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East African Medical Journal Vol. 82 No. 12 December 2005DRUG SUSCEPTIBILITY PATTERN OF HELICOBACTER PYLORI IN PATIENTS WITH DYSPEPSIA AT THE KENYATTA NATIONAL HOSPITAL,NAIROBIL. Lwai-Lume, MBChB, (MUK), MMed (UON), Senior House Officer, E. O. Ogutu, MBChB, MMed (UON), Consultant Physician/Gastroenterologist,E. O. Amayo, MBChB, MMed (UON), Consultant Physician/Neurologist, Senior Lecturer (at time of study), Department of Medicine, College of Health Sciences,University of Nairobi, P.O. Box 19676, Nairobi, Kenya and S. Kariuki, BVMed. (UON) MSc (Nor.) PhD (UK), Consultant Microbiologist, Senior Researcher,Centre for Microbiology Research, Kenya Medical Research Institute, P. O. Box 43610, Nairobi, Kenya Request for reprints to: Dr. L. Lwai-Lume, Department of Medicine, College of Health Sciences, University of Nairobi, P.O. Box 19676, Nairobi, Kenya DRUG SUSCEPTIBILITY PATTERN OF HELICOBACTER PYLORI IN PATIENTS WITH
DYSPEPSIA AT THE KENYATTA NATIONAL HOSPITAL, NAIROBI
L. LWAI-LUME, E. O. OGUTU, E. O. AMAYO and S. KARIUKI ABSTRACT
Objective: To determine drug susceptibility pattern of Helicobacter pylori to metronidazole,
clarithromycin, amoxicillin and tetracycline in patients presenting with dyspepsia at the
Kenyatta National Hospital.
Design: Cross-sectional descriptive study.
Setting: Kenyatta National Hospital, Nairobi.
Subjects: Two hundred and sixty-seven patients aged 15 to 85 years, presenting with
dyspepsia and referred for upper gastro-intestinal endoscopy were recruited into the
study.
Results: Between October 2003 and April 2004, 138 male and 129 female patients aged
15-85 years, with a mean age of 45.4 years were studied. Gastritis was the most common
endoscopic finding, occurring in 55%, followed by normal-looking mucosa in 27% and
peptic ulcer disease in 16% of the patients. The rapid urease test was positive in 184
patients (69%). The culture yield was 62% of these CLO (Campylobacter like organisms)
positive biopsies. The MIC (minimum inhibitory concentration) was 256 mg/l for
metronidazole, 1.5mg/l for clarithromycin, 1.5mg/l for tetracycline and 0.75 mg/l for
amoxicillin. The MIC values for amoxicillin were significantly higher in the female
patients (p = 0.02) but showed no significant variation for age. The MIC values for
metronidazole, tetracycline and clarithromycin showed no significant difference for age
or gender. MIC values for tetracycline were significantly higher for patients with
duodenitis and duodenal ulcer p = 0.009 and 0.02, respectively.
Conclusion: All isolated H. pylori organisms were resistant to metronidazole. The
susceptibility of the H. pylori isolates was 93.6% for clarithromycin, 95.4% for
amoxicillin and 98.1% for tetracycline. The MIC90 for amoxicillin and clarithromycin
were found to be close to the upper limit of the susceptibility range. There was a
rising MIC90 for tetracycline and metronidazole compared to that found in a previous
study in 1991.
gastric outlet obstruction. A local study by Lule et alin 1989 reported a 90% prevalence of H. pylori in Helicobacter pylori (H. pylori) is a gram-negative, peptic ulcer disease (3), while Ogutu et al in 1994 spiral, flagellate bacilli affecting more than one billion isolated H. pylori in 100% of patients presenting to the people worldwide. Warren and Marshall, two Australian Kenyatta National Hospital with peptic ulcer disease investigators, first described the organisms in 1983, in (4). H. pylori is classified as a group 1 (or definite) gastric mucosal biopsies of patients with chronic active carcinogen by the World Health Organisation's gastritis (1). The gastric antrum is the most favoured International Agency for Research on Cancer. Infected site, but any part of the stomach may be colonised.
persons have an increased risk of developing gastric About 80% of those who are infected are asymptomatic.
cancer and mucosa-associated lymphoid type (MALT) However H. pylori can cause acute gastritis, chronic active, chronic persistent and chronic atrophic gastritis.
Treatment of the diseases associated with H. pylori The organism is also strongly associated with peptic involves eradication of the organism as the cornerstone ulcer disease (2), and ensuing complications of upper for disease clearance. The recommendations for gastrointestinal bleeding, iron deficiency anaemia and (i) Triple therapy regimens with a combination Ethical considerations: The study was carried out with the of any proton pump inhibitor (PPI) with two antibiotics approval of the Kenyatta National Hospital Scientific and Ethical or ranitidine bismuth citrate in combination with two Review Committee. The patient or parent/ guardian (if the patient antibiotics. The former option has been adopted in was under 18 years old) gave informed, written consent.
most countries due to better efficacy and tolerability.
Clinical procedures and laboratory methods: All patients The antibiotics used include tetracycline, amoxicillin, completed a questionnaire on demographic features and were clarithromycin, metronidazole (or other nitroimidazole), and more recently rifabutin (a semi synthetic The patients underwent upper gastrointestinal endoscopy ansamycin). The drug regimens are recommended for using an Olympus gastro intestinal fibreoptic scope Q20, Q40 or XQ20 (Olympus Keymed, Southend-on-Sea, Essex, U.K.).
(ii) Quadruple therapy, which combines a PPI Six biopsies were taken, two from each of the following sites: with a bismuth-based regimen including any two of gastric body, the incisura and the antrum.
the recommended antibiotics. The duration of One biopsy specimen from each site was used for the CLO test (a rapid urease test), which is a marker of the treatment is again 7-14 days, with eradication rates presence of viable H. pylori organisms and detects the of over 95% being achieved, although the amoxicillin- conversion of urea to ammonia by bacterial urease. The three based regimens have not been as effective (6, 7).
biopsies for culture (one from each site) were immersed in This is mainly used as second line treatment in tryptone soy broth with 15% glycerol, for transportation to patients with failed response to triple therapy. Audit the laboratory at the Kenya Medical Research Institute standards for eradication of H. pylori have been set (KEMRI). To increase the concentration of the organisms, variously at 80% for intention-to-treat results only 1ml of broth was used. The CLO test was read up to (Maastricht consensus meeting, 1996), and 90% for six hours post biopsy, which enabled us to include some of the low yielders of H. pylori. The CLO positive specimenswere cultured within six hours of harvesting (11).
(iii)There are other antibiotics being recommended Brain heart infusion agar (Oxoid, Basingstoke, U.K.), for resistance to the above two regimes. These include supplemented with 7% defibrinated horse blood was used for levofloxacin, furazolidone, and an amoxicillin- culture. The modified Thayer- Martin's recommended antibiotics for Campylobacter (vancomycin 3mg/l, trimethoprim 5mg/l, The prevalence of H. pylori drug resistance varies colistin 7.5 mg/l and nystatin 12.50 IU/I) were added to enhance in different countries. Metronidazole resistance varies selectivity (11). The culture plates were pre-warmed to 37° C from 10-30% in the United States and Western Europe and 1ml of broth was then poured onto the agar. A sterile swab, (2), to 73% in Hong Kong (9) and 74% in Malawi (10).
moistened with some of the transport broth, was used to spread Culture studies are not routinely done in any part of the sample over the 90mm plate for culture. Thc culture plateswere covered and placed in a moistened jar.
the world. They are expensive and require a high degree Microaerophilic conditions were obtained by the candle of expertise. Local research laboratories need to keep extinction method and were maintained by use of a Campy surveillance of the susceptibility pattern of H. pylori pack (B.D.L, USA). Once opened, the Campy pack released so as to be able to recommend treatment regimens. Data hydrogen radicals. These formed water molecules from the on the susceptibility of H. pylori in our local population ambient oxygen, ensuring a persistent moist yet microaerophilic were only available up to 1991. There was no reported environment. The plates were incubated at 37°C for a resistance to the recommended antibiotics at that time.
maximum of seven days. Suspected colonies of H. pylori were This study was therefore designed to determine the gram stained and further identified by standard catalase, current H. pylori drug susceptibility pattern and to make oxidase and urease tests (11). The H. pylori isolates wereimmersed in 20% glycerol and stored at -70°C until further appropriate recommendations for eradication treatment Sensitivity testing: The isolated strains of H. pylori were thawed and subcultured on Mueller-Hinton agar plates.
Bacterial suspensions of approximately 107-109 cfu/ml were Study design: The study design was a cross-sectional, prepared according to the McFarland turbidity standard (the test strains) (12). These were then inoculated into the plateswith a multi-point inoculator delivering 7µl. The final Study population: The study was carried out between inoculums on the agar surface were 106 cfu/ml. Epsilometer- October 2003 and April 2004 at the Kenyatta National test strips (E-test) were used to determine the minimum Hospital, a teaching and referral hospital in Nairobi, Kenya.
inhibitory concentration (MIC in mg/l) of the antibiotics: Patient recruitment: Consecutive patients with past or present clarithromycin, metronidazole, amoxicillin, and tetracycline history of dyspepsia, and having been referred for upper (13). The plates were incubated under microaerophilic gastrointestinal tract (GIT) endoscopy were included in the study. Also included were patients with a complication The lowest concentration of antimicrobial agent that relating to a disease state known to cause dyspepsia. Patients inhibited growth in 90% of the isolates is the MIC90.
were excluded if they were found to have predominantly Resistance was considered with an MIC90 > 8mg/l for reflux symptoms or if they failed to give written consent.
metronidazole, > 2mg/l for clarithromycin, > 2mg/l for The minimum sample size was 92 H. pylori positive isolates.
amoxicillin and > 2mg/l for tetracycline (13).
Data collection and analysis: Data were collected and culture yield from CLO positive biopsies was 62% in coded. It was verified, cleaned and entered into the social sciences software package (SPSS Version 12), beingsummarised into means and standard deviations. The data Susceptibility testing/minimum inhibitory were presented in histograms, tables, and pie charts. The chi- concentration of anti-microbial: One hundred and eight square test was used to analyse categorical variables and the strains were tested for susceptibility to metronidazole, student's t-test for continuous variables. A p-value < 0.05 wasconsidered significant.
amoxicillin, clarithromycin and tetracycline. There wasequal distribution of isolates between male and female Metronidazole: The isolated strains of H. pylori Two hundred sixty seven consecutive patients with showed no susceptibility to metronidazole. The MIC dyspepsia and having been referred for upper GIT was ≥ 256mg/l for all isolates. An MIC ≥ 8mg/l endoscopy at the Kenyatta National Hospital were indicates resistance: There was no significant variation in MIC values for age, gender or upper GIT endoscopic The age range was 15 to 85 years, with a mean age of 45.4 years (SD ± 17.6 years). There were 138 male (52%) and 129 female (48%) patients, giving a clarithromycin ranged from 0.016 mg/l to 2mg/l.
There was no statistically significant difference between The most frequent upper GIT endoscopic finding MIC values for clarithromycin with regards to was gastritis (55%) (Table 1). Several patients with endoscopic findings. This also applied to gender. The gastritis also had reflux oesophagitis. Those patients older age groups (45 - 74 years) had MIC values > whose sole upper GIT endoscopic finding was 1.5mg/l for clarithromycin (p<0.05). The MIC values gastro-oesophageal reflux were not included in this for the H. pylori isolates showed susceptibility to clarithromycin. The MIC50 was 0.38mg/l. The MIC90was 1.5mg/l (Figure 1). An MIC > 2mg/l indicates resistance. The isolates showed 93.6% susceptibilityto clarithromycin.
Endoscopic findings of 267 patients with dyspepsia at the MIC values for clarithromycin (mg/l) in patients with dyspepsia at the Kenyatta National Hospital Antibiotic/ other drugs used prior to endoscopy: Antibiotic or other drug use, in the four weeks priorto upper GIT endoscopy, was noted in 158 patients(59%). Of the 158 patients 87 were male (55%) and71 were female (45%). Amoxicillin was the most Amoxicillin: The MIC values for amoxicillin ranged commonly used antibiotic (41%), followed by from 0.016mg/l to 2mg/l. There were significantly more metronidazole (28%), clarithromycin (25%) and female with an MIC of over 0.38 mg/l, (p=0.02). The tetracycline (6%). PPI use was noted in 25%, H MIC values did not show significant variation for upper receptor antagonists in 20% and antacids in 32%.
GIT endoscopic findings or age. The MIC values for Increased antibiotic use was noted in those patients in the H. pylori isolates showed susceptibility to the 35-44 year age group (clarithromycin 31%, amoxicillin. The MIC50 was 0.38mg/l. The MIC90 was amoxicillin 25%, metronidazole 20%).
0.75mg/l. (Figure 2). An MIC ≥ 2mg/l indicates Culture: Of the total patient population, 133 resistance. The isolates showed 95.4% susceptibility to patients (50%) had positive H. pylori cultures. The Minimum inhibitory concentrations (MIC's) of four antimicrobial agents tested against 108 H. pylori isolates MIC values for amoxicillin (mg/l) in patients with Eradication of H. pylori remains the cornerstone dyspepsia at Kenyatta National Hospital for reduction of morbidity and mortality associated withdyspepsia and the ensuing complications.
Drug resistance has now emerged as the most limiting factor to H. pylori eradication.
Becx et al reported metronidazole resistance as early as 1990, predominantly in females (14).
et al reported 27% metronidazole resistance in Belgium in the same year (15). Harries et al reporteda 74% metronidazole resistance in Malawi in 1992 (10) and Ling et al reported resistance at 73.2% in HongKong (9). Perez Aldana et al reported a significant rise in metronidazole resistance from 6.6% (1997- 98) to 12% (1999-2000) (p=0.02). The prevalence ofmetronidazole and clarithromycin resistance in this Tetracyclin: The MIC values for tetracycline ranged study was related to the annual consumption of these from 0.016mg/l to 2mg/l.The MIC values for tetracycline did not show a statistically significant variation for age Using the E-test for 62 isolates, Garza-Gonzalez group or gender. Higher MIC values (over 0.5mg/l) et al reported metronidazole resistance at 38% (17). A showed association with duodenitis (p=0.009) and recent Iranian study reported a 75.4% metronidazole duodenal ulcer (p=0.02). The MIC values for the H. resistance for 120 H. pylori strains by the disk diffusion pylori isolates showed susceptibility to tetracycline. The test (18). The observed in vitro resistance to metronidazole can however be overcome when this 50 was 0.5mg/l. The MIC90 was 1.5mg/l. An MIC ≥ 2mg/l indicates resistance (Figure 3). The isolates drug is used in combination therapy regimes. Longer showed 98.1% susceptibility to tetracycline.
eradication courses have also been found to lead toincreased clinical response (19). The overuse ofmetronidazole in the treatment of amoebiasis and other anaerobic infections has greatly contributed to the risein drug resistance in populations where these infections MIC values for tetracycline (Mg/l) in patients with dyspepsia at Kenyatta National Hospital Resistance to clarithromycin, metronidazole and amoxicillin can decrease rates of cure by up to 62% (20), 36% (7) and 20% (8) respectively. Resistance to tetracycline and bismuth salts have not been reported There was no statistically significant difference between MIC values for clarithromycin as regardsgender or upper GIT endoscopic findings. However, the older age groups (45 - 74 years) had MIC values > 1.5mg/l for clarithromycin (p<0.05). Clarithromycin is used to treat atypical pneumonia, which is more common in the older age group. Our study could suggest increased exposure to clarithromycin in this age group or may be an indicator of primary resistance, since clarithromycin is a relatively recent antibiotic on selection and transmission of resistant organisms.
the Kenyan market. Cross-resistance with erythromycin We recommend that metronidazole should not be considered as first line drug therapy in triple There were significantly more females with regimens for H. pylori eradication in Nairobi and it's H. pylori isolates with an MIC of over 0.38 mg/l for environments. This could probably be extrapolated amoxicillin, p=0.02. This could be due to increased use to most of the Kenyan population. We should still of this antibiotic in the female population especially incorporate amoxicillin, clarithromycin and in the treatment of pelvic inflammatory disease and tetracycline in H. pylori eradication regimens. Future other gynaecological conditions. The MIC values did studies are required to evaluate in vivo susceptibility not show significant variation for upper GIT endoscopic of H. pylori to metronidazole in the Kenyan population findings or age. Perez Aldana et al reported growing and to determine H. pylori susceptibility to the newer drug resistance, albeit to metronidazole and recommended antibiotics, which were not included clarithromycin, and related it to the annual consumption There were a number of limitations of our study.
A tetracycline MIC > 0.5 mg/l showed a The study population may not have been representative significant association with duodenitis (p= 0.009) of the general Kenyan population. The Kenyatta and duodenal ulcer (p= 0.02) but did not show National Hospital is a referral hospital, drawing statistical significance for gender or age group. H. patients from all over the country, but the majority pylori infection is specific for gastric mucosa and of the patients are however from the greater Nairobi areas of gastric metaplasia. Our study could suggest area and may not be representative of the general that H. pylori isolates that have higher MlC's for population. Patients living in the environments of a tetracycline are more likely to induce gastric hospital or pharmacy may have had greater exposure metaplasia within the duodenum. It is now known to antibiotics, which may have a bias on the drug that the virulence of H. pylori is affected more by resistance profile. Patients with recurrent dyspepsia the vacuolating cytotoxin (VacA) and the cag are more likely to be referred to a tertiary facility.
pathogenicity island (CagA) evidenced by genotypic These may represent a select group of patients with markers, than it is by the presence of drug resistance a high rate of drug resistance, which may not be (2,21-22). Drug resistance as evidenced by a rise in reflective of the resistance pattern in the general MIC90 level could not clearly be related to the population. Screening for H. pylori is usually on the severity of disease expression at upper GIT endoscopy.
strength of two tests such as histology and CLO Histology may have answered this more appropriately.
testing. The CLO test is 85-90% sensitive if read up The future for therapy is uncertain. There are to 24 hours. This was the only screening test in our no new antimicrobials that would have a major study and was read up to six hours post-biopsy. We impact on the current position. Predictions that may therefore have missed many H. pylori positive knowledge of the genome would lead to more patients. Further delay in reading the CLO tests appropriate therapy have not materialised. Drugs would not have been beneficial, as it would have under investigation to try and increase the increased the chances of overgrowth of oral and susceptibility of H. pylori to antibiotics include the stomach contaminants and hence reduced the use of aspirin, but the results are controversial and sensitivity of the culture results. However, the major the benefits have not been noted in vivo (23).
objective of this study was susceptibility testing and Prolonging therapy to 10 to 14 days should be not H. pylori prevalence; the latter would have been greatly affected by the use of one screening test. This In conclusion all our isolates showed in vitro was an in vitro study, which may not be fully resistance to metronidazole, MIC > 256mg/ml, but reflective of the in vivo situation showed susceptibility to clarithromycin (93.6%),amoxicillin (95.4%) and tetracycline (98.1%). Higher MIC values for tetracycline were significantly associated with duodenitis and duodenal ulcer. H. Microbiology laboratory for the culture and sensitivity pylori showed a rising MIC90 for metronidazole and testing; Mr Waweru, Department of Medicine, KNH tetracycline as compared to a similar study carried and the endoscopy room nurses for their technical out in Kenyatta National Hospital by Sang et al in assistance; Dr. Mutie, Dr. Kioko, Dr. Musau, (KNH), Dr. Lodenyo (KEMRI) and Mr. Okumu (Department metronidazole of 4.0mg/l, for tetracycline 0.12mg/l, of Surgery, KNH) for the upper GIT endoscopies and for ampicillin 0.03mg/l and for erythromycin 0.25mg/ Lords Pharmaceuticals and AstraZeneca for financial l. A rising MIC90 is a good indicator of growing assistance. Funds from these companies were used to purchase E-test strips from an independent In this light, all attempts should be made to company, AB BIODISK (Solna, Sweden).
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