The authors have declared that no competing interests exist.
Traditional diagnostic assays for
There are many testing methods available for the detection of
The aim of this study was to develop a proprietary quantitative real time PCR, analyse its utility and compare it with culture and RUT methods for the detection of
This study comprised samples from 199 symptomatic patients attending the Gastroenterology Unit of the Hospital Universitario San Agustín (Avilés, Spain). Written informed consent was obtained from each patient, and the protocol was approved by the Research Ethics Committee of the Hospital. At the time that samples were collected, patients had not received proton-pump inhibitor drugs (PPIs) or antibiotics for at least 2 weeks. Clinical features of the patients recruited are presented in
Male |
Female |
Total |
|
---|---|---|---|
Age (mean ± SD (range)) | 56.1±16.3 (14–87) | 57.6±18.6 (15–92) | 57.0±17.7 (14–92) |
Normal/Gastritis | 30 (40.0%) | 72 (58.1%) | 102 (51.3%) |
Gastric ulcer | 9 (12.0%) | 23 (18.5%) | 23 (16.1%) |
Duodenal ulcer | 29 (38.7%) | 27 (21.8%) | 56 (28.1%) |
Healed ulcer | 7 (9.3%) | 2 (1.6%) | 9 (4.5%) |
All the data were anonymised.
All the samples were taken from stomach antrum. All samples were transported to the Microbiology Laboratory in
DNA was extracted from the biopsies after an overnight enzymatic digestion with 0.25% trypsin at 37°C. A proteinase K method was used: 200μL of lysis solution (10mM Tris-HCl pH 8.3, 50mM potassium chloride, 2.5mM magnesium chloride, 0.5% Igepal, 0.5% Tween 20 and 10μg of proteinase K) was added to each sample, mixed and incubated for 40 minutes at 60°C, then 10 minutes at 96°C to inactivate proteinase K. Samples were then stored at -20°C until use.
An in-house system was designed to amplify a fragment of the
Primers and Probes | Sequences | |
---|---|---|
UreA |
Forward target DNA primer | |
Reverse target DNA primer | ||
Target DNA probe | ||
Beta | Forward target DNA primer | |
Reverse target DNA primer | ||
Target DNA probe |
A standard curve was performed using serial dilutions from 10 to 1010 of amplicon-based positive controls in order to determine the sensitivity of the assay.
The specificity was evaluated using bacterial strains of various microorganisms frequently found in the human gastrointestinal microbiome:
In addition, the human ß-globine gene was quantified in each sample in order to evaluate sample cellularity and correct for the different sizes of gastric biopsies in order to normalise bacterial load (
Bacterial count as copies/105cells and log was calculated using the quotient between the standard curve of the controls and the curve for the β-globine gene described in Álvarez-Argüelles et al.[
A patient was considered to be infected with
Statistical tests were performed using
A ten-fold dilution series of
By plotting the log DNA copies count against threshold cycle (Ct) values, a standard curve was performed (
Specificity of q-PCR was proven using DNA from several other bacteria, none of which gave any amplification signal.
Amplification of human ß-globine gene was positive in all the biopsies, indicating successful DNA extraction.
In terms of the diagnostic method used and endoscopy results,
Endoscopic findings | Normal/Gastritis |
Gastric ulcer |
Duodenal ulcer |
Healed ulcer |
TOTAL |
---|---|---|---|---|---|
q-PCR | 42 (41%) | 15 (47%) | 31 (55%) | 2 (22%) | 90 (45%) |
Culture | 37 (36%) | 14 (44%) | 28 (50%) | 2 (22%) | 81 (41%) |
RUT | 30 (29%) | 10 (31%) | 22 (39%) | 2 (22%) | 64 (32%) |
Culture+RUT | 41 (40%) | 15 (47%) | 31 (55%) | 2 (22%) | 89 (45%) |
The percentage of biopsies testing positive for
A total of 196 patients gave results which were concordant using both traditional methods and PCR. Of the non-concordant results, ten biopsies were positive by PCR although showed negative in culture, eight of which were RUT positive. One biopsy was negative by PCR, although positive by both culture and RUT. PCR and culture had the highest agreement (kappa = 0.887), then PCR and RUT (kappa = 0.709) and finally culture and RUT (kappa = 0.645).
Culture | RUT | Culture+RUT | ||||
---|---|---|---|---|---|---|
Positive | Negative | Positive | Negative | Positive | Negative | |
80 (40%) | 10 (5%) | 63 (32%) | 27 (14%) | 88 (44%) | 2 (1%) | |
1 (0.5%) | 108 (54%) | 1 (0.5%) | 108 (54%) | 1 (0.5%) | 108 (54%) |
The average
With respect to the different diagnostic tests, the average number of copies of
Out of 91 positive
Although both q-PCR and culture have high detection rates, while PCR gives results quickly, only 38 (42%) patients gave positive culture results within seven days. Speed of culture results however had no bearing on number of copies, average number for positive culture results before 7 days being 3.37±0.93 log (1.76–5.93) and those after 7 days being 3.32±1.15 log (0.75–5.67) (p = ns) (
Traditional diagnosis of
What is more, this method showed total specificity since it did not detect any of the other colonising bacteria from the same habitat as
This q-PCR also allows quantification of bacterial load expressed per 105 cells. The use of normalized counts, focusing on sample quality rather than absolute numbers enables comparisons between samples to be made. The possibility of quantifying bacterial load has been proven to be useful in previous studies, where the variability of
Of the 199 patients included in this study,
One of the great advantages of q-PCR over traditional methods is the decrease in response time. Indeed, culturing
It must be recognised, however, that traditional culture techniques are able to provide information about antimicrobial susceptibility, which is essential in order to establish the appropiate antibiotic treatment, and ensure local resistance data is taken into account in adjusting the empirical treatments. This technique could be improved by also considering with the addition of detection of genomic resistance by searching for specific mutations in the
We found differences between bacterial load in samples testing positive in two or three traditional diagnostic tests and those which gave only one positive result. Quantification of microorganisms in the gastric mucosa by q-PCR would complement the limitations of traditional methods making it possible to differentiate between asymptomatic colonisation and infection [
No bacterial load difference was associated with clinical symptoms. As said above, further work focused on more specific clinical symptoms and using a greater number of patients could help to clarify this point.
In conclusion, we developed a q-PCR with satisfactory results in terms of sensitivity, specificity, low cost and easy processing, though of course, supplementary studies with a greater number of samples would provide stronger data. The good results achieved in this study, coupled with the low cost and easy processing make this technique useful for both large and small microbiology laboratories, both public and private. It might even be possible to look for a non-invasive technique, such as pharyngeal swabs or stool samples where this technique could be used. Nevertheless, culture still remains an important tool as it provides information in terms of antimicrobial susceptibility which is necessary to choose the right treatment option [
We thank Ronnie Lendrum for her help with correcting the English.