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ABSTRACT |
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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INTRODUCTION |
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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ETEC causes diarrhea by colonizing the small intestine and produces one or two enterotoxins, a heat-labile enterotoxin (LT) and a heat-stable enterotoxin (ST).4 Previous methods for the detection of ETEC and other E. coli pathogens causing diarrhea included several types of DNA hybridization assays, also known as colony hybridizations.5–7 Currently, polymerase chain reaction (PCR) techniques are commonly used in enteric diagnostic laboratories for the detection of ETEC.8–11 Iijima and others recently suggested that quantitative real-time PCR (qPCR) will better identify diarrhea-producing E. coli (DEC).12
Conventionally, five E. coli-like colonies selected from stool cultures of patients with TD are tested for ETEC and other DEC. 1,2 This procedure has been standardized to use five E. coli colonies for ETEC toxin assay without scientific validation. 13,14 The underlining assumption has been that most E. coli present in stool specimens of patients with acute diarrhea are of the pathogenic form. This assumption may or may not be correct because the extent of DEC present may be related to duration of illness at time of study. A study showed that ratios of DEC to non-pathogenic E. coli in stools of patients with diarrhea were low, with the exception of ETEC and Shiga toxin–producing E. coli (STEC), which were present in proportions as low as 40% of total E. coli.12 Moreover, more than one-third of patients with TD studied remain negative for all known enteropathogens.1 We hypothesized that some of these pathogen-negative patients have ETEC that simply go undetected. We proposed that an increase in number of E. coli colonies selected and tested will more accurately detect cases of TD caused by ETEC. Our proposition was further supported by studies showing a positive antibacterial drug effective for in the treatment of pathogen-negative TD. 15,16
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MATERIALS AND METHODS |
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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The collected stool samples were processed in our field laboratory for enteric bacterial pathogens (including Salmonella spp., Shigella spp., Vibrio spp., Campylobacter jejuni, Yersinia enterocolitica, Aeromonas spp., and Plesiomonas shigelloides) and parasites (Giardia lamblia, Cryptosporidium species, and Entamoeba histolytica) by previously described methods. 17
After we cultured these stools separately on selective MacConky agar, 20 colonies of E. coli for each patient were selected and saved in peptone stubs. The study sample consisted of 116 sets of 20 E. coli colonies. The sets of 20 E. coli colonies for each stool specimen were randomly numbered from 1 to 20 and divided into four sub-groups of five colonies: 1–5, 6–10, 11–15, and 16–20.
Testing of 20 colonies per subject for ETEC. Each patient with TD was tested for ETEC in four steps, corresponding to the four sub-groups of five E. coli colonies. First, five colonies (colonies 1–5) of all 116 stool cultures were tested for ETEC, by identifying ST or LT toxin–encoding genes using PCR (see below for methods). Patients who were positive for ST, LT, or ST/LT for these five E. coli colonies were considered positive for ETEC. The remaining 15 E. coli colonies for each of these patients positive for ETEC were not tested further. Patients who were negative for ST and LT for the first five E. coli colonies were taken to the second step, where the next five colonies (colonies 6–10) of each patient were tested for ETEC using PCR. Similarly, those patients positive for ST, LT, or ST/ LT in the second five E. coli colonies were considered positive for ETEC and was not tested beyond the first 10 E. coli colonies. The patients that remained negative for ETEC were taken to the third step. The same process was repeated for the third set of five E. coli colonies (colonies 11–15) and then for the fourth set (colonies 16–20). After testing for ETEC in up to 20 E. coli colonies, patients negative for ST and LT toxin–encoding genes were considered to be truly negative for ETEC.
DNA extraction and pooled DNA preparation. In groups of five colonies per patient, E. coli from peptone stubs were grown separately on MacConky agar plates at 37°C overnight and collected individually into 200 µL of deionized water in 1.5-mL microfuge tubes. 18 Each sample in microfuge tubes were heated at 100°C for 10 minutes, subjected to vertex for 10 seconds, and centrifuged at 12,000 rpm for 2 minutes. Supernatants containing the DNA extracts were transferred into separate 0.5-mL microfuge tubes and labeled. Pooled DNA samples were prepared by mixing 10 µL of each of the five DNA extractions corresponding to a patient into another 0.5-mL microfuge tube. Six DNA extracts for each patient, five for the individual E. coli colonies, and one for the pooled DNA sample were stored in their 0.5-mL microfuge tube at –20°C.
PCR amplification and ETEC identification. Conventional PCR was used to amplify the ST and LT toxin–encoding genes in the pooled DNA extracts of E. coli colonies prepared for each patient. The PCR mixture per DNA extract contained 2.0 µL of 10x buffer, 4.0 µL of 5Q buffer, 0.8 µL of 2.5 mM MgCl2, 1.5 µL of 2.5 µM dNTPs, 1.0 µL of forward primer, 1.0 µL of reverse primer, and 6.7 µL of double-distilled deionized water. A total of 0.5 µL of Taq DNA polymerase was used per 100 µL of PCR mixture. In each PCR tube, 3 µL of pooled DNA extract was combined with 17 µL of PCR reaction mixture. Amplification of ST and LT were done separately using only one set of primers at a time. The primer sequences used were ST-F 5'-TGGATGCCATGTCCGGAGGT-3', ST-R 5'-CAACAGGTACATACGTTACAGAC-3',LT-F5'-GAGT-ACTTCGATAGAGGAACTCA-3',andLT-R5'-GATCCGGT-GGGAAACCTGCT-3'. Two thermocycler were used to run the PCR starting at 95°C for 2 minutes, then 10 cycles at 94°C for 30 seconds, 62°C for 30 seconds, and 72°C for 1 minute, then 30 cycles at 94°C for 30 seconds, 56°C for 30 seconds, and 72°C for 1 minute, a final step at 72°C for 1 minute, and holding the PCR products at 4°C until removed. A positive control, E. coli H10407 (no. 35401; American Type Culture Collection, Manassas, VA) and a negative control (without a DNA extract) were used with each PCR cycle. Gel electrophoresis was used to separate amplified ST and LT toxin–encoding gene sequences present. Running gels were prepared with 1.5% agarose containing trace amounts of ethidium bromide (3 µL per 100-mL gel). Gel lanes contained a mixture of 3 µL of PCR product, 2 µL of 6x loading buffer (0.5 mM EDTA, 20% sodium dodecyl sulfate, 5% bromophenol blue, 1% glycerol), and 5 µL of 1 M Tris, pH 8.0, 0.5 M EDTA. Gels were run for 30 minutes, at 100V, and digital images of the bands were taken under ultraviolet light.
Confirmation and detection of ETEC-positive colonies. The PCR was conducted with pooled DNA extracts of groups of five E. coli colonies per patient. Once an ST- or LT-positive sample was detected in one of the pooled DNA extracts, this result was confirmed and determined as to which individual colonies were positive. This confirmation was conducted by repeating the PCR for either ST or LT on individual DNA extracts of the pooled DNA extracts that were positive for ST or LT. This method indicated how many ST- or LT-positive colonies were present within a sample of five E. coli colonies tested. In cases with an ST- and an LT-positive pooled DNA extract, the PCR for both ST and LT was repeated separately on individual DNA extracts. The same colony or DNA extract had to be positive for ST and LT to be identified as positive for ST/LT.
Statistical analysis and assay comparison. The results were grouped into four groups: colonies 1–5, colonies 1–10, colonies 1–15, and colonies 1–20. The PCR assay using 20 E. coli colonies had the best probability of detecting all cases with diarrhea caused by the ETEC. Therefore, the proportion of ETEC detected by the 20 E. coli colony assay was compared with that of the 5-, 10-, and 15-colony assays. Patients positive for ST, LT, ST/LT, or ETEC (total) in the first five colonies were assumed to be positive in the 10-, 15-, and 20-colony assays. Likewise, this assumption was also made for those positive for the second five colonies and the third five colonies. The sample of 116 case-patients with TD was large enough for a normal approximation, and the z-test was used to compare population proportions. After we applied the Bonferroni correction for multiple comparisons of data, the significance level (P value) for the comparison was set at 0.0167. There were 12 patients with one missing E. coli colony, 2 with 2 missing E. coli colonies, and 1 with 3 missing E. coli colonies of their sets of 20 E. coli colonies saved. This finding was caused either by drying-up of the peptone media or the absence of an E. coli colony in the peptone media. These missing colonies had no significant affect on the data and were not included in the statistical analysis.
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RESULTS |
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INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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). The 10-colony assay detected 37 (31.9%) patients positive for ETEC: 26 positive for ST, 10 positive for LT, and 1 positive for ST and LT. The 15-colony assay detected 45 (38.8%) patients positive for ETEC: 26 positive for ST, 17 positive for LT and 2 positive for ST and LT. The 20-colony assay detected 46 (39.7%) patients positive for ETEC: 26 positive for ST, 17 positive for LT, and 3 positive for ST and LT. The 5-colony assay had the lowest sensitivity, and sensitivities increased with use of more E. coli colonies. The 10-, 15-, and 20-colony assays were 1.68, 2.05, and 2.09 times, respectively, more sensitive than the 5-colony assay. If the 20-colony assay was assumed to have a sensitivity of 100%, the sensitivities of the 5-, 10-, and 15-colony assays would be 47.8%, 80.4% and 97.8%, respectively.
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Proportion of ETEC to E. coli stool cultures. The proportions (95% confidence intervals [CIs]) of ST, LT, and ST/LT toxin–encoding genes detected in patients positive for ETEC to total E. coli colonies present in these patients were 0.39 (0.29–0.49), 0.22 (0.18–0.32), and 0.11 (0.00–0.31), respectively. The proportion (95% CI) of ETEC to total E. coli was 0.31 (0.24–0.39) for the sample of patients in this study. It should be noted that in calculating the proportion estimates, the probability samples were not the same size, having 5, 10, 15, and at times, 20 colonies, depending upon the step at which the first positive sample was identified.
Mixed infection. Eight patients (7%) with ETEC infections were co-infected with another pathogen in this study. Co-infection with EAEC and ETEC accounted for six of the eight mixed infections. One patient with ETEC infection was co-infected with Cryptosporidium parvum and another was co-infected with Salmonella spp.
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DISCUSSION |
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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The sensitivity of the 20-colony assay was 1.24 times higher than that of the 10-colony assay. However, the 31.9% of ETEC-positive patients detected by the 10-colony assay was not significantly different from the 39.7% of ETEC-positive patients detected by the 20-colony assay (P = 0.0438). There was also no statistically significant difference between the 10- and 20-colony assays for detection of ST, LT, and ST/LT individually. Increasing the number of colonies from a 15 to 20 detected only one more ST/LT-positive patient, but this difference was not statistically significant.
The low proportion of DEC found by Iijima and others 12 when five E. coli colonies were tested is supportive of our findings. Their study also showed that ETEC and STEC were present in a proportion of approximately 40% among non-pathogenic E. coli, which was much higher than the observed proportions for non-ETEC DEC. Their study and ours provided evidence that ETEC could be detected with less than 20 colonies. Their suggestion that real-time PCR is needed for effective detection of DEC may not be true for ETEC because we could identify most ETEC-positive sample with less than 20 E. coli colonies.
Use of the conventional assay with five E. coli colonies would have categorized the ETEC diarrhea that we detected in colonies 6–20 as pathogen-negative after screening for other known enteropathogens. Our findings may at least partially explain a portion of the pathogen-negative TD cases found in studies of etiology of TD.1 Our results also help explain in part the effectiveness of antibacterial drug therapy used to treat pathogen-negative TD. 15
Although increasing the number of colonies tested increased the sensitivity and the percentage of ETEC detected, the specificity of the assays may decrease. The more colonies used, ETEC will be detected even when present in low quantities. Thus, this assay may detect ETEC that are not etiologically important in the acute diarrhea episode, which represent false-positives samples. Therefore choosing an optimum number of colonies for detection of ETEC is important. However, in our study, most ETEC-positive samples wee detected with the first 15 colonies.
We recently reported that fecal DNA remained stable on hemoccult cards stored at room temperature for up to 14 months and could be used for PCR amplification for detection of diarrheagenic E. coli.19 Significantly more ETEC cases were detected from hemoccult card DNA (38%) than from fecal DNA (30%) or by culture that was followed by hybridization (10%) (P < 0.001). These results support those of the current study, which indicate that ETEC may go undetected when standard assay methods are used.
We recommend the use of at least 10 E. coli colonies for detection of ETEC in enteric diagnostic laboratories instead of the five E. coli colonies often used. This modification will increase the sensitivity of the current PCR assay and the number of ETEC detected in patients with diarrhea patients and lead to a lower percentage of unexplained pathogen-negative TD.
Received July 23, 2008. Accepted for publication September 28, 2008.
Acknowledgments: We thank Dr. Asha S. Kapadia (The University of Texas School of Public Health [UTSPH]) for assisting with the statistical analyses, Dr. Ismail M. Meraz (UTSPH) for his contributions to the initial laboratory procedures, and the students of The University of Texas Medical School for collecting stool specimens used in this study. This project fulfilled the requirement for MPH for Thushara Galbadage at the University of Texas School of Public Health.
* Address correspondence to Zhi-Dong Jiang, 1200 Herman Pressler, RAS E-739, Houston, TX 77030. E-mail: Zhi-Dong.Jiang{at}uth.tmc.edu 
Authors’ addresses: Thushara Galbadage sand Zhi-Dong Jiang, The University of Texas School of Public Health at Houston, Houston, TX 77225. Herbert L. DuPont, The University of Texas School of Public Health at Houston, Houston, TX 77225; The University of Texas Medical School at Houston, Houston, TX 77030; St. Luke’s Episcopal Hospital, Houston, TX 77030; Baylor College of Medicine, Houston, TX 77030.
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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