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Previous Article | Next Article 
Journal of Clinical Microbiology, October 2001, p. 3778-3780, Vol. 39, No. 10
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.10.3778-3780.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Magnetic Immuno-PCR Assay with Inhibitor Removal for Direct
Detection of Helicobacter pylori in Human Feces
Lurdes
Monteiro,1,2
Nathalie
Gras,1 and
Francis
Megraud1,*
Laboratoire de Bactériologie,
Université Victor Segalen Bordeaux 2, Bordeaux,
France,1 and Instituto Nacional de
Saúde Dr. Ricardo Jorge, Lisbon, Portugal2
Received 23 February 2001/Returned for modification 30 May
2001/Accepted 23 July 2001
 |
ABSTRACT |
A PCR protocol was developed to detect Helicobacter
pylori in human stool specimens. This protocol was based on the
association of a magnetic immuno-PCR assay with a technique to remove
inhibitors (agarose-embedded DNA preparation). Of the 47 H. pylori-positive and 57 H. pylori-negative patients
included in this study, 38 were positive and 66 were negative by this
new protocol. The sensitivity, specificity, and predictive values for a
positive or a negative result were 80.9% (95% confidence interval
[CI], 66.3 to 90.4), 100% (95% CI, 92.1 to 100), 100% (95% CI,
88.6 to 100), and 86.4% (95% CI, 75.2 to 93.2), respectively.
| |
TEXT |
The high clinical relevance of
Helicobacter pylori infection has stimulated the development
of numerous diagnostic methods. These methods can be classified as
invasive, (i.e., those that require an endoscopy to detect H. pylori directly in gastric biopsy specimens) or noninvasive (i.e.,
based on the study of various samples (serum, breath air, urine,
etc.)), which indirectly indicate the presence of H. pylori.
Stool specimens constitute a sample of easy, noninvasive access and
consequently of high potential interest for the development of a direct
method of H. pylori detection. PCR is a powerful technique for the detection of target DNA in various clinical specimens, but its
application to stool specimens has been limited due to the presence of
substances inhibiting the reaction. Numerous attempts to detect
H. pylori by PCR in stool samples have been made, with controversial results (5, 12, 17; N. P. Mapstone,
F. A. Lewis, D. S. Tompkins, D. A. F. Lynch,
A. T. R. Axon, M. F. Dixon, and P. Quirke, Letter,
Lancet 341:447, 1993).
Difficulty in eliminating PCR inhibitors from stool specimens has been
extensively reported (4, 5, 8, 12). Some authors have
claimed to be successful, but usually only after applying complex
procedures that are difficult to implement routinely.
The aim of this study was to develop a PCR protocol to detect H. pylori in human stool specimens that is simple enough to be used
routinely. For this purpose, we associated a magnetic immuno-PCR assay
(MIPA) with a technique to remove inhibitors that was recently
described (11).
Clinical samples.
One hundred four consecutive, untreated,
dyspeptic patients (69 male, 35 female; mean age, 50 years; range, 17 to 87 years) who were consulting gastroenterologists for dyspepsia in
Bordeaux, France, were included in the study. Exclusion criteria were
as follows: H. pylori eradication treatment in the previous
6 months; consumption of antibiotics in the previous month; or
consumption of antisecretory drugs, bismuth salts, or sucralfate in the
previous 2 weeks. A history of coagulopathy or other disorders that are contraindications for endoscopy and/or biopsy sampling was also a
reason for exclusion. For each patient, biopsies were taken for
culture, histological examination, urease test (UT), and PCR. A urea
breath test (UBT) and a serological test were also performed as
previously described (9).
A patient was classified as being H. pylori positive on the
basis of (i) a positive culture; or, in the case of a negative culture, (ii) both a positive histological examination and a positive UT (CLOtest; TriMed Specialities, Osborne Park, Western
Australia); or (iii) positive noninvasive tests (serology and
UBT) and biopsy PCR. Stool specimens were collected within 1 week of
the time of endoscopy in sterile containers and kept at 
80°C until analysis.
Immunomagnetic separation of H. pylori from human feces
and DNA extraction.
Stool specimens were suspended at 1.5:5
(wt/vol) for solid and semisolid samples and 1.5:5 (vol/vol) for liquid
in phosphate-buffered saline and incubated overnight, under agitation
at room temperature. The suspension was then filtered through three
layers of cotton gauze and used for immunomagnetic separation of
H. pylori. Briefly, magnetic uncoated beads (Dynabeads
M-450; Dynal, Oslo, Norway) were coated with rabbit anti-H.
pylori immunoglobulin (Dako, Glostrup, Denmark) at a
concentration of 5 µg of antibody for 107 Dynabeads
according to the procedure recommended by Dynal. A 60-µl volume of
coated Dynabeads was mixed with 1 ml of fecal suspension and incubated
at 4°C with continuous shaking for 2 h. The coated Dynabeads
were recovered by magnetic force with a Dynalmagnet and then suspended
in the lysis buffer of the QIAamp tissue kit (Qiagen, Hilden, Germany)
for DNA extraction (9). Twenty microliters of a proteinase
solution (20 mg/ml) was then added, followed by incubation at 56°C
for 2 h. A second buffer provided in the kit was added, and the
sample was incubated at 70°C for 10 min. Next, 200 µl of ethanol
was added, and the suspension was loaded on the QIAamp spin column
followed by a centrifugation at 6,000 × g for 1 min.
The QIAamp spin column was placed in a 2-ml collection microtube, and
the tube containing the filtrate was discarded. The column material was
washed twice (250 µl each) with the first washing buffer and twice
(250 µl each) with the second washing buffer provided in the kit.
Finally, the DNA was eluted with 100 µl of distilled water preheated
to 70°C (2 × 50 µl).
PCR.
PCR was performed as described previously
(7). Briefly, reactions were carried out in a volume of 50 µl with a combination of 10× PCR buffer [670 mM Tris-HCl (pH 8.8),
160 mM (NH4)SO2, 0.1% Tween]; 1.5 mM
MgCl2; 200 µM (each) nucleotides dATP, dTTP, dGTP, and
dCTP; and 3 U of Taq polymerase (Eurobio, Les Ulis, France),
with 5 µl of purified sample and with specific primers (HPU1 and
HPU2) for the ureA gene of H. pylori
(1) at 0.4 µM each. PCR consisted of 35 cycles of 1 min
at 94°C, 1 min at 45°C, and 1 min at 72°C with a first cycle of 5 min at 95°C and a final cycle of 5 min at 72°C. Bands were
visualized on a 1% agarose gel stained with ethidium bromide.
The size of the PCR product obtained when using the HPU1 and HPU2
primers was 411 bp. In each experiment, a positive control consisting
of 50 ng of reference strain DNA and a negative control (distilled
water) were performed.
Removal of PCR inhibitors.
The PCR inhibitors present in stool
samples were removed by using an agarose-embedded DNA preparation
described previously (9, 11). Briefly, 1 volume of 1.6%
melted agarose was added to each DNA aliquot; these were then poured
into molds (Disposable Plug Molds; 1.5 by 10 by 5 mm; Bio-Rad,
Richmond, Calif.) until solidification. Agarose blocks were removed
from molds and washed in Tris-EDTA (10 ml per block) overnight with
gentle shaking. A second wash with 5 ml of distilled water was
performed (2 h with gentle shaking). PCR was subsequently carried out
with DNA-containing agarose slices as templates.
Statistics.
Standard methods were used to calculate
sensitivity, specificity, predictive values of positive and
negative results, and the 95% confidence intervals (CI) of these values.
Ethics.
This study received the approval of the Ethics
Committee of Bordeaux. The informed consent of the patients was also obtained.
According to the case definition, 47 of the 104 patients tested were
H. pylori positive, and 57 were negative. The five patients for which doubtful results were found based on invasive tests were
evaluated according to the results of noninvasive tests and biopsy PCR
(Table 1). Two were considered
H. pylori positive, and three were considered H. pylori negative.
View this table:
[in this window]
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|
TABLE 1.
Diagnostic test results for the five patients for whom
the results of invasive tests for Helicobacter pylori
diagnosis were doubtful
|
|
According to this new PCR protocol to detect H. pylori in
human feces (Fig. 1), 38 of the 47 H. pylori-positive patients were H. pylori
positive (giving 9 false negatives), and all 57 H. pylori-negative patients were also H. pylori negative
with the new protocol (no false positive). The sensitivity,
specificity, and predictive values for a positive or a negative result
were 80.9% (95% CI, 66.3 to 90.4), 100% (92.1 to 100), 100% (88.6 to 100), and 86.4% (75.2 to 93.2), respectively. Most of the cases
(n = 32) were detected before agarose blocks were
performed. However, six additional cases were detected with this
method. The overall correlation between biopsy PCR and stool PCR was
good (90 of 104). There were 11 cases positive only by biopsy PCR, and
there were 3 cases positive only by stool PCR.
Detection of H. pylori from stool specimens as a diagnostic
tool for detecting H. pylori infection is attractive because
it is noninvasive. However, H. pylori has never been
reliably detected from stools (3). The first reason is probably the
lack of viability of these bacteria in an environment of competing
flora under normal conditions in the bowel. In addition, selective
culture media for the isolation of H. pylori are frequently
contaminated with other, faster-growing gram-negative bacteria. The
only positive attempts reported used feces obtained from patients
experiencing spontaneous (15) or induced diarrhea
(13).
Similarly, detection of H. pylori in stool specimens by
using PCR after standard extraction methods has proved to be difficult (5, 12, 17; Mapstone et al., Letter), producing erratic results due to a variety of fecal inhibitors, such as acid
polysaccharides, metabolic products, and large amounts of irrelevant
DNA (8).
In this study, we developed a new PCR protocol to detect H. pylori in human feces. For this purpose, a MIPA was associated with an agarose-embedded DNA preparation to remove inhibitors (9). Successful amplification and specific detection of
H. pylori DNA directly from stool samples in most of
the infected patients indicate that MIPA, in association with the
agarose-embedded DNA block method, is a promising procedure to detect
H. pylori in stool specimens, but requires further
development before it will be suitable for routine clinical use,
because the sensitivity obtained is not sufficient. However, it is
easier to apply than previously published methods (4, 5;
Mapstone et al., Letter), avoiding the use of nested PCR. While nested
PCR is supposed to increase sensitivity, it also has some limits,
especially in routine use. The high risk of contamination makes it
difficult to implement, and for this reason, such a procedure has not
been recommended in the past (2).
Another stool test, based on the detection of H. pylori
antigens to diagnose infection, is also currently available
(16). However, despite the fact that this H. pylori antigen stool assay is reliable and easy to perform, it
only indicates the presence or absence of H. pylori based on
the detection of antigens the exact character of which is unknown. In
contrast, PCR can provide additional information, principally related
to the presence of pathogenicity factors (cagA or
vacA alleles) or susceptibility to macrolides, antibiotics
commonly used to treat this infection (6, 14). This
subject will be of great interest for future investigations.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire de
Bactériologie, Hôpital Pellegrin, Place Amélie Raba
Léon, 33076 Bordeaux Cedex, France. Phone: (33) 5 56 79 59 77. Fax: (33) 5 56 79 60 18. E-mail:
francis.megraud{at}chu-bordeaux.fr.
| |
REFERENCES |
| 1.
|
Clayton, C. L.,
H. Kleanthous,
P. J. Coates,
D. D. Morgan, and S. Tabaqchali.
1992.
Sensitive detection of Helicobacter pylori by using polymerase chain reaction.
J. Clin. Microbiol.
30:192-200[Abstract/Free Full Text].
|
| 2.
|
European Helicobacter pylori Study Group.
1997.
Guidelines for clinical trials in Helicobacter pylori infection.
Gut
41(Suppl. 2):8-13[Abstract/Free Full Text].
|
| 3.
|
Kelly, S. M.,
M. C. L. Pitcher,
S. M. Farmery, and G. R. Gibson.
1994.
Isolation of Helicobacter pylori from feces of patients with dyspepsia in the United Kingdom.
Gastroenterology
107:1671-1674[Medline].
|
| 4.
|
Lantz, P.-G.,
M. Matsson,
T. Wadström, and P. Rådstrom.
1997.
Removal of PCR inhibitors from human faecal samples through the use of an aqueous two-phase system for sample preparation prior to PCR.
J. Microbiol. Methods
28:159-167[CrossRef].
|
| 5.
|
Makristathis, A.,
E. Pasching,
K. Schütze,
M. Wimmer,
M. L. Rotter, and A. M. Hirschl.
1998.
Detection of Helicobacter pylori in stool specimens by PCR and antigen enzyme immunoassay.
J. Clin. Microbiol.
36:2772-2774[Abstract/Free Full Text].
|
| 6.
|
Marais, A.,
L. Monteiro,
A. Occhialini,
M. Pina,
H. Lamouliatte, and F. Mégraud.
1999.
Direct detection of Helicobacter pylori resistance to macrolides by a polymerase chain reaction/DNA enzyme immunoassay in gastric biopsy specimens.
Gut
44:463-467[Abstract/Free Full Text].
|
| 7.
|
Monteiro, L.,
C. Birac, and F. Mégraud.
1995.
Detection of Helicobacter pylori in gastric biopsy by polymerase chain reaction, p. 112-120.
In
A. Lee, and F. Mégraud (ed.), Helicobacter pylori: techniques for clinical diagnosis and basic research. W. B. Saunders Co., Ltd., London, England.
|
| 8.
|
Monteiro, L.,
D. Bonnemaison,
A. Vekris,
K. G. Petry,
J. Bonnet,
R. Vidal,
J. Cabrita, and F. Mégraud.
1997.
Complex polysaccharides as PCR inhibitors in feces: Helicobacter pylori model.
J. Clin. Microbiol.
35:995-998[Abstract].
|
| 9.
|
Monteiro, L.,
N. Gras,
R. Vidal,
J. Cabrita, and F. Mégraud.
2001.
Detection of Helicobacter pylori DNA in human feces by PCR: DNA stability and removal of inhibitors.
J. Microbiol. Methods
45:87-94.
|
| 10.
|
Monteiro, L.,
A. de Mascarel,
A. M. Sarasqueta,
B. Bergey,
C. Barberis,
P. Talby,
D. Roux,
L. Shouler,
D. Goldfain,
H. Lamouliatte, and F. Mégraud.
2001.
Diagnosis of Helicobacter pylori infection: non-invasive methods compared to invasive methods and evaluation of two new tests.
Am. J. Gastroenterol.
96:353-358[CrossRef][Medline].
|
| 11.
|
Moreira, D.
1998.
Efficient removal of PCR inhibitors using agarose-embedded DNA preparations.
Nucleic Acids Res.
26:3309-3310[Abstract/Free Full Text].
|
| 12.
|
Notarnicola, M.,
F. Russo,
A. Cavallini,
M. Bianco,
E. Jirillo,
S. Pece,
C. Leoci,
G. di Matteo, and A. di Leo.
1996.
PCR identification of Helicobacter pylori DNA in faeces from patients with gastroduodenal pathology.
Med. Sci. Res.
24:785-787.
|
| 13.
|
Parsonnet, J.,
H. Shmuely, and T. Haggerty.
1999.
Fecal and oral shedding of Helicobacter pylori from healthy infected adults.
JAMA
23:2240-2245.
|
| 14.
|
Pina, M.,
A. Occhialini,
L. Monteiro,
H.-P. Doermann, and F. Mégraud.
1998.
Detection of point mutations associated with resistance of Helicobacter pylori to clarithromycin by hybridization in liquid phase.
J. Clin. Microbiol.
36:3285-3290[Abstract/Free Full Text].
|
| 15.
|
Thomas, J. E.,
G. R. Gibson,
M. K. Darboe,
A. Dale, and L. T. Weaver.
1992.
Isolation of Helicobacter pylori from human feces.
Lancet
340:1194-1195[CrossRef][Medline].
|
| 16.
|
Vaira, D.,
P. Malfertheiner,
F. Mégraud, and A. T. Axon.
1999.
Diagnosis of Helicobacter pylori infection by HpSA test.
Lancet
354:1732[Medline].
|
| 17.
|
van Zwet, A. A.,
J. C. Thijs,
A. M. D. Kooistra-Smid,
J. Schirm, and J. A. M. Snijder.
1994.
Use of PCR with feces for detection of Helicobacter pylori infections in patients.
J. Clin. Microbiol.
32:1346-1348[Abstract/Free Full Text].
|
Journal of Clinical Microbiology, October 2001, p. 3778-3780, Vol. 39, No. 10
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.10.3778-3780.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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