Detection of Campylobacter in Stool and Determination of Significance by Culture,Enzyme Immunoassay,and PCR in Developing Countries

Campylobacter is a common bacterial enteropathogen that can be detected in stool by culture, enzyme immunoassay (EIA), or PCR. We compared culture for C. jejuni/C. coli, EIA (ProSpecT), and duplex PCR to distinguish Campylobacter jejuni/C. coli and non-jejuni/coli Campylobacter on 432 diarrheal and matched control stool samples from infants in a multisite longitudinal study of enteric infections in Tanzania, Bangladesh, and Peru. The sensitivity and specificity of culture were 8.5% and 97.6%, respectively, compared with the results of EIA and 8.7% and 98.0%, respectively, compared with the results of PCR for C. jejuni/C. coli. Most (71.6%) EIA-positive samples were positive by PCR for C. jejuni/C. coli, but 27.6% were positive for non-jejuni/coli Campylobacter species. Sequencing of 16S rRNA from 53 of these non-jejuni/coli Campylobacter samples showed that it most closely matched the 16S rRNA of C. hyointestinalis subsp. lawsonii (56%), C. troglodytis (33%), C. upsaliensis (7.7%), and C. jejuni/C. coli (2.6%). Campylobacter-negative stool spiked with each of the above-mentioned Campylobacter species revealed reactivity with EIA. PCR detection of Campylobacter species was strongly associated with diarrhea in Peru (odds ratio [OR] = 3.66, P < 0.001) but not in Tanzania (OR = 1.56, P = 0.24) or Bangladesh (OR = 1.13, P = 0.75). According to PCR, Campylobacter jejuni/C. coli infections represented less than half of all infections with Campylobacter species. In sum, in infants in developing country settings, the ProSpecT EIA and PCR for Campylobacter reveal extremely high rates of positivity. We propose the use of PCR because it retains high sensitivity, can ascertain burden, and can distinguish between Campylobacter infections at the species level.     

Development and evaluation of immunochromatographic assay for simple and rapid detection of Campylobacter jejuni and Campylobacter coli in human stool specimens

An immunochromatographic assay (Campy-ICA) using a newly generated single monoclonal antibody against a 15-kDa cell surface protein of Campylobacter jejuni was developed. When cell suspensions of 86 C. jejuni strains and 27 Campylobacter coli strains were treated with a commercially available bacterial protein extraction reagent and the resulting extracts were tested with the Campy-ICA, they all yielded positive results. The minimum detectable limits for the C. jejuni strains ranged from 1.8 x 10(4) to 8.2 x 10(5) CFU/ml of cell suspension, and those for the C. coli strains ranged from 1.4 x 10(5) to 4.6 x 10(6) CFU/ml of cell suspension. All 26 non-Campylobacter species tested yielded negative results with the Campy-ICA. To evaluate the ability of the Campy-ICA to detect C. jejuni and C. coli in human stool specimens, suspensions of 222 stool specimens from patients with acute gastroenteritis were treated with the bacterial protein extraction reagent, and the resulting extracts were tested with the Campy-ICA. The Campy-ICA results showed a sensitivity of 84.8% (28 of 33 specimens) and a specificity of 100% (189 of 189 specimens) compared to the results of isolation of C. jejuni and C. coli from the stool specimens by a bacterial culture test. The Campy-ICA was simple to perform and was able to detect Campylobacter antigen in a fecal extract within 15 min. These results suggest that Campy-ICA testing of fecal extracts may be useful as a simple and rapid adjunct to stool culture for detecting C. jejuni and C. coli in human stool specimens.     

Use of an alkaline phosphatase-labeled synthetic oligonucleotide probe for detection of Campylobacter jejuni and Campylobacter coli.

A commercially available synthetic nucleic acid probe (SNAP) conjugated to alkaline phosphatase was compared with standard culture techniques for detecting Campylobacter species. The SNAP was able to detect either 5 ng of C. jejuni DNA or 10(5) CFU of bacteria. The SNAP could also detect DNA extracted from 10(5) CFU in mock-infected stool samples. The SNAP detected C. jejuni and C. coli but showed no reactivity with C. laridis, C. fetus subsp. fetus, C. fetus subsp. venerealis, C. fennelliae, "C. upsaliensis," C. cinaedii, C. fecalis, C. hyointestinalis, C. mucosalis, or Helicobacter (Campylobacter) pylori. The SNAP also showed no cross-reactivity with other enteric pathogens. When applied to pure cultures, the SNAP detected 55 clinical isolates of C. jejuni and 11 clinical isolates of C. coli, with an accuracy of 100%. When applied directly to clinical specimens, the SNAP detected Campylobacter spp. in 19 of 23 culture-positive stool specimens (sensitivity, 82.6%; specificity, 100%). Pure cultures of the Campylobacter strains isolated from the four probe-negative, culture-positive stool specimens gave positive reactions with the SNAP. While the SNAP had excellent sensitivity and specificity for isolated bacterial colony isolates, the main limitation to the Campylobacter probe detection kit may be the sensitivity limit on direct detection of Campylobacter organisms in stools.     

Evaluation of a PCR/DNA Probe Colorimetric Membrane Assay for Identification of Campylobacter spp. in Human Stool Specimens

DNA was extracted from 50 human stool specimens using the QIAamp DNA stool minikit. PCR amplification was followed by post-PCR hybridization to DNA probes specific for the Campylobacter genus, Campylobacter jejuni, and Campylobacter coli in a colorimetric membrane assay. Thirty-two of 38 culture-positive specimens were PCR/DNA probe positive for C. jejuni. The assay is rapid and simple and can be applied to stool specimens for the detection of Campylobacter.     

Use of tri-gas incubator for routine culture of Campylobacter species from fecal specimens.

We evaluated a tri-gas incubator for Campylobacter isolation to be used instead of an anaerobic jar. Fecal specimens were cultured in duplicate onto charcoal selective medium and incubated at 43 degrees C for 48 h in two different environments: a tri-gas incubator (Forma Scientific) adjusted to provide an atmosphere of 10% CO2, 10% O2, and the balance N2; and evacuated anaerobic jars with a replacement gas mixture of 10% CO2, 5% O2, and 85% N2. A total of 106 Campylobacter jejuni and 8 Campylobacter coli isolates were obtained from 2,348 stool specimens. Of the positive specimens, 113 isolates came from the incubator and 111 isolates came from the anaerobic jars. An additional 32 previously positive specimens were replated onto charcoal selective medium and retested by both methods. We recovered 27 C. jejuni isolates, 26 isolates by each method. The isolates from the incubator typically produced discrete colonies, while the isolates from the anaerobic jar showed some degree of swarming in colony formation. The tri-gas incubator provided a cost-effective method for culturing Campylobacter spp.     

Evaluation of Four Enzyme Immunoassays for the Detection of Giardia lamblia Antigen in Stool Specimens

 Four enzyme immunoassays for the detection of Giardia lamblia antigen in stool specimens were evaluated: the ProSpecT Giardia Microplate Assay (Alexon, USA), the Giardia CELISA (Cellabs, Australia), the DSl-Giardia-ELISA (DSL, Germany), and the Melotest Giardiasis Ag (Melotec, Spain). Microscopic examination and enzyme immunoassays were performed on 168 stool specimens collected from 168 patients suspected to have giardiasis. All assays were easy to perform. The ProSpecT Giardia assay had the highest sensitivity of the assays evaluated (91%), and its interpretation was the easiest. The sensitivity of the three other assays ranged from 63 to 81%. The ProSpecT Giardia assay can be useful to detect Giardia lamblia and may replace microscopic examination in areas of high endemicity.     

Use of culture, PCR analysis, and DNA microarrays for detection of Campylobacter jejuni and Campylobacter coli from chicken feces

A DNA microarray for detection of Campylobacter spp. was recently developed and applied to detect Campylobacter spp. directly from chicken feces. Sixty-five pooled chicken cloacal swab samples from 650 individual broiler chickens were included in the study. The results of Campylobacter sp. detection obtained with DNA microarrays were compared to those obtained by conventional culture and gel electrophoresis. By conventional culture, 60% of the samples were positive for either Campylobacter jejuni or Campylobacter coli. By PCR and capillary electrophoresis, 95% of the samples were positive for Campylobacter spp., whereas with DNA microarrays all samples were positive for Campylobacter spp. By application of DNA microarray analysis, the isolates in 4 samples (6%) could not be identified to the species level, whereas by PCR-capillary electrophoresis, the isolates in 12 samples (19%) remained unidentified. Interestingly, PCR-capillary electrophoresis analysis revealed that two (3%) of the samples were positive for both C. jejuni and C. coli, while DNA microarray analysis revealed that nine (14%) of the samples were positive for both species. Of 65 samples, 2 samples were identified to contain C. coli by conventional culture but were positive for C. jejuni by both PCR-capillary electrophoresis and DNA microarray analysis. The discrepancy between the methods is discussed.     

Comparison of conventional stool concentration and preserved-smear methods with Merifluor Cryptosporidium/Giardia Direct Immunofluorescence Assay and ProSpecT Giardia EZ Microplate Assay for detection of Giardia lamblia.

Giardiasis is the most common parasitic infection in the United States. Variation in the numbers of cysts and/or trophozoites that are present along with the need for a skilled microscopist offer challenges in diagnosis. We compared the sediment wet preparation and permanent stained smear results (concentration in formalin-ethyl acetate and preparation of a smear prepared from a polyvinyl alcohol-preserved specimen) from 512 consecutive specimens with the results obtained by using the Merifluor Cryptosporidium/Giardia Direct Immunofluorescence Assay (DFA; Meridian Diagnostics, Inc., Cincinnati, Ohio) and the ProSpecT Giardia EZ Microplate Assay (EIA; Alexon, Inc., Sunnyvale, Calif.). The Merifluor DFA detected 33 of 33 positive specimens, and the ProSpecT EIA detected 32 of 33 positive specimens. The diagnostic sensitivities of the Merifluor DFA and the ProSpecT EIA were 100 and 97%, respectively. The specificities of the assays were 99.8%. The Merifluor DFA and the ProSpecT EIA appear to be equally sensitive, and both are more sensitive than conventional microscopy.     

Comparison of a blood-free medium and a filtration technique for the isolation of Campylobacter spp. from diarrhoeal stools of hospitalised patients in central Australia.

Single specimens of diarrhoeal stool from 676 patients, mostly aboriginals aged less than 5 years, admitted to Alice Springs Hospital, central Australia, for diarrhoea between Sept. 1988 and Feb. 1989, were examined for Campylobacter spp. by culture on a blood-free medium with selective supplement (BFM; Oxoid) and blood agar overlaid with a membrane filter (FM). Campylobacter spp. were isolated on either BFM or FM or both from 225 patients. Campylobacter spp. were isolated on BFM alone from 75 patients and on FM alone from 213 patients (p less than 0.001; chi 2 test). Most campylobacters isolated on BFM were C. jejuni. All C. jejuni subsp. doylei, all "C. upsaliensis" except one, all C. laridis, C. fetus subsp. fetus and several uncharacterised Campylobacter isolates were isolated on FM only. C. jejuni was isolated on BFM but not FM from several patients, and vice versa. Serotyping of C. jejuni and C. coli isolated from both media showed the serotypes recovered from the two media to be different in some patients. In some patients concurrent infection with several species or serotypes (up to five) of Campylobacter, or both, was shown for the first time by the use of FM. We conclude that the use in combination of a selective medium and a non-selective medium with a filtration technique are better than either medium alone for the isolation of Campylobacter spp.     

Detection of Campylobacter species by using polymerase chain reaction and nonradioactive labeled DNA probe]

We have detected Campylobacter species which are now recognized as major pathogens of acute diarrheal disease in humans using polymerase chain reaction (PCR) and a nonradioactive labeled DNA probe. Diagnosis of Campylobacter enteritis without doing culture from stool samples is of great benefit in the laboratory. Two oligonucleotide primers (20 mer) complementary to a unique sequence of the DNA encoding ribosomal RNA (rRNA) of Campylobacter jejuni for PCR were synthesized by solid-phase phosphoamidite method. Amplified target DNA of 275 base pairs could be resolved on ethidium bromide-stained gels, and hybridized with an oligodeoxynucleotide probe (28 mer) conjugated to alkaline phosphatase. In identification experiments, it was shown that the nonradioactive probe was hybridized to clinical strains of C. jejuni (104), C. coli (5), C. laridis (5), C. hyointestinalis (1) and C. fetus subsp. fetus (1) with an accuracy of 99-100%, while it was not for Helicobacter pylori. Further, there was no evidence of amplification in strains of K. pneumoniae, S. marcescens and E. coli. Using direct detection to stool specimens, this method could be performed in C. jejuni in 39 of 43 culture-positive specimens (91%), and in 19 of 141 culture-negative specimens (13.5%), respectively. The results of this comparative study suggested that the DNA probe assay became a rapid and reliable technique to confirm culture of Campylobacter species.     

Evaluation of a New Commercial Immunoassay >for Rapid Detection of Campylobacter jejuni in Stool Samples

 In order to evaluate a new commercial enzyme immunoassay (ProspecT Campylobacter Microplate Assay; Alexon-Trend, USA) for the detection of Campylobacter jejuni and Campylobacter coli in stool samples, 30 faecal specimens known to be culture-positive for Campylobacter jejuni were tested with the new assay. The detection limit was approximately 3×10⁶/ml in faecal suspensions. The sensitivity relative to culture was 80% (24/30). All of the 24 positive samples, except for one, remained positive after being stored at –20  °C for 60 days. The specificity of the test was 100%. Interestingly, 6 of 11 additional Campylobacter jejuni culture-positive samples that had been obtained from patients with Guillain-Barré syndrome and stored at –20  °C for periods of up to 5 years tested positive in the assay. The performance of the assay indicates that it has potential value for use in future early intervention studies.     

Evaluation of performance and potential clinical impact of ProSpecT Shiga toxin Escherichia coli microplate assay for detection of Shiga Toxin-producing E. coli in stool samples

Shiga toxin-producing Escherichia coli bacteria (STEC) are emerging pathogens capable of producing sporadic and epidemic diarrhea, hemorrhagic colitis, and potentially life-threatening hemolytic-uremic syndrome. Although the presence of E. coli O157 can be readily detected in stool by sorbitol-MacConkey agar culture (SMAC), STEC non-O157 serotypes cannot. In contrast to culture, testing for the presence of Shiga toxins 1 and 2 in stool detects both O157 and non-O157 STEC serotypes capable of causing disease. Over two consecutive summers, we evaluated the performance of the ProSpecT Shiga toxin E. coli Microplate assay (Alexon-Trend, Ramsey, Minn.), an enzyme immunoassay for the detection of Shiga toxins 1 and 2, on all stools submitted for culture of enteric pathogens, and the potential clinical impact of Shiga toxin detection. Twenty-nine stool specimens were STEC positive by ProSpecT assay. Twenty-seven of 29 STEC-positive isolates were confirmed by SMAC and serotyping or by a second enzyme immunoassay and PCR (positive predictive value, 93%). Thirteen of 27 confirmed Shiga toxin-producing strains were serotype O157. The remaining 14 strains represented 8 other serotypes. The ProSpecT assay was 100% sensitive and specific for detection of E. coli O157 in stool (7 of 7) compared to SMAC. In addition, the ProSpecT assay detected twice as many STEC as SMAC. Fifty-two percent of confirmed STEC-positive stools were nonbloody. Thus, in our population, screening strategies that test only visibly bloody stools for STEC would miss a majority of cases. Eleven (41%) STEC-positive patients were hospitalized, and eight (30%) developed severe disease (two developed hemolytic-uremic syndrome, and six developed hemorrhagic colitis). Prior to detection of STEC infection, seven (26%) and eight patients (30%) underwent unnecessary diagnostic procedures or received potentially deleterious empirical treatment, respectively. We propose that establishing a specific diagnosis of STEC may have prevented these potentially harmful interventions. We conclude that the ProSpecT assay is sensitive and specific for the detection of Shiga toxins 1 and 2 in stool and has potentially significant clinical impact for the individual patient and public health. Shiga toxin assays should be considered for routine use in settings where prevalence of STEC disease warrants testing.     

CASA chromogenic medium for enteric Campylobacter species

We prospectively assessed stool samples from 370 patients for Campylobacter species by comparing three selective agar media incubated at two temperatures: 42°C and 37°C. Twenty patients (5.4%) were found positive. The chromogenic medium CASA (AES Chemunex, France) proved highly efficient for C. jejuni and C. coli recovery, while lessening the workload in the lab.     

Performance of three enzyme immunoassays and two direct fluorescence assays for detection of Giardia lamblia in stool specimens preserved in ECOFIX

ECOFIX is a single-vial stool preservative that is both formalin- and mercury-free. We evaluated the abilities of three commercial Giardia lamblia-specific enzyme immunoassays (EIAs) (ProSpecT Giardia Microplate Assay [Alexon-Trend Inc.], Giardia Test [Techlab], and Premier Giardia lamblia [Meridian Diagnostics, Inc.]) and two commercial direct fluorescent-antibody (FA) assays for G. lamblia (Crypto/Giardia IF Test [Techlab] and Merifluor Cryptosporidium/Giardia [Meridian Diagnostics, Inc.]) to detect G. lamblia in 34 G. lamblia-positive and 44 G. lamblia-negative stool specimens (determined by traditional examination for ova and parasites) preserved in ECOFIX compared to their abilities to detect G. lamblia in the same specimens preserved in formalin as the "gold standard" for each assay. Of the 34 formalin-fixed positive specimens, the number detected by each assay was as follows:, Alexon EIA, 34; Meridian EIA, 27; Techlab EIA, 29; Meridian FA assay, 31; and Techlab FA assay, 28. Both FA tests and the Alexon EIA performed well with ECOFIX, but the other two EIAs detected fewer positive specimens (the difference was statistically significant with the Techlab EIA) when ECOFIX was the preservative. Use of G. lamblia cyst antigen from cultured organisms preserved in formalin and ECOFIX demonstrated that the Alexon EIA could detect smaller amounts of antigen in ECOFIX than the other two EIAs could and suggested that cyst antigen is more stable in formalin. We recommend that laboratories use an FA assay or the Alexon EIA if they plan to use ECOFIX as their stool preservative.     

Rapid detection of Campylobacter jejuni in stool specimens by an enzyme immunoassay and surveillance for Campylobacter upsaliensis in the greater Salt Lake City area

The Alexon-Trend, Inc. (Ramsey, Minn.), ProSpecT Campylobacter microplate assay was compared with culture on a Campy-CVA plate (Remel, Lenexa, Kans.) and blood-free campylobacter agar with cefoperazone (20 microg/ml), amphotericin B (10 microg/ml), and teicoplanin (4 microg/ml) (CAT medium; Oxoid Limited, Hampshire, England) with 631 patient stool samples. The CAT medium was used to isolate Campylobacter upsaliensis. The enzyme immunoassay (EIA) had a sensitivity and a specificity of 89 and 99%, respectively, and the positive and negative predictive values were 80 and 99%, respectively. Even though we extensively looked for C. upsaliensis in stool samples from patients from the greater Salt Lake City area, we did not isolate this species during the study period. The overall excellent specificity of the EIA allows rapid detection and treatment of positive patients; however, a negative result should be confirmed by culture when clinical suspicion is high.     

Evaluation of antibodies reactive with Campylobacter jejuni in Egyptian diarrhea patients.

Serum and stool samples were collected from 128 individuals: 96 diarrhea patients and 32 apparently healthy controls. Stool specimens were cultured for enteric bacterial pathogens, while sera were screened by enzyme-linked immunosorbent assay for Campylobacter jejuni-reactive antibodies. Of 28 diarrhea patients who demonstrated C. jejuni-reactive antibodies (titers, > 100), 14 were culture positive for this organism. The 32 healthy controls showed significantly lower antibody titers (P < 0.05) with the exception of 10 subjects who were culture positive for C. jejuni and had reactive immunoglobulin M (IgM) (6 subjects) and IgG (7 subjects). IgA was not detected in those 10 individuals (asymptomatic). Avidity was expressed as the thiocyanate ion concentration required to inhibit 50% of the bound antibodies. The avidity was higher in symptomatic patients than asymptomatic healthy controls. IgG was less avid (0.92 M) compared to IgM (0.1 M) and IgA (1.1 M), with no correlation between antibody titer and avidity. However, the thiocyanate ion concentration required for the complete inhibition of IgG (5 M)-bound antibodies was higher than that of IgA (2 M) and IgM (3 M). This study also shows that C. jejuni antibodies were variably cross-reactive with Escherichia coli, Shigella flexneri, Shigella sonnei, and Neisseria meningitidis in addition to Campylobacter coli and Campylobacter rectus.     

Specific detection of Campylobacter jejuni and Campylobacter coli by using polymerase chain reaction.

Development of a routine detection assay for Campylobacter jejuni and Campylobacter coli in clinical specimens was undertaken by using the polymerase chain reaction (PCR). An oligonucleotide primer pair from a conserved 5' region of the flaA gene of C. coli VC167 was used to amplify a 450-bp region by PCR. The primer pair specifically detected 4 strains of C. coli and 47 strains of C. jejuni; but it did not detect strains of Campylobacter fetus, Campylobacter lari, Campylobacter upsaliensis, Campylobacter cryaerophila, Campylobacter butzleri, Campylobacter hyointestinalis, Wolinella recta, Helicobacter pylori, Escherichia coli, Shigella spp., Salmonella spp., Vibrio cholerae, Citrobacter freundii, or Aeromonas spp. By using a nonradioactively labeled probe internal to the PCR product, the assay could detect as little as 0.0062 pg of purified C. coli DNA, or the equivalent of four bacteria. In stools seeded with C. coli cells, the probe could detect between 30 and 60 bacteria per PCR assay. The assay was also successfully used to detect C. coli in rectal swab specimens from experimentally infected rabbits and C. jejuni in human stool samples.     

New methods for detection of campylobacters in stool samples in comparison to culture

Campylobacter species, especially Campylobacter jejuni and Campylobacter coli, are a major cause of human bacterial enteritis. Current detection in stools is done essentially by culture on selective and nonselective media with filtration. These methods were compared to 2 molecular biology methods, an in-house real-time PCR and a multiplex PCR named Seeplex Diarrhea ACE Detection, and 3 immunoenzymatic methods, Premier Campy, RidaScreen Campylobacter, and ImmunoCard Stat!Campy. Out of 242 stool specimens tested, 23 (9.5%) fulfilled the positivity criteria, i.e., they were positive by one or both culture methods or, in case of a negative culture, by a positive molecular method and a positive immunoenzymatic method. The striking feature of this study is the low sensitivity of culture, in the range of 60%, in contrast to immunoenzymatic and molecular tests.     

Laboratory and clinical evaluation of isolation media for Campylobacter jejuni.

Six selective isolation media were evaluated for their ability to support the growth of Campylobacter jejuni. Colony counts of 70 isolated strains of C. jejuni and recovery studies on these strains in simulated positive feces samples demonstrated that Bolton and Hutchinson' charcoal, cefoperazone, deoxycholate agar and Karmali's charcoal-based selective medium produced the highest recovery rates with the greatest suppression of other fecal flora. C. jejuni colonies were more easily recognized on charcoal-based selective medium. A clinical evaluation performed on 2,780 human, animal, and avian feces specimens confirmed the results of the laboratory investigation. From human samples, 4 more strains of C. jejuni were isolated on charcoal-based selective medium than were isolated on Skirrow medium, and 19 more strains of C. jejuni or C. coli were isolated on charcoal-based selective medium from animal specimens. Suppression of normal fecal flora was also greater on charcoal-based selective medium.     

2010年广州某医疗中心儿童夏季细菌性腹泻的病原学调查

目的研究广州地区小儿夏季细菌性腹泻的病原菌分布。方法采集2010年5～7月广州市妇女儿童医疗中心珠江新城院区腹泻患儿的大便标本进行常规病原菌的分离培养,通过生化反应和血清凝集试验进行鉴定和分型,并使用金标法对空肠弯曲菌抗原进行检测。结果从110份标本中检出44株病原菌,检出率为40.0%。其中致病性大肠埃希菌17株,2岁以下患儿检出15株;空肠弯曲菌12株,2岁以下患儿检出10株;沙门菌6株;念珠菌纯生长6株;产气荚膜杆菌3株。结论广州地区夏季儿童细菌性腹泻的主要病原菌是致病性大肠埃希菌及空肠弯曲菌,两者的易感人群以2岁以下婴幼儿为主。