Research Article
Helicobacter pylori from Peptic Ulcer Patients in
Uganda Is Highly Resistant to Clarithromycin and
Fluoroquinolones: Results of the GenoType HelicoDR
Test Directly Applied on Stool

Denish Calmax Angol,1,2 Ponsiano Ocama,3 Tess Ayazika Kirabo,1

Alfred Okeng,4 Irene Najjingo,4 and Freddie Bwanga1,4

1Department of Medical Microbiology, Makerere University College of Health Sciences, P.O. Box 7072, Kampala, Uganda
2Department of Midwifery, Faculty of Health Sciences, Lira University, P.O. Box 1035, Lira, Uganda
3Department of Medicine, Makerere University College of Health Sciences, P.O. Box 7072, Kampala, Uganda
4MBN Clinical Laboratories, Plot 28 Nakasero Road, Kampala, Uganda

Correspondence should be addressed to Freddie Bwanga; fxb18@case.edu

Received 30 September 2016; Revised 13 January 2017; Accepted 13 February 2017; Published 7 May 2017

Academic Editor: Frederick Adzitey

Copyright © 2017 Denish Calmax Angol et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.

Background. Around 70–90% of peptic ulcer disease (PUD) is due toHelicobacter pylori and requires treatment with antimicrobials
to which these bacteria are susceptible. Common H. pylori diagnostic tests do not provide drug susceptibility data. Using
the GenoType HelicoDR PCR test designed for gastric biopsies for simultaneous detection of H. pylori and its resistance to
clarithromycin (CLA)/fluoroquinolones (FLQ), we present evidence for stool as an optional test specimen and also provide data
on prevalence of H. pylori resistance to CLA and FLQ in Uganda. Methods. Stool from 142 symptomatic PUD patients at three
hospitals in Kampala was screened for H. pylori using a rapid antigen test. The GenoType HelicoDR test was run on all H. pylori
antigen positives to determine PCR positivity and resistance to CLA/FLQ. Results.Thirty-one samples (22%) wereH. pylori antigen
positive, and 21 (68%) of these were H. pylori PCR positive. Six of the 21 (29%) were resistant to CLA and eight to FLQ (42%),
while two gave invalid FLQ resistance results. Conclusion. Stool is a possible specimen for the GenoType HelicoDR test for rapid
detection of H. pylori and drug resistance. In Uganda, Helicobacter pylori is highly resistant to CLA and FLQ.

1. Introduction

Helicobacter pylori is a motile Gram-negative, oxidase-,
catalase-, and urease-positive, and microaerophilic bacillus
[1]. Beingmicroaerophilic, it needs only about 4%oxygen, 5%
carbon dioxide, and 5% hydrogen for its growth and survival.
This bacterium has been reported to be the cause of 90%
of duodenal and 70% of gastric ulcer cases and may lead to
stomach and/or duodenal perforation and fatal bleeding, and
it is also associated with gastric cancer [2–4]. It is estimated
that half of the world’s population is infected with H. pylori,
making it the most widespread infection in the world [5–
7]. The actual burden of infection varies from nation to

another. In Western Europe, North America, Australia, and
parts of Asia, the proportion of infected people is estimated
to be about 25% [7]. Developing countries have much higher
infection rates, and in Africa, it is estimated that 61–100% of
the population is infected and at risk of PUD and the other
associated diseases [2, 8–11].

Effective management of PUD caused by H. pylori
requires a combination of antimicrobials and proton pump
inhibitors (PPIs, which inhibit secretion of acid by the
stomach). The antimicrobial agents commonly used in first-
line treatment include clarithromycin (CLA), metronidazole,
and amoxicillin, while others such as fluoroquinolones (FLQ)
and tetracycline are used in second-line regimens. A number

Hindawi
BioMed Research International
Volume 2017, Article ID 5430723, 9 pages
https://doi.org/10.1155/2017/5430723

https://doi.org/10.1155/2017/5430723


2 BioMed Research International

of studies have shown thatH. pylori has developed resistance
to the most commonly used antimicrobial agents used in
PUD treatment. Work reported by Lee et al. from 2003 to
2012 found that resistance ofH. pylori to CLA increased from
17 to 24% while for the FLQ (levofloxacin and moxifloxacin)
from5% to 28%and amoxicillin from6% to 15% [12]. Another
study by Kumala and Rani in 2006 found that H. pylori
resistance to CLAwas 28%, to amoxicillin at 19%, and to FLQ
(ciprofloxacin, norfloxacin, and ofloxacin at 7%, sparfloxacin
and gatifloxacin at 3%, and levofloxacin and moxifloxacin
at 1%) [13]. Similarly, the prevalence of resistance in 128 H.
pylori strains isolated in 2004-2005 to FLQ (ciprofloxacin
MIC > 1mg/L) was 17% (22 strains) [14], while for CLA
the pretreatment and posttreatment prevalence of resistance
were 19% (23/123) and 69% (9/13), respectively [15]. This
CLA resistance was relatively high compared to that reported
as the national prevalence of resistance in France, which
stood at 13% [16]. Results of a study of 2204 patients in
Europe showed that H. pylori resistance rates were 16%
to CLA, 14% to levofloxacin, and 35% to metronidazole.
Resistance was significantly higher for CLA and levofloxacin
in Western, Central, and Southern Europe (>20%) than in
Northern European countries (<10%) [17]. Most of these
studies recommended that in addition to accurate detection
of H. Pylori, susceptibility tests (resistance diagnosis) should
be done prior to treatment.

The diagnosis of H. pylori infection is currently carried
out using four major methods [18, 19]: (i) breath test, in
which the patient drinks 13C- or 14C-labelled urea, which
the bacteriummetabolizes producing labelled carbon dioxide
that can be detected in the breath (the test is quick in
the initial detection of the bacteria but requires complex
equipment and cannot be used to detect drug resistance);
(ii) stool antigen test, which detects H. pylori in the faeces
for the purpose of initial detection of the bacteria and
recurrences after antibiotic therapy (just like breath test, the
H. pylori stool antigen test cannot detect drug resistance);
(iii) blood antibody test, which detects antibodies to H. pylori
in blood samples for purpose of detecting prior exposure
but cannot also detect drug resistance; and (iv) gastric biopsy
examination test (for culture, rapid urease test, or histological
examination), which confirms H. pylori infection in PUD
cases. These test approaches have before been regarded as
reference tests but they are invasive, expensive, and not easily
adopted in routine diagnostics in resource-poor settings.
Culture and phenotypic antimicrobial susceptibility testing
of H. pylori requires up to 10–14 days, on expensive special
media and special microaerophilic environments, and it
is hardly performed in routine clinical laboratories in the
resource-poor settings [20].

The GenoType HelicoDR test (GTHDT, Hain Lifescience
Nehren, Germany) was developed recently.This is a multiplex
PCR-based assay for simultaneous detection of H. pylori and
its resistance to two-key antibiotics, CLA and FLQ [21]. The
test was originally designed for use on gastric biopsy or
culture materials to detect mutations in gyrA gene codons
87 and 91 for resistance to FLQ and in the 23S rRNA genes
for resistance to CLA. The test involves DNA extraction,

amplification, and solid-phase reverse hybridization onto
probes fixed on a nitrocellulose membrane. Several studies
evaluating the GTHDT for diagnosis of CLA/FLQ-resistant
H. pylori have been conducted on biopsy specimens or cul-
tured isolates, but as a diagnostic test forH. pylori performed
directly on stool specimens the data is limited. In 2009,
Cambau et al. evaluated the GTHDT using gastric biopsy
and clinical strains, and the sensitivity and specificity for
detecting resistance were high, that is, 94% and 99% for CLA
and 87% and 98.5% for FLQ, respectively [22].

However, the requirement of stomach or duodenal biopsy
specimen is a major limitation to use of this test in poor
countries where endoscopic facilities as well as the other
tests remain extremely scarce and expensive. Obtaining
these biopsy specimens involves an invasive and expensive
endoscopic procedure not usually acceptable by patients.
Consequently, this potentially useful test has attracted little
use in routine patient care, particularly in the developing
countries where H. pylori infection is highest, yet resources
for endoscopy are very limited. Hence, very few data on
H. pylori resistance have come from these resource-poor
countries. An alternative noninvasive, less costly, easy to
collect specimen such as stool, which is also more acceptable
to patients, would be needed for use with the GTHDT in
routine PUD patient care particularly in the detection of
H. pylori and diagnosis of drug resistance. This study was
undertaken to determine the performance of the GTHDT
on stool as a starting material for molecular diagnosis of H.
pylori and its resistance to CLA and FLQ simultaneously and
determine the prevalence ofH. pylori resistance to CLA/FLQ
in Uganda.

2. Materials and Methods

2.1. Study Design and Approval. This was a cross-sectional
study carried out fromNovember 2012 to July 2013.This study
was approved by the School of Biomedical Sciences Research
and Ethics Committee (SBS-REC) of Makerere University
College of Health Science (SBS 076) and by the Mulago
Hospital Research and Ethics Committee (MREC: 374).

2.2. Study Site and Setting. The study was conducted at three
sites (Mulago, Mengo, and Case Medical Centre Hospitals)
in Kampala, Uganda. The Mulago National Referral Hos-
pital has a gastrointestinal (GI) unit with both inpatient
and outpatient services. The outpatient service runs weekly
with about 40 patients seen every week, about one-half
due to either established or suspected PUD cases, some of
which get admitted into the wards. Mengo Hospital and
Case Medical Centre are tertiary referral Nongovernmental
Organisation and private hospitals within Kampala City,
respectively.They provide care formany patients with various
ailments including gastroenterological cases. Recruitment of
the study participants and sample collections were done at
these three hospitals. All laboratory assays were performed
at MBNClinical Laboratories, Kampala, Uganda, which offer
advanced medical laboratory diagnostic services for patient
care and research.



BioMed Research International 3

2.3. Study Population and Inclusion Criteria. The study
included patients with suspected PUD who were at least 13
years old attending the GIT clinics at the study hospitals.
Inclusionwas based on having symptoms and signs of PUDas
judged by the attending physician and consent to participate
in the study. These symptoms included burning pain in the
epigastrium with or without any of the following: bloating,
nausea, dark or black stool, vomiting blood, or weight
loss. The patients were individually approached to obtain
informed consent (or assent with legal guardian consent)
and consecutively recruited into the study. A predesigned
case report form was used to collect both demographic and
clinical data.

2.4. Sample Collection. Upon consenting, each participant
was provided with a sterile dry stool container and instructed
to collect three scoopful of the stool into the stool container.
The stool specimen was then placed in a cool box and
transported to the laboratory within 2 hours of sample
collection.

2.5. Laboratory Methods

2.5.1. H. pylori Stool Antigen Test. All stool samples were test-
ed for H. pylori antigen using an immunochromatopgraphy
assay, the Taytec H. pylori stool antigen rapid diagnostic kit
(Taytec Enterprises Canada) whose manufacturer’s claimed
sensitivity and specificity were above 95% [23, 24]. In the
assay, the H. pylori antigen in stool reacts with conjugated-
red latex particles sensitised with anti-H. pylori monoclonal
antibodies. The H. pylori-conjugate complex migrates along
the membrane by capillarity and binds to the specific anti-
body molecules fixed at the reaction zone. The excess of
the complex keeps migrating through the membrane until
reaching the control (C) zone where it binds to another
specific antibody coated to the membrane forming a green
band. Presence of the green band confirms the functionality
of the test. In the procedure, about 50mg of each stool sample
was picked using the applicator stick and emulsified in the
buffer and four drops of the emulsionwere dispensed into the
well of the cassette. The interpretations of the results (bands)
for the H. pylori stool antigen test were as follows: a positive
result was indicated by two bands (control = green and test
= red), negative by only the control band (green), and invalid
by either only red or no band [24]. The detailed procedure is
found in the Taytec, Aldercrest Dr. Mississauga, Canada, H.
pylori stool antigen test kit insert [24].

2.5.2. GTHDT. OnlyH. pylori antigen positive stool samples
were subjected to this test.

DNA Extraction from Stool. This was performed using the
QIAamp� DNA Stool Mini Kit (Qiagen, Hilden Germany,
Catalogue Number: 51504). The procedures are detailed in
the QIAamp DNA Stool Handbook [25]. Briefly, 180 to
220mg of each H. pylori antigen positive stool specimen was
transferred to 1.5mLmicrocentrifuge tube and theASL buffer
was added to lyse the bacterial cells.The suspension was then
heated for 5min at 70∘C and then vortexed and centrifuged

to pellet debris. The supernatant was then transferred into
a new 1.5mL microcentrifuge followed by addition of the
InhibitEX tablet and incubated at 65∘C for 5 minutes to
allow inhibitors to be adsorbed to the InhibitEX matrix.
After centrifugation, 200𝜇L of the resultant supernatant
was transferred to 15 𝜇L proteinase K in another 1.5mL
microcentrifuge tube. Then 200 𝜇L of absolute ethanol was
added to the lysate to purify the DNA.This was then washed
with 500 𝜇L of buffer AW1 followed by the same amount of
buffer AW2. It was then eluted using 50 𝜇L of elution buffer
AE after incubation at 65∘C for 10 minutes. Eight microlitres
of the pure DNA was added to 45 𝜇L PCR reagent mix ready
for amplification.

Multiplex PCR Amplification of Extracted DNA. As rec-
ommended by the manufacturer, the composition of each
reaction mix was 35 𝜇L of Primer Nucleotide Mix (PNM,
containing biotin labelled primer sequences), 5𝜇L of 10x
PCR buffer, 5𝜇L of 25mM MgCl

2
, 0.2𝜇L of HotStarTaq

polymerase (1 U), and 8 𝜇L of the extracted DNA solutions.
The PCR reaction tubes were loaded and amplified on the
GTQ Cycler 96 (Hain LifeScience Nehren, Germany). The
amplification program included phase 1, one cycle at 95∘C for
15min; phase 2, 10 cycles at 95∘C for 30 s and at 58∘C for 2min;
phase 3, 25 cycles at 95∘C for 30 s, 53∘C for 40 s, and 70∘C for
40 s; and phase 4, one cycle at 70∘C for 8min. Details of the
PCR reagent recipe, primer sequences, what they detect, and
detailed procedure are found in the GTHDT kit insert, Lot
number JF0045 [21].

Detection of Amplification Products.This was performed using
a solid-phase reverse hybridization technique on the TwinCu-
bator according to themanufacturer’s instructions outlined in
theGTHDTkit insert [21].Thehybridization process entailed
chemical denaturation of double stranded DNA at room
temperature for 5min, hybridization of single stranded DNA
with complementary DNA probes fixed on a nitrocellulose
membrane strip in the hybridization buffer (HYB, green)
at 45∘C for 30min, stringent washing, conjugation at room
temperature for 30min, and enzymatic detection of the
reaction products [21].

Quality Control and Interpretation of the Results. During
the entire experimental processes, measures were taken
to avoid contamination and ensure validity of the results.
Also, positive and negative controls were included at all
steps from sample processing to hybridization. Data were
cleaned before analysis. A result was considered valid if the
Helicobacter pylori (HP), amplification control (AC), and
conjugate control (CC) bands were all present. In addition,
the locus controls (gyrA, 23S and RNA) must have stained
positive when the HP zone indicated the presence of H.
pylori. If neither the locus control probe nor the respective
wild type or mutation probes of one of the two genes
examined had developed, the result was considered invalid
for interpretation of resistance to the respective drug. The
bands formed were interpreted accordingly as shown in the
prototype in Figure 1, and details are outlined in the GTHDT
kit insert [21].



4 BioMed Research International

FLQS FLQS

CLAS CLAS CLAS CLAS
FLQR FLQR FLQR

CLAR

Conjugate control [CC]
Ampli�cation control [AC]

Helicobacter pylori [HP]

gyrA locus control [gyrA]
gyrA 87 wild type probe 1 [gyr87 WT1]
gyrA 87 wild type probe 2 [gyr87 WT2]
gyrA 87 wild type probe 3 [gyr87 WT3]
gyrA 87 wild type probe 4 [gyr87 WT4]

gyrA 91 wild type probe [gyr91 WT]
gyrA 87 mutation probe [gyr87 MUT]

gyrA 91 mutation probe 1 [gyr91 MUT1]
gyrA 91 mutation probe 2 [gyr91 MUT2]
gyrA 91 mutation probe 3 [gyr91 MUT3]

23S locus control [23S] 
23S wild type probe [23S WT]
23S mutation probe 1 [23S MUT1]
23S mutation probe 2 [23S MUT2]
23S mutation probe 3 [23S MUT3]

FLQS
= �uoroquinolone sensitive

FLQR
= �uoroquinolone resistant

CLAS
= clarithromycin sensitive

CLAR
= clarithromycin resistant

1 2 3 4 5

Figure 1: Prototype of the GTHDT result (downloaded from http://www.hain-lifescience.de/en/products/microbiology/helicobacter/geno-
type-helicodr.html).

2.5.3. DataManagement. The collected data was entered into
a Microsoft excel spread sheet and exported to SPSS software
v.11 for the analysis. The proportions of positivity of each test
and the proportion of H. pylori resistance to CLA and FLQ
were computed using SPSS v.16 software.

3. Results

3.1. Baseline Characteristics of the Studied Participants. A
total of 142 participants, 105 (74%) from Mulago, 22 (15%)
from Mengo, and 15 (1%) from Case Medical Centre, were
recruited. Of these, 79/142 (55.6%) were females. The partic-
ipants were aged between 13 and 85 (mean age = 40, SD =
18). A total of 135 participants had had the symptoms of PUD
for more than two weeks. In addition, at least 42% (59/142)
admitted to have taken one or more antibiotic(s) during the
course of illness. Furthermore, 114/142 (80%) had taken a
drug within one week before the screening. Detailed data are
shown in Table 1.

3.2. Taytec H. pylori Antigen Test Results. Out of the 142 stool
samples, 31 were positive for the H. pylori antigen giving a
prevalence of 22% in patients clinically diagnosed with PUD
as shown in Figure 2.

3.3. GTHDT Results. In Figure 3 in the supplementary mate-
rials (available online at https://doi.org/10.1155/2017/5430723),

we have shown representative results of hybridization on the
nitrocellulose strips from our laboratory.

Of the 31 H. pylori stool antigen positive samples, 21
(68%) tested positive with the GTHDT, as shown in Table 2
and Figure 3 in the supplementary materials. Among the 10
GTHDT negative samples, 7 were from patients who had
taken antibiotics such as amoxicillin, metronidazole, and
ceftrioxone, and one of these had also taken ciprofloxacin.

3.4. H. pylori Resistance to CLA and FLQ

3.4.1. CLA. All the 21 samples which tested positive for H.
pylori with the GTHDT gave valid CLA test results, and 6
(29%) were resistant to CLA as shown in Figure 3 in the
supplementary materials.

3.4.2. FLQ. Nineteen of the 21 antigen positive samples gave
interpretable results for FLQ resistance testing, of which 8
(42%) were resistant as shown in Table 2 and Figure 2. Invalid
results were due to lack of the gyrA gene locus control bands
for H. pylori.

4. Discussion

In this study, we explored new simple to collect stool
specimens as starting materials for use with the GTHDT for
simultaneous detection forH. pylori and its resistance to CLA

http://www.hain-lifescience.de/en/products/microbiology/helicobacter/genotype-helicodr.html
http://www.hain-lifescience.de/en/products/microbiology/helicobacter/genotype-helicodr.html
https://doi.org/10.1155/2017/5430723


BioMed Research International 5

H. pylori antigen positive
31 (22%)

H. pylori PCR Negative
10 (32%)H. pylori PCR Positive

21 (68%)

Resistant to FLQ
8 (42%)

Sensitive to FLQ
11 (58%)

Resistant to CLA
6 (29%)

Sensitive to CLA
15 (71%)

H. pylori antigen negative
111 (78%)

Clinically diagnosed with PUD 
(n = 142)

GenoType HelicoDR PCR test
(n = 31)

GenoType HelicoDR PCR test for FLQ 
(n = 21)

Valid results
(n = 21)

Invalid results
(n = 2)

Valid results
(n = 19)

GenoType HelicoDR PCR test for
CLA (n = 21)

Figure 2: Study flowchart and summary of the results. Invalid results: lack of H. pylori control bands in the gyrA gene locus, CLA:
clarithromycin, and FLQ: fluoroquinolones. Of the two FLQ invalid results, one was CLA susceptible and the other was resistant.

and FLQ. We also determined the proportions of H. pylori
resistance to FLQ and CLA. We anticipate that our findings
have the potential to inform clinicians on the level of H.
pylori resistance to the selected antibiotics, which will help to
improve on overall management of patients with suspected
peptic or gastroduodenal ulcers. Additionally, with stool as
specimen for this test, we anticipate high patient acceptability
and reduce the costs to the patient/health systems of testing
for H. pylori.

In this preliminary study only H. pylori antigen positive
stool samples were subjected to the study test. Out of the
H. pylori stool antigen positive specimens, the GTHDT
showed a high positivity score of 68% (21/31). By study
design,we did not aim at determining sensitivity or specificity
of the GTHDT at this stage of new test evaluation, and
another study to do this has been planned. However, the
high positivity score obtained in this study provides strong
evidence for stool as a possible specimen for same day
molecular diagnosis ofH. pylori and its resistance to CLA and
FLQ.Our results are similar to those obtained in other studies
on H. pylori diagnostics. In a study in 2016, Brennan et al.
used the GenoType HelicoDR test to detect 80.3% (53/66) of
H. pylori infection in stool samples [26]. In another study by

Lottspeich et al., 63% of the confirmed stool samples turned
positive for H. pylori when a real-time PCR assay was used
[23]. Data from various studies indicate that antigen tests
for H. pylori on stool samples have a high sensitivity and
specificity [23].The low performance of the PCR-based test in
our study compared with the antigen test is disappointing but
might be improved if antigen positive PCR-negative samples
were retested, and/or the amount of stool/DNA used for the
PCR-based test was increased as reported by Schabereiter-
Gurtner et al. 2004 [27]. By repeating the stool antigen tests
before PCR, Schabereiter-Gurtner and colleagues reported
that the real-time PCR test was highly accurate in the detec-
tion of H. pylori infection in stool and allowed for culture-
independent clarithromycin susceptibility testing [27]. Fur-
thermore, in a systematic review and meta-analysis study in
patients with bleeding peptic ulcer by Gisbert et al. in 2006
[28], the sensitivities of the different test methods were stool
antigen test 87% (6 studies and 377 patients), rapid urease
test 67% (16 studies and 1,417 patients), urea breath test 93%
(8 studies and 520 patients), serology 88% (9 studies and
803 patients), histology 70% (10 studies and 827 patients),
and culture 45% (3 studies and 314 patients). While our
study did not perform formal computation of sensitivity



6 BioMed Research International

Table 1: Baseline characteristics of studied patients (𝑁 = 142).

ITEM Number
(𝑛 = 142) %

Hospitals of recruitment
Mulago 105 74
Mengo 22 15
Case Medical Centre 15 1
Gender
Female 79 56
Male 62 44
Not given 1 7
Duration of illness
Up to 2 weeks 7 5
3-4 weeks 8 6
2 months to 1 year 40 28
2–5 years 61 43
More than 5 years 26 18
Taken drug(s)
Yes 140 99
No 2 1
The time the drug was last taken
Up to one week 114 83
Within a month 16 11
More than a month 10 7
NA 2 1
Drug(s) taken
One antibiotic plus proton pump inhibitors
(PPI) 41 29

Proton pump inhibitors (PPI) only 38 27
Triple therapy 28 20
Single antibiotic 19 13
Two antibiotics 12 9
Never taken peptic ulcer drugs 3 2
Herbal 1 0
Specific antibiotics taken in last 2 weeks
FLQ taken

No 135 95
Yes 7 5

CLA taken
No 138 97
Yes 4 3
∗Other antibiotics taken

Yes 94 66
No 48 34

CLA = clarithromycin, FLQ = fluoroquinolones. ∗Other antibiotics included
amoxicillin, metronidazole, and ceftrioxone.

of the GTHDT, the high positivity rate of 68% among the
H. pylori antigen positive stool samples indicates this test
to be technically promising and warranties well-designed
prospective studies to determine the diagnostic accuracy

of the GTHDT on stool specimens against an established
gold standard. In our study, 32% of the hitherto H. pylori
Ag positive stool samples remained negative at PCR. It is
likely that nonspecific PCR inhibitors were responsible for
this negativity. Optimization of DNA extraction methods or
repeat testing as proposed by Makristathis and Hirschl may
help to solve this false negativity problem but it should be
noted that repeat testing is an expensive approach in routine
patient care [29]. This could also be associated with mixed
populations of the bacteria as reported by Schabereiter-
Gurtner where it led to failure of PCR to detect the resistant
genotype in the biopsy DNA, stool DNA, or both in one case
[27].

4.1. Drug Resistant H. pylori. The 4th edition of the Maas-
tricht consensus recommended a threshold of 15–20% to
separate regions of high and low CLA resistance [30]. In our
study, H. pylori resistance to CLA was high at 29%. Uganda
can thus be considered an area of high CLA resistance. Our
findings are in conformity with several global reports. The
study by Lee et al. reported H. pylori resistance to CLA at
24.7% [12]. Another study by Kumala and Rani in 2006 found
that H. pylori resistance to CLA was 28% [13]. In addition,
CLA resistance in children is reported to be at 34% inAustria,
37% in France, 39% in Portugal, and 49.2% in Spain [31]. Our
findings also agree with the 37% resistance to CLA reported
in a Pakistan study [32], 20% in Southern European studies
[33], 52% in Brazil [34], and 28% in Japan [35]. However,
in USA, CLA resistance was found in only 8.6% [36, 37].
Some researchers have reported that a higher prevalence of
resistance to both CLA and FLQ was detected in samples
from stool compared to biopsy, and they suggested that this
could be related to the existence of mixed infections with
both resistant and susceptible strains [38].Whereas this could
partly explain our findings, it also could imply that when
patients harbouring mixed infections receive antibiotics, the
resistant strains naturally become selected for making the
drug resistant problem even worse.

In Uganda clarithromycin is often used in first-line anti-
PUD regimens, the so-called triple therapy without any
susceptibility data or sometimes even a prescription. It is
most likely that patients who tend to get recurrent disease
might be harbouring super selected drug resistant H. pylori.
Future studies need to focus on this area. In low clar-
ithromycin resistance regions, treatment regimens contain-
ing clarithromycin are recommended as a first-line empirical
treatment. In areas of high clarithromycin resistance such as
in Uganda as from this study, first-line empirical treatment
should utilize quadruple treatment regimens, that is, bismuth
subsalicylate, proton pump inhibitors (PPI), tetracycline, and
metronidazole [39]. After failure of the latter regimen, a
fluoroquinolone-containing triple therapy should ideally be
recommended [39].

The FLQ are usually considered safe oral antibiotics used
as second-line drug forH. pylori infection treatment.The42%
resistance to FLQ in Uganda is not only very high but also
worrying. In Uganda and many other developing countries
with insufficient controls on sale of antibiotics, FLQ are used
as first-line antibiotics for other diseases such as enteric



BioMed Research International 7

Table 2: Results of the Taytec H. pylori stool antigen and GTHDT (𝑁 = 31).

Serial number Sex Age (years) Antibiotics taken within last two weeks Taytec antigen test GTHDT results
FLQ CLA ∗Other antibiotics H. pylori CLA FLQ

(1) M 30 No No Yes P P R S
(2) M 29 No No No P P S S
(3) M 40 No No No P P S R
(4) M 76 No No No P N NA NA
(5) M 35 No No Yes P N NA NA
(6) M 45 No No Yes P P R Invalid
(7) M 25 No No No P P S S
(8) F 58 No No Yes P N NA NA
(9) F 23 No No No P P S S
(10) F 85 No No Yes P P S S
(11) M 20 Yes No Yes P N NA NA
(12) F 80 No No Yes P P S S
(13) F 16 No No Yes P N NA NA
(14) F 44 No No Yes P P R R
(15) F 32 No No No P P S S
(16) F 75 No No No P N NA NA
(17) M 57 No No Yes P P S R
(18) F 55 No No Yes P P S S
(19) M 24 No No No P P R R
(20) F 35 No No Yes P N NA NA
(21) F 42 No No Yes P P S R
(22) F 43 No No No P P R R
(23) M 17 No No No P P S S
(24) M 33 No No No P P S Invalid
(25) M 40 No No Yes P N NA NA
(26) UNK UNK No No No P P R R
(27) F 28 No No No P N NA NA
(28) F 42 No No Yes P N NA NA
(29) M 38 No No No P P S S
(30) F 17 No No No P P S S
(31) M 20 No No Yes P P S R

Total
M = 15 No = 30 No = 31 Yes = 16

P = 31
P = 21 R = 6 R = 8

S = 15 S = 11

F = 15 Yes = 1 Yes = 0 No = 15 N = 10 NA = 10 Invalid = 2
NA = 10

CLA = clarithromycin, F = female, FLQ = fluoroquinolone, M =male, N = negative, NA = not tested, P = positive, S = sensitive, and UNK = unknown. ∗Other
antibiotics included amoxicillin, metronidazole, and ceftrioxone.

upsets, cough, and urinary tract infections (UTIs).This could
have led to super selection of the resistant strains and thus the
relatively high resistance of H. pylori (42%) reported in the
current study. Our findings are in agreement with previous
studies where resistance to FLQ was found in 48%, 56%, and
62%, in Japan, China, and Pakistan, respectively [32, 40–42].
Furthermore, it is in agreement with the Lee et al. work
carried out from 2003 to 2012 which reported an increased
resistance rate of H. pylori to FLQ (levofloxacin and moxi-
floxacin) from 5% to 28% [12]. However, Hung et al. in their
2009 study in Taiwan found resistance in only 11% [43]. Addi-
tionally, Kumala and Rani study of 2006 also reported lower
resistance of H. pylori to FLQ (ciprofloxacin, norfloxacin,

and ofloxacin at 6.9%, sparfloxacin and gatifloxacin at
2.8%, and levofloxacin and moxifloxacin at 1.4%) [13]. In
the developed countries, FLQ are reserved as second-line
antibiotics for H. pylori treatment. In Africa, FLQ are first-
line antibiotics for many other ailments increasing antibiotic
resistance selection pressure, and it remains worrying what
options will be there for the 42% patients with FLQ-resistant
H. pylori.

5. Conclusions

This preliminary study has shown that stool is a possible
specimen for use with the GTHDT in direct detection of H.



8 BioMed Research International

pylori and its resistance to CLA/FLQ.We have further shown
that H. pylori resistance to CLA and FLQ in Uganda is high.
We recommend a prospectively designed study to determine
the sensitivity and specificity of the GTHDT for diagnosis
of H. pylori in stool samples against an established standard
reference test and also determine the prevalence of H. pylori
resistance to CLA and FLQ on larger sample sizes to guide
patient care.

6. Limitations

The main limitation of our study is that we did not have
resources for endoscopic biopsy and confirmatory diagnosis
of H. pylori as we could not carry out the urease breath
test, and we studied a biased population of stool antigen
positive samples. However, we strongly believe that the
evidence presented in this paper will ignite more studies to
be conducted on this topic.

Conflicts of Interest

All authors declare that they have no conflicts of interest.

Authors’ Contributions

The authors made the following contributions: Freddie
Bwanga conceived the idea, contributed to the development
of the study protocol, interpreted results, analysed data,
interpreted the data, and prepared the manuscript. Denish
Calmax Angol contributed to the development of the study
protocol, collected samples, ran the laboratory experiments,
participated in data analysis, interpreted results, and prepared
the manuscript. Tess Ayazika Kirabo contributed to the
development of the study protocols, collection of samples,
and laboratory experiments. Alfred Okeng helped in fixing
the experimental issues and ran some of the PCR tests.
Irene Najjingo ran part of the PCR test. Ponsiano Ocama
contributed to the development of the study protocol, was the
lead clinician, and critically reviewed the manuscript.

Acknowledgments

The authors acknowledge the management and clinicians
of Mulago National Referral, Mengo, and Case Medical
Centre Hospitals for allowing them to collect samples from
their patients. The authors recognize the contribution of
the research assistants: Richard Okillan, Emmanuel Oumo,
John Enzama, and Christopher Muchwa for recruiting the
study participants and collection of samples. Deep and
sincere gratitude goes to the Management of MBN Clinical
Laboratories for partial sponsorship of this research project
and for the excellent laboratory services.

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