Helicobacter pylori Primary Antibiotic Resistance in 2015 in Morocco: A Phenotypic and Genotypic Prospective and Multicenter Study

Abstract

Knowledge of local antibiotic resistance is crucial to adaption of the choice of effective empirical first-line treatment for Helicobacter pylori infection. The aim of this study was to evaluate, for the first time in Morocco, the prevalence of the primary resistance of H. pylori to clarithromycin, metronidazole, amoxicillin, levofloxacin, tetracycline, and rifamycin. We conducted a 1-year prospective study (2015), including 255 Moroccan patients referred for gastro-duodenal endoscopy to two hospitals of Rabat (Morocco) and never previously treated for H. pylori infection. Three gastric biopsies were collected: one for histology, one for culture, and one for molecular detection of H. pylori and the mutations in 23S rRNA genes that confer resistance to clarithromycin. Antimicrobial susceptibility testing was performed on isolated strains by Etest and disk diffusion methods. One hundred seventy-seven patients were infected (69.4%). The prevalence of primary resistances of H. pylori to clarithromycin was 29%, 40% to metronidazole, 0% to amoxicillin, tetracycline, and rifamycin, and 11% to levofloxacin. Only four isolates (2%) were resistant to both clarithromycin and metronidazole. The high level of primary clarithromycin resistance in the H. pylori strains infecting the Moroccan population leads us to recommend the abandonment of the standard clarithromycin-based triple therapy as a first-line treatment in Morocco and to prefer a concomitant quadruple therapy.

Introduction

H elicobacter pylori infection is one of the most common chronic bacterial infections in the world.¹ Eradication therapy is recommended for peptic ulcer disease, mucosa-associated lymphoid tissue lymphoma, atrophic gastritis, first-degree relatives of gastric cancer patients, unexplained iron deficiency anemia, and chronic idiopathic thrombocytopenic purpura.²

It was 20 years ago that clarithromycin-based triple therapy was established in clinical practice for the eradication of the bacteria. The efficacy of triple therapy for H. pylori infection has dramatically declined over the past decade. In Morocco as in many parts of the world, the efficacy of triple therapy is seriously challenged, and eradication rates have declined to unacceptably low levels reaching 65.9%.^3,4 Clarithromycin resistance has been increasing in many countries, and it constitutes the main risk factor for treatment failure: it reduces the efficacy of first-line therapy by up to 70%.^5–8

We previously conducted a prospective study from 2010 to 2015 on 112 Moroccan patients and determined by PCR that primary resistance to clarithromycin was 28.2%, largely above the 15–20% threshold put forward by the Maastricht IV consensus report, which leads to recommending abandonment of standard clarithromycin-based triple therapy as a first-line treatment.^2–9 Since bismuth is not available in Morocco, determination of primary resistance to the other antimicrobial agents that are effective against H. pylori, particularly to metronidazole, is crucial to adaption of the choice of effective empirical first-line treatment for H. pylori infection.

Resistance of H. pylori can be detected either by phenotypic susceptibility testing or by molecular techniques based on PCR for clarithromycin and levofloxacin.^10–14 Phenotypic susceptibility testing requires isolation of the bacterium, which is cultured from gastric biopsy. Culture is known to be difficult to achieve, but it is the only method that can determine antimicrobial susceptibility to all antibiotics used in H. pylori eradication. The molecular techniques detecting mutations that confer resistance to clarithromycin are easier to perform, faster, and more efficient than antimicrobial susceptibility testing.^13–14 In 2008, we developed a quadruplex real-time PCR assay using four scorpion primers that were specific to each of the mutations that confer resistance to clarithromycin, and performances were excellent (Se 98.3, Sp 98.0).¹³ The Scorpion PCR has been used in several molecular studies in France, Tunisia, Senegal, Algeria, and Morocco.^5,9,15–17

The aim of this study was to evaluate, for the first time in Morocco, the prevalence of primary resistance of H. pylori to clarithromycin, metronidazole, amoxicillin, levofloxacin, tetracycline, and rifamycin and to determine the mutations in the 23S rRNA gene that confer resistance to clarithromycin.

Patients and Methods

This prospective multicenter study was conducted from January 2015 to January 2016 in two hospitals of Rabat, Morocco (University Hospital Cheikh-Zaid and Military Teaching Hospital Mohammed V). Inclusion criteria were adult patients referred for gastro-duodenal endoscopy and never treated for H. pylori infection. Exclusion criteria were a previous attempt at H. pylori eradication, and administration of proton pump inhibitors (PPI), non-steroidal anti-inflammatory drugs, or antibiotics within the previous 4 weeks. Consecutive patients were given notice, and those who gave informed consent were included. Three gastric biopsies were collected in the antrum during upper gastrointestinal endoscopy: one for histology, one for culture, and one for molecular detection of H. pylori and the mutations in 23S rRNA genes that confer resistance to clarithromycin.

Histology

One antral biopsy was fixed in buffered formalin (10%). The presence or absence of H. pylori was determined on histological slides stained with Giemsa.

Culture

Pre-analytical conditions were optimized to shorten the delay from biopsy collection to plating and incubation to <2 h. Biopsies were transported to the laboratory in 0.5 ml of sterile saline solution. The biopsy specimens were ground with a sterile scalpel in a Petri dish. Half of the grinding material was spread on a Columbia agar plate supplemented with 10% (vol/vol) horse blood and Skirrow antibiotic supplement (Oxoid, Basingstoke Hampshire, United Kingdom). The inoculated plates were placed at 37°C for 5–10 days under microaerobic conditions by using campyGen generator (Oxoid) in an anaerobic jar. Identification of H. pylori was based on colony morphology; typical appearance on Gram staining; and positive urease, catalase, and oxidase activities.

Determination of antibiotic susceptibilities by phenotypic methods

Two phenotypic methods were used to determine the antimicrobial susceptibility to the six antibiotics tested. Clarithromycin, amoxicillin, and metronidazole susceptibility was tested by determination of the minimum inhibitory concentration (MIC) using Etest (bioMérieux, Marcy-l'Etoile, France). Levofloxacin, tetracycline, and rifamycin susceptibility was tested by using the disk diffusion method because of availability in Morocco, for economic reasons, and because of the usual low MIC easily detected in this way.⁸

A bacterial suspension was prepared to a McFarland 3 turbidity standard (∼10⁹ colony-forming unit/ml of H. pylori), spread with a sterile swab on a Mueller-Hinton agar plate supplemented with 10% horse blood. Etests and disks were placed on agar plates. Plates were placed at 37°C for 48 h under microaerobic conditions. EUCAST criteria were used to determine resistance to clarithromycin, amoxicillin, and metronidazole (www.eucast.org). For the other antibiotics, the following cut-off diameters (mm) were used: levofloxacin: resistant <17, susceptible ≥20; tetracycline: resistant <17, susceptible ≥19; and rifamycin: resistant <14, susceptible ≥19. The H. pylori strain CCUG 17874 was used for quality control.

Molecular analysis

DNA was isolated from gastric biopsies by using QIAamp DNA mini-kit (Qiagen, Courtaboeuf, France) according to the manufacturer's instructions. The isolated DNA was eluted in 100 μl of 1× TE buffer (10 mmol/L Tris-HCl, 1 mmol/L ethylenediaminetetraaceticacid (pH 8.0)) and stored at −20°C until use.

Detection of H. pylori infection and determination of point mutation in the 23S rRNA gene by Scorpion PCR was performed according to Burucoa et al. in a single-vessel multiplex real-time PCR assay that detects H. pylori infection and identifies four alleles of the 23S rRNA gene of H. pylori: the wild-type sequence and three mutations conferring clarithromycin resistance (A2142G, A2143G, and A2142C), using allele-specific Scorpion primers directly on biopsy specimen DNA extracts.¹³ Positive (extracted DNA of four strains sequenced into 23S rRNA genes) and negative (molecular quality distilled water) controls were used in each run of the Scorpion PCR assay. To control the absence of PCR inhibitors, all negative Scorpion PCR extracts were systematically tested for the detection of a 110-bp fragment of a human housekeeping gene encoding for β-hemoglobin.¹³

Statistics

Categorical data were analyzed by the χ² and Fisher's exact test. Continuous variables were analyzed by the Student t test. p-values lower than 0.05 were considered statistically significant.

To determine the clarithromycin resistance rate, we considered the results of the Scorpion PCR because of its well-known better sensitivity.¹³

Results

A total of 255 Moroccan adult patients referred for gastro-duodenal endoscopy and never previously treated for H. pylori infection were recruited in the two hospitals of Rabat (Morocco): 238 in the Military Hospital Mohamed V, and 17 in the Cheikh Zaid Hospital. They were 103 (40%) men and 152 (60%) women aged from 19 to 80 years, with a mean age of 47.5 years. Sex ratio and mean age were not statistically different in the two inclusion centers. Endoscopic examination detected 19 (7.5%) patients with a duodenal ulcer and 13 patients (5.1%) with a gastric ulcer. Histology detected H. pylori in 176 biopsies (69%). Culture detected H. pylori in 177 biopsies (69.4%). The Scorpion PCR detected H. pylori DNA in the same 177 biopsies (69.4%). All negative Scorpion PCR extracts were positive for β-hemoglobin gene PCR detection, thereby confirming the absence of PCR inhibitors. The infection rate in the studied population was 69.4%. Patients with ulcer disease were all, except four, infected by H. pylori (87.5%). Infection rate was not significantly different according to gender or age.

The results of antimicrobial susceptibility testing as determined by phenotypic and genotypic methods are presented in Table 1, indicating a high level of primary clarithromycin resistance (28.8%) and metronidazole resistance (40.1%) and a relatively low level of primary levofloxacin resistance (10.7%). We did not detect resistance to amoxicillin, tetracycline, and rifamycin. Only 4 among the 177 infected patients harbored a strain that was resistant to both clarithromycin and metronidazole (2%). Resistance rates were not significantly different according to gender or age. MIC50 and MIC90 were 0.03 and 64; 1.5 and 256 for clarithromycin and metronidazole, respectively (Table 1).

Table 1.

Primary Resistance Rate of Helicobacter pylori to Antibiotics in Morocco in 2014 (n = 177)

Antibiotic	No. resistant	% Resistant	MIC50 (mg/L)	MIC90 (mg/L)
Clarithromycin Etest	45	25.4	0.03	64
Clarithromycin PCR	51	28.8
Metronidazole	71	40.1	1.5	256
Levofloxacin	19	10.7
Amoxicillin	0	0
Tetracycline	0	0
Rifamycin	0	0

MIC, minimum inhibitory concentration; MIC50, concentration of antibiotic inhibiting 50% of the studied isolates; MIC90, concentration of antibiotic inhibiting 90% of the studied isolates.

A mutation conferring resistance to clarithromycin was detected in 51 of the 177 positive samples: six more than the phenotypic method by Etest. These six isolates were detected by Etest as susceptible to clarithromycin (MICs from <0.016 to 0.064), and the Scorpion PCR detected a mix of a wild-type allele (susceptible) and a mutated allele (resistant: three A2142G, two A2142C, and one A2143G). Among the 51 biopsies detected positive for clarithromycin resistance by Scorpion PCR, 29 (56.8%) harbored a A2142G mutation, 20 (39.2%) a A2143G mutation, and 4 (8%) a A2142C mutation (Table 2). One harbored two different mutations (A2142G and A2142C), and one harbored three different alleles of the 23SrRNA genes (wild-type, A2143G, and A2142C). In 27 (53%) biopsies, Scorpion PCR revealed a mixed infection with the concomitant detection of a wild-type (clarithromycin-susceptible) allele and a mutation (clarithromycin-resistant) allele.

Table 2.

Distribution of the Point Mutations Among Clarithromycin-Resistant H. pylori Isolates from Morocco 2014

Resistance	Mutations	No.	%
Susceptible	Wild-type	126	72.2
Resistant		51	28.8
	A2142G	17
	A2143G	6
	A2142C	0
	A2142G + wild-type	11
	A2143G + wild-type	13
	A2142C + wild-type	2
	A2142G + A2142C	1
	A2143G + A2142C + wild-type	1

Discussion

Knowledge of local antibiotic resistance rate is crucial to adaptation of an appropriate local choice for an effective empirical first-line treatment leading to eradication of H. pylori infection.² The standard triple therapy containing clarithromycin proposed at the first Maastricht conference in 1997 to treat H. pylori infection has become universal. The loss of effectiveness found in many countries since the beginning of the 21th century is due to the increasing prevalence of H. pylori clarithromycin resistance.⁷ In the most recent Maastricht IV consensus conference, it was recommended that “PPI–clarithromycin-containing triple therapy without prior susceptibility testing should be abandoned when the clarithromycin resistance rate in the region is more than 15–20%.”²

In Morocco, as in many countries, clarithromycin-based empirical triple therapy is widely used as first-line eradication treatment of H. pylori infection. The primary clarithromycin resistance rate remained unknown in Morocco until a previous work in which we conducted a prospective multicenter study (2011–2014) and reported a primary resistance rate reaching 28.2%, largely over the 15–20% threshold put forward by the Maastricht IV consensus report as a reason to abandon the clarithromycin-based first-line treatment. Two possibilities could then be proposed to Moroccan clinicians for treatment of their H. pylori infected patients: either a new regimen involving a combination of more antibiotics for the first-line empirical treatment or a new therapeutic strategy guided by antimicrobial susceptibility testing. In areas of high clarithromycin resistance, bismuth-containing quadruple treatments are recommended for first-line empirical treatment.² In Morocco, however, bismuth is not available. As a result, concomitant or sequential treatments are recommended in spite of obvious ecological consequences on gastroenterological micro-flora and adverse effects. A recent analysis indicated that 14-day concomitant therapy should be preferred, because it is the most effective in overcoming dual resistance (clarithromycin and metronidazole resistance).¹⁸ Although we have observed a high level of metronidazole resistance (40%), dual resistance (clarithromycin and metronidazole resistance) is surprisingly rare in our study (four cases, 2%). With high prevalence of resistance to clarithromycin (29%), and to metronidazole (40%), we would expect 11.6% of dual resistance. We did not find any significant demographic difference between patients infected with a clarithromycin-resistant strain and patients infected with a metronidazole-resistant strain to explain this surprising result. In this situation, the alternative is to use a treatment guided by the results of resistance detection. Several studies have shown the medical and economic benefits of a phenotypic resistance-guided therapeutic strategy.^19–21 Phenotypic susceptibility testing of H. pylori is considered as complex and, therefore, seems difficult to apply on a large scale in a developing country. We demonstrate here that obstacles to the installation of the culture and antimicrobial susceptibility testing for H. pylori are not insurmountable in a North African country and that in 1 year we have achieved a very satisfactory technological transfer, since our molecular results are very similar to phenotypic results. We obtained excellent results for culture with 100% concordance with PCR. Usually, PCR is slightly superior (3–5%) to culture in sensitivity to detect H. pylori.^8,13 The total concordance between culture and PCR observed in this study could be explained by the optimized pre-analytical conditions for culture (<2 h between biopsy collection and incubation of grinding material plated on culture media).

The 28.8% clarithromycin resistance rate reported in this study confirms the 28.2% clarithromycin resistance rate reported in our previous molecular study performed from 2011 to 2014. This clarithromycin resistance level is high compared with the levels observed around the world. It has been shown that the level of H. pylori primary clarithromycin resistance in a country is positively correlated with macrolide use in the outpatient community of the country.⁶ In Africa, very few available studies have reported low clarithromycin resistance: 0% in Gambia, Nigeria, and Ethiopia, 1% in Senegal, 1.7% in Congo, 4% in Egypt, 6.4% in Kenya, and 20% in South Africa.^16,22–28 In Maghreb, such data are scarce, and the only two published studies conducted from 2005 to 2007 in Tunisia and more recently from 2008 to 2014 in Algeria reported a prevalence of primary resistance to clarithromycin reaching 33%.^15,17 Across the Mediterranean Sea, Spain and France, which have maintained a special relationship with Morocco, exhibit lower primary resistance rates: 19–22.2% in France and 13.3–14.7% in Spain.^5,6,8,29 But similar clarithromycin resistance rates were also observed in other Southern European countries (Greece 24.7%, Italy 26.7%, and Portugal 31.5%).⁶

Two recent clinical trials conducted in Morocco suggested a high rate of clarithromycin resistance. The first, conducted in 2009, showed eradication rates lower than 80% for clarithromycin-based triple therapy (intention to treat [ITT] 78.2, per protocol [PP] 79.6).³ The second, conducted in 2012, showed a decline in the eradication rates achieved by this regimen (ITT 65.9, PP 71), thereby suggesting an increase of the resistance rate to clarithromycin.⁴

In this study, six isolates were detected as susceptible to clarithromycin by Etest and the Scorpion PCR detected a mix of a wild-type allele (susceptible) and a mutated allele (resistant) characteristic of the concomitant presence in the biopsy of two strains with different susceptibilities to clarithromycin. In each of these six cases, the Cycle Threshold (number of PCR cycles to reach the positive cut-off) was higher for the resistant allele than for the susceptible allele, thereby indicating a subpopulation of resistant isolate in lower quantity than the susceptible allele. We have already demonstrated the good sensitivity of this molecular method to detect such a mix, enabling detection of a resistant subpopulation up until 1% of an inoculum containing 99% of the susceptible strain.¹³ We report a distribution of mutations with a predominance of the A2142G mutation similar to our previous Moroccan results,⁹ whereas A2143G predominates in France, Algeria, and Tunisia, thereby confirming the local emergence of this resistance under selection pressure by macrolide consumption rather than the epidemic spread of resistance genes.^5,15,17

Among the 51 patients with a resistant strain, 27 (53%) were infected with at least two different alleles of the 23S rRNA gene in the same biopsy, and one of these 27 patients harbored a mix of three alleles: one susceptible wild-type and two different mutations, thereby suggesting either the presence of different strains in the same biopsy specimen (multiple infection) or the presence of different clones of the same strain harboring different mutations (mixed infection). Multiple infections are more frequent in countries with a high prevalence of infection and resistance such as Tunisia and Morocco.³⁰

In conclusion, we report a high level of primary clarithromycin resistance in the Moroccan population (28.8%), which leads us to recommend either abandonment of standard clarithromycin-based triple therapy as a first-line treatment in favor of a quadruple concomitant treatment or an attempt to develop a guided strategy based on phenotypic or genotypic susceptibility testing.

Footnotes

Acknowledgment

The authors gratefully acknowledge Jeffrey Arsham, an American translator, for his rereading and revision of the original English-language article.

This study was supported in part by the CHU of Poitiers.

Disclosure Statement

No competing financial interests exist.

References

Suerbaum

, and Michetti

. 2002. Helicobacter pylori infection. N. Engl. J. Med., 347:1175–1186.

Malfertheiner

, Mégraud

, C.O'Morain , Atherton

, Axon

A.T.

, Bazzoli

, Gensini

G.F.

, Gisbert

J.P.

, Graham

D.Y.

, Rokkas

, El-Omar

E.M.

, and Kuipers

E.J.

. 2012. Management of Helicobacter pylori infection—The Maastricht IV/Florence Consensus Report. Gut, 61:646–664.

Lahbabi

, Alaoui

, El Rhazi

, El Abkari

, Nejjari

, Amarii

, Bennani

, Mahmoud

, Ibrahimi

, and Benajah

D.A.

. 2013. Sequential therapy versus standard triple-drug therapy for Helicobacter pylori eradication: result of the HPFEZ randomized study. Clin. Res. Hepatol. Gastroenterol., 37:416–421.

Seddik

, Ahid

, El Adioui

, El Hamdi

F.Z.

, Hassar

, Abouqal

, Cherrah

, and Benkirane

. 2013. Sequential therapy versus standard triple-drug therapy for Helicobacter pylori eradication: a prospective randomized study. Eur. J. Clin. Pharmacol., 69:1709–1715.

Raymond

, Lamarque

, Kalach

, Chaussade

, and Burucoa

. 2010. High level of antimicrobial resistance in French Helicobacter pylori isolates. Helicobacter, 15:21–27.

Mégraud

, Coenen

, Versporten

, Kist

, Lopez-Brea

, Hirschl

A.M.

, Andersen

L.P.

, Goossens

, and Glupczynski

. 2013. Helicobacter pylori resistance to antibiotics in Europe and its relationship to antibiotic consumption. Gut, 62:34–42.

Mégraud

2004. H pylori antibiotic resistance: prevalence, importance, and advances in testing. Gut, 53:1374–1384.

Ducournau

, Bénéjat

, Sifré

, Bessède

, Lehours

, and Mégraud

. 2016. Helicobacter pylori resistance to antibiotics in 2014 in France detected by phenotypic and genotypic methods. Clin. Microbiol. Infect. [Epub ahead of print]; DOI: 10.1016/j.cmi.2016.06.003

Bouilhat

, Burucoa

, Benkirane

, El Idrissi-lamghari

, Al Bouzidi

, El feydi

, Elouenas

, and Benouda

. 2015. High-level primary clarithromycin resistance of Helicobacter pylori in Morocco: a prospective multicenter molecular study. Helicobacter, 20:422–423.

10.

Mégraud

, and Lehours

. 2007. Helicobacter pylori detection and antimicrobial susceptibility testing. Clin. Microbiol. Rev., 20:280–322.

11.

Burucoa

, Landron

, Garnier

, and Fauchere

J.L.

. 2005. T2182C mutation is not associated with clarithromycin resistance in Helicobacter pylori. Antimicrob. Agents Chemother., 49:868.

12.

Burucoa

, and Mégraud

. 2011. Helicobacter pylori. In Cornaglia

, Courcol

, and Herrmann

J.L.

(ed.), European manual of clinical microbiology, first edition. European Society of Clinical Microbiology and Infectious Diseases, Basel, Switzerland, pp. 283–286.

13.

Burucoa

, Garnier

, Silvain

, and Fauchere

J.L.

. 2008. Quadruplex real-time PCR assay using allele-specific scorpion primers for detection of mutations conferring clarithromycin resistance to Helicobacter pylori. J. Clin. Microbiol., 46:2320–2326.

14.

Cambau

, Allerheiligen

, Coulon

, Corbel

, Lascols

, Deforges

, Soussy

C.J.

, Delchier

J.C.

, and Megraud

. 2009. Evaluation of a new test, genotype HelicoDR, for molecular detection of antibiotic resistance in Helicobacter pylori. J. Clin. Microbiol., 47:3600–3607.

15.

Ben Mansour

, Burucoa

, Zribi

, Masmoudi

, Karoui

, Kallel

, Chouaib

, Matri

, Fekih

, Zarrouk

, Labbene

, Boubaker

, Cheikh

, Hriz

M.B.

, Siala

, Ayadi

, Filali

, Mami

N.B.

, Najjar

, Maherzi

, Sfar

M.T.

, and Fendri

. 2010. Primary resistance to clarithromycin, metronidazole and amoxicillin of Helicobacter pylori isolated from Tunisian patients with peptic ulcers and gastritis: a prospective multicentre study. Ann. Clin. Microbiol. Antimicrob., 9:22.

16.

Seck

, Burucoa

, Dia

, Mbengue

, Onambele

, Raymond

, and Breurec

. 2013. Primary antibiotic resistance and associated mechanisms in Helicobacter pylori isolates from Senegalese patients. Ann. Clin. Microbiol. Antimicrob., 12:3.

17.

Djennane-Hadibi

, Bachtarzi

, Layaida

, Ali Arous

, Nakmouche

, Saadi

, Tazir

, Ramdani-Bouguessa

, and Burucoa

. 2016. High-level primary clarithromycin resistance of Helicobacter pylori in Algiers, Algeria: a prospective multicenter molecular study. Microb. Drug Resist., 22:223–226.

18.

Molina-Infante

, and Gisbert

. 2014. Optimizing clarithromycine-containing therapy for Helicobacter pylori in the era of antibiotic resistance. World J. Gastroenterol., 20:10338–10347.

19.

Lee

H.J.

, Kim

J.I.

, Cheung

D.Y.

, Kim

T.H.

, Jun

E.J.

, Oh

J.H.

, Chung

W.C.

, Kim

B.W.

, Kim

S.S.

, Park

S.H.

, and Kim

J.K.

. 2013. Eradication of Helicobacter pylori according to 23S ribosomal RNA point mutations associated with clarithromycin resistance. J. Infect. Dis., 208:1123–1130.

20.

Wenzhen

, Yumin

, Quanlin

, Kehu

, Donghai

, and Lijuan

. 2012. Is antimicrobial susceptibility testing necessary before first-line treatment for Helicobacter pylori infection? Meta-analysis of randomized controlled trials. Intern. Med., 49:1103–1109.

21.

Romano

, Marmo

, Cuomo

, De Simone

, Mucherino

, Iovene

M.R.

, Montella

, Tufano

M.A.

, Del Vecchio Blanco

, and Nardone

. 2003. Pretreatment antimicrobial-susceptibility testing is cost saving in the eradication of H. pylori. Clin. Gastroenterol. Hepatol., 4:273–278.

22.

Secka

, Berg

D.E.

, Antonio

, Corrah

, Tapgun

, Walton

, Thomas

, Galano

J.J.

, Sancho

, Adegbola

R.A.

, and Thomas

J.E.

. 2013. Antimicrobial susceptibility and resistance patterns among Helicobacter pylori strains from The Gambia, West Africa. Antimicrob. Agents Chemother., 57:1231–1237.

23.

Oyedeji

K.S.

, Smith

S.I.

, Coker

A.O.

, and Arigbabu

A.O.

. 2009. Antibiotic susceptibility patterns in Helicobacter pylori strains from patients with upper gastrointestinal pathology in western Nigeria. Br. J. Biomed. Sci., 66:10–13.

24.

Asrat

, Kassa

, Mengistu

, Nilsson

, and Wadstrom

. 2004. Antimicrobial susceptibility pattern of Helicobacter pylori strains isolated from adult dyspeptic patients in Tikur Anbassa University Hospital, Addis Ababa, Ethiopia. Ethiop. Med. J., 42:79–85.

25.

Sherif

, Mohran

, Fathy

, Rockabrand

D.M.

, Rozmajzi

P.J.

, and Frenck

R.W.

. 2004. Universal high-level primary metronidazole resistance in Helicobacter pylori isolated from children in Egypt. J. Clin. Microbiol., 42:4832–4834.

26.

Lwai-Lume

, Ogutu

E.O.

, Amayo

E.O.

, and Kariuki

. 2005. Drug susceptibility pattern of Helicobacter pylori in patients with dyspepsia at the Kenyatta National Hospital, Nairobi. East Afr. Med. J., 82:603–608.

27.

Tanih

N.F.

, and Ndip

R.N.

. 2013. Molecular detection of antibiotic resistance in South African isolates of Helicobacter pylori. Gastroenterol. Res. Pract., 2013:259457.

28.

Ontsira Ngoyi

E.N.

, Atipo Ibara

B.I.

, Moyen

, Ahoui Apendi

P.C.

, Ibara

J.R.

, Obengui

, Ossibi Ibara

R.B.

, Nguimbi

, Niama

R.F.

, Ouamba

J.M.

, Yala

, Abena

A.A.

, Vadivelu

, Goh

K.L.

, Menard

, Benejat

, Sifre

, Lehours

, and Megraud

. 2015. Molecular detection of Helicobacter pylori and its antimicrobial resistance in Brazzaville, Congo. Helicobacter, 20:316–320.

29.

Cuadrado-Lavin

, Salcines-Caviedes

J.R.

, Carrascosa

M.F.

, Mellado

, Monteagudo

, Llorca

, Cobo

, Campos

M.R.

, Ayestaran

, Fernandez-Pousa

, and Gonzalez-Colominas

. 2012. Antimicrobial susceptibility of Helicobacter pylori to six antibiotics currently used in Spain. J. Antimicrob. Chemother., 67:170–173.

30.

Ben Mansour

, Fendri

, Battikh

, Garnier

, Zribi

, Jlizi

, and Burucoa

. 2016. Multiple and mixed Helicobacter pylori infections: comparison of two epidemiological situations in Tunisia and France. Infect. Genet. Evol., 37:43–48.