main species infecting humans, have been positively assessed.     

Evaluation of a New Chromogenic Medium for the Isolation and Presumptive Identification of Salmonella Species from Stool Specimens

The performance of BBL CHROMagar Salmonella (Becton Dickinson, France), a new selective chromogenic medium for the isolation and presumptive identification of Salmonella spp., was evaluated. On this medium, which is a modification of CHROMagar Salmonella (CHROMagar Microbiology, France) with enhanced selectivity, the colonies of Salmonella are stained in mauve (rose-violet), while those of other organisms appear in blue-green or are not stained by any of the chromogens of the medium. The medium was evaluated with a total of 176 strains of Salmonella and other organisms, consisting of 18 reference strains and 158 clinical isolates. All Salmonella strains except subspecies IIIa and IIIb strains and Salmonella Gallinarum yielded typical mauve colonies. During the evaluation with 107 known positive and 332 unknown stool specimens in a clinical laboratory, a total of 115 and 105 Salmonella isolates were obtained on BBL CHROMagar Salmonella and Hektoen enteric agar, respectively. From the known positive stool specimens, 92 true positive cultures were obtained on BBL CHROMagar Salmonella and 89 on Hektoen enteric agar, yielding sensitivities of 86 and 83%, respectively. From the unknown stool specimens, a total of 27 Salmonella isolates were obtained, with 23 isolated from BBL CHROMagar Salmonella and 16 from Hektoen enteric agar by direct plating (sensitivity 85 and 59%, specificity 99 and 97%, respectively). Seroagglutination tests could be performed directly from BBL CHROMagar Salmonella. Compared to conventional isolation media, the time needed for confirmatory biochemical and serological tests was shortened by about 1 day when BBL CHROMagar Salmonella was used. On the basis of these results, the medium can be recommended for the primary isolation and presumptive identification of Salmonella spp. from clinical stool specimens. Electronic Publication     

Blinded, Externally Controlled Multicenter Evaluation of Light Microscopy and PCR for Detection of Microsporidia in Stool Specimens

The quality parameters for the detection of microsporidia in identical sets of 50 stool samples were determined for six laboratories where technicians used light microscopy and for six laboratories where technicians used PCR. The average overall sensitivities were 67% (89% for patient samples only) for the PCR laboratories and 54% (80% for patient samples only) for the light microscopy laboratories. Specificities were 98 and 95%, respectively. Differences in results were most apparent between the individual laboratories rather than between the two major methods used.     

Multicenter Clinical Evaluation of the Portrait Toxigenic C. difficile Assay for Detection of Toxigenic Clostridium difficile Strains in Clinical Stool Specimens

We compared the Portrait Toxigenic C. difficile Assay, a new semiautomated sample-to-result molecular test, to a toxigenic bacterial culture/cell cytotoxin neutralization assay (TBC/CCNA) for the detection of toxigenic Clostridium difficile in 549 stool specimens. Stool specimens were also tested by one of three alternative FDA-cleared molecular tests for toxigenic C. difficile (Xpert C. difficile, Illumigene C. difficile, or GeneOhm Cdiff). The sensitivities and specificities of the molecular tests compared to TBC/CCNA were as follows: 98.2% and 92.8% for the Portrait assay, 100% and 91.7% for the Xpert assay, 93.3% and 95.1% for the Illumigene assay, and 97.4% and 98.5% for the GeneOhm assay, respectively. The majority of Portrait false-positive results (20/31; 64.5%) were also positive for C. difficile by an alternative molecular test, suggesting an increased sensitivity compared to the culture-based “gold standard” method. The Portrait test detected an assay input of 30 CFU in 100% of spiked samples and detected an input of 10 CFU in 96.7% of samples tested.     

Pentaplex PCR Assay for Detection of Hemorrhagic Bacteria from Stool Samples

Diarrheal diseases cause illness and death among children younger than 10 years in developing countries. Conventional testing for the detection of hemorrhagic bacteria takes 2 to 5 days to yield complete information on the organism and its antibiotic sensitivity pattern. Hence, in the present study, we developed a molecular-based diagnostic assay that identifies common hemorrhagic bacteria in stool samples. A set of specific primers were designed for the detection of Salmonella spp., Shigella spp., enterohemorrhagic Escherichia coli (EHEC), and Campylobacter spp., suitable for use in a one-tube PCR assay. The assay in the present study simultaneously detected five genes, namely, ompC for the Salmonella genus, virA for the Shigella genus, eaeA for EHEC, 16S rRNA for the Campylobacter genus, and hemA for an internal control. Specific primer pairs were successfully designed and simultaneously amplified the targeted genes. Validation with 20 Gram-negative and 17 Gram-positive strains yielded 100% specificity. The limit of detection of the multiplex PCR assay was 1 × 10³ CFU at the bacterial cell level and 100 pg at the genomic DNA level. Further evaluation of the multiplex PCR with 223 bacterium-spiked stool specimens revealed 100% sensitivity and specificity. We conclude that the developed multiplex PCR assay was rapid, giving results within 4 h, which is essential for the identification of hemorrhagic bacteria, and it might be useful as an additional diagnostic tool whenever time is important in the diagnosis of hemorrhagic bacteria that cause diarrhea. In addition, the presence of an internal control in the multiplex PCR assay is important for excluding false-negative cases.     

Performance of a PCR Assay for Detection of Pneumocystis carinii from Respiratory Specimens

This study evaluates the performance of a PCR assay for the detection of Pneumocystis carinii from respiratory specimens that has been designed for use in the clinical microbiology laboratory. The test includes a simple method for nucleic acid extraction and amplification, a colorimetric probe hybridization technique for detection of amplicons, and an internal control to evaluate for the presence of inhibitors of amplification. Two hundred thirty-two clinical specimens (120 induced-sputum [IS] and 112 bronchoalveolar lavage [BAL] specimens) from 168 patients were tested by both immunofluorescent (direct fluorescent-antibody [DFA]) staining and PCR. Of the 112 BAL specimens, 17 were positive for P. carinii by DFA staining and PCR. An additional two specimens were DFA negative and PCR positive. For BAL specimens, the sensitivity and specificity of PCR compared to DFA were 100 and 98%, respectively. Eighteen IS specimens were positive for P. carinii by DFA, and 27 were positive by PCR. One of the 18 DFA-positive IS specimens was negative by PCR; this patient had just completed therapy for P. carinii pneumonia. Of the 10 specimens that were PCR positive and DFA negative, 4 were from patients who had a subsequent BAL specimen that was positive by DFA and PCR. For IS specimens, the sensitivity of DFA and PCR was 82 and 95%, respectively. The specificity of PCR for IS specimens was 94%. Due to the high sensitivity of PCR for the detection of P. carinii from IS specimens, a PCR-based diagnostic test may be a useful screening test and may alleviate the need for bronchoscopy in some patients.     

Detection of Helicobacter pylori in Stool Specimens by PCR and Antigen Enzyme Immunoassay

A highly sensitive seminested PCR assay to detect Helicobacter pylori DNA in feces was developed. PCR with stool specimens and a novel antigen enzyme immunoassay (EIA) for H. pylori detection in feces were evaluated as diagnostic tools and in follow-up with samples from 63 infected and 37 noninfected persons. Infected individuals received eradication therapy followed by endoscopic follow-up 35 days after the start of treatment. At that time, a second stool specimen was obtained from 55 of these patients. Before eradication, the sensitivity of PCR was 93.7% and that of EIA 88.9%. Specificities were 100 and 94.6%, respectively. Of the 55 follow-up specimens, 41 originated from patients from whom H. pylori had been eradicated. Of these, 21 were still positive by PCR and 13 were positive by EIA, indicating that 1 month may be too short a period for follow-up evaluation of stool specimens by these tests.     

PCR–Enzyme-Linked Immunosorbent Assay for Detection and Identification of Campylobacter Species: Application to Isolates and Stool Samples

We report a PCR–enzyme-linked immunosorbent assay which identifies Campylobacter species by capture hybridization of a single-stranded 16S rRNA gene amplicon with species-specific probes in a microtiter plate format. Specificities were confirmed for both reference and field strains, but the type strain of Campylobacter coli was atypical. The assay was rapid, informative, and usable with stool-extracted DNA.     

PCR for Detection and Identification of Abiotrophia spp.

Members of the genus Abiotrophia, formerly known as nutritionally variant streptococci, are important pathogens causing septicemia and endocarditis. Cultivation and biochemical differentiation of Abiotrophia spp. are often difficult. Based on 16S rRNA sequences, two PCR assays for detection and identification of Abiotrophia spp. were developed. The first PCR assay was positive for all Abiotrophia spp. Subsequently performed restriction fragment length polymorphism analysis allowed the verification of the PCR amplicons and the differentiation of the three species. The second PCR assay was positive only for A. elegans, the most fastidious species of Abiotrophia. Both PCR assays were shown to be specific and sensitive and should facilitate the identification of Abiotrophia spp.     

Strategy for Optimizing DNA Amplification in a Peripheral Blood PCR Assay Used for Diagnosis of Human Brucellosis

We studied two of the possible factors which can interfere with specific DNA amplification in a peripheral-blood PCR assay used for the diagnosis of human brucellosis. We found that high concentrations of leukocyte DNA and heme compounds inhibit PCR. These inhibitors can be efficiently suppressed by increasing the number of washings to four or five and decreasing the amount of total DNA to 2 to 4 μg, thereby avoiding false-negative results.     

Genus Level Identification of Mycobacteria from Clinical Specimens by Using an Easy-To-Handle Mycobacterium-Specific PCR Assay

An easy-to-handle Mycobacterium-specific PCR assay for detection of the presence of a wide range of mycobacterial species in clinical samples was evaluated. The performance of the genus probe was compared with the performance of probes specific for Mycobacterium tuberculosis and Mycobacterium avium and with that of standard culture. In addition, the utility of an internal control in monitoring amplification inhibitors was studied. Of 545 respiratory and 325 nonrespiratory specimens (a total of 870 specimens), 58 (6.7%) showed the presence of amplification inhibitors, as determined by a negative result for the internal control. Of these 58 specimens, 31 (53%) were stool specimens; other material, even citrate blood after lysis of erythrocytes, did not pose a problem with regard to inhibition of PCR amplification. Eighty-one of the remaining 812 specimens had a positive Mycobacterium culture result. Of these culture-positive specimens, 58 (71.6%) showed a positive result with the Mycobacterium genus-specific probe. Seventy-two samples had a positive result with the Mycobacterium-specific probe but a negative culture result. Of these 72 samples, 26 samples were regarded as true positive, either because the M. tuberculosis- or M. avium-specific probe was also positive at the same time or because other specimens from the same patient taken at the same time were culture positive. The sensitivity of the Mycobacterium-specific probe was 78.5% and the specificity was 93.5%. This study showed that pretesting of clinical specimens for mycobacteria to the genus level with a Mycobacterium-specific probe offers the routine clinical laboratory the possibility of detecting tuberculous and nontuberculous mycobacteria with one test. Furthermore, specimens testing positive with the genus-specific probe can be immediately identified with species-specific probes.     

Clinical Evaluation of a New PCR Assay for Detection of Coxiella burnetii in Human Serum Samples

A nested PCR method was developed for the detection of Coxiella burnetii in human serum samples. Two pairs of oligonucleotide primers were designed to amplify a 438-bp fragment of the com1 gene encoding a 27-kDa outer membrane protein of C. burnetii. The primers amplified the predicted fragments of 21 various strains of C. burnetii but did not react with DNA samples from other microorganisms. The 438-bp amplification products could be digested with restriction enzymes SspI and SalI. The utility of the nested PCR was evaluated by testing human serum samples. The com1 gene fragment was amplified from 135 (87%) of 155 indirect immunofluorescence test (IF)-positive serum samples and from 11 (11%) of 100 IF-negative serum samples. The nested PCR with primers targeted to the com1 gene appeared to be a sensitive, specific, and useful method for the detection of C. burnetii in serum samples.     

Detection of Campylobacter spp. in chicken fecal samples by real-time PCR

A real-time PCR assay for detecting thermophilic Campylobacter spp. directly in chicken feces has been developed. DNA was isolated from fecal material by using magnetic beads followed by PCR with a prealiquoted PCR mixture, which had been stored at -18 degrees C. Campylobacter could be detected in less than 4 h, with a detection limit of 100 to 150 CFU/ml, in a fecal suspension. A bacterial internal control was added before DNA extraction to control both DNA isolation and the presence of PCR inhibitors in the samples. The assay was performed on 111 swab samples from a Danish surveillance program and compared to conventional culturing using selective enrichment. There was no statistically significant difference in performance between real-time PCR and culture by selective enrichment, and the diagnostic specificity was 0.96 with an agreement of 0.92. Therefore, the assay should be useful for screening poultry flocks for the presence of Campylobacter.