Journal of Infection (1992) 24, 269--276

Helicobacter pyrlori in M a l a w i , C e n t r a l A f r i c a

A. D. Harries,* M. Stewart,T K. M. Deegan,$ G. K. Mughogho,w J. J. Wirima,w M. Hommel* and C. A. Hart t II

* Liverpool School of Tropical Medicine, t Department of Medical Microbiology, University of Liverpool, $ Department of Histopathology,

Fazakerley Hospital, UK and w Kamuzu Central Hospital, Lilongwe, Malawi

Accepted for publication 23 October I991

Summary

A total of I6O adult Malawians with epigastric pain for longer than 2 weeks was investigated by endoscopy and serologically for evidence of infection with Helicobacter pylori. The organism was demonstrated histologically and/or by culture in I4I (88 %) patients. With histological means and/or culture as the 'gold standard', the histological technique was Ioo % sensitive while culture was only 8I % sensitive. All isolates tested were sensitive to amoxycillin and tetracycline; 74 % were resistant to metronidazole. Endoscopic findings were normal in IO4 (65 %) patients (86"5 % H. pylori positive). Evidence of duodenal ulcer was found in 4I (25 %) patients (95 % H. pylori positive). Histologically, gastritis was common, severe gastritis being associated with increased colonisation by H. pylori. Two kinds of urease test were found to be Ioo% specific for the presence of H. pylori. The sensitivity of the serological test (Helico-G test) was 98 % but its specificity was only 27 %. These results provide important background information for planned therapeutic studies in patients with upper gastro-intestinal disease in Malawi.

Introduction

Epigastr ic pain is very common among the indigenous populat ions of sub- Saharan Africa. In some regions it accounts for up to IO% of hospital admissions 1 and many outpat ient a t t endances? In patients assessed by upper gastro-intestinal endoscopy, the commones t findings are a normal mucosa, evidence of gastritis or duodenal u l ce r? -5 Studies in Rwandaf l Ghana, 6 Kenya , 7 Uganda , 8 Z imbabwe 8 and Nigeria 5 have shown a high prevalence of Helicobacter pylori in Africans with dyspepsia, associated in part icular with histological evidence of gastritis and duodenal ulcer.

In Malawi, Central Africa, medical t reatment of duodenal ulcer is often l imited to administrat ion of non-absorbab le antacids or to short courses of H2- receptor antagonists. Persistence of ulcers or their relapse is common and often leads to complications, part icularly pyloric stenosis 4 and haemorrhage. 9 Sustained symptomat ic improvement in patients with dyspepsia wi thout an ulcer is also disappointing with conventional t reatment. I f H. pylori were s trongly and causally related to these conditions, its eradication would represent an important therapeutic advance. We decided (i) to investigate the prevalance of H. pylori and its relation to disease in Malawian adults with

[[ Address correspondence to: Professor C. A. Hart, Department of Medical Microbiology, University of Liverpool, P.O. Bo x47, Liverpool L69 3BX, U.K.

oi63-4453/92/o3o269+o8 $03.00/0 �9 I992 The British Society for the Study of Infection

270 A. D. H A R R I E S E T A L .

epigastric pain, (ii) to determine antibiotic sensitivities of the bacterium in vitro with reference to antibiotics commonly used in Malawi, and (iii) to examine methods of diagnosis.

Patients and methods

Patients

The study took place in Kamuzu Central Hospital, Lilongwe, Malawi, over a period of 5 weeks during June and July I99O. Malawian adults aged I8-7o years with epigastric pain of more than 2 weeks' duration and which was considered clinically to be caused by gastritis or peptic ulcer were invited to participate in the study. Those taking non-steroidal anti-inflammatory drugs or anti-ulcer drugs other than antacids were excluded. Patients were referred from outpatient clinics in the hospital as well as from district hospitals in the Central and Northern regions of the country. The study was approved by the Malawi Ethical and Research Committee. Informed consent was obtained from each patient.

Demographic and endoscopic features

A questionnaire on demographic features was completed by all patients under medical supervision. From each, a IO ml sample of blood was taken and the serum stored at - 2 o ~ Upper gastro-intestinal endoscopy was performed with topical anaesthesia and use ofa Fujinon UGI-PF3 endoscope with a F IL- I5O EEH Fujinon light-source. Macroscopic findings were recorded and samples of all gastric and oesophageal ulcers were obtained for histological assessment. At least four gastric mucosal biopsy specimens were taken from the antrum 1-2 cm from the pylorus. Two specimens were placed in IO % buffered formalin for histological examination, one was taken for micro- biological examination and one/two for urease testing. The instrument was thoroughly washed, then sterilised in a 2 % solution of glutaraldehyde after use in each patient. Patients with macroscopic gastritis or peptic ulcer received conventional treatment.

Urease production

Each specimen was placed immediately in I ml of a IO % solution of urea in deionised water (pH 6"8) to which two drops of 1% phenol red solution had been added and incubated at 37 ~ This unbuffered, so-called 'one minute ' urease test was read at 15 min and again at 24 h. Specimens from some patients were also placed in I ml of 2 % Christensen's urea broth, and results read after 4 and 24 h incubation at 37 ~

Microbiology

One specimen was immediately inoculated on brain-heart infusion agar containing 1% isovitalex, 7% horse blood and Skirrow's recommended antibiotics for Campylobacter fetus (vancomycin 5 rag, trimethoprim 2"5 mg and polymyxin B 125o IU). Plates of medium were freshly prepared before each endoscopy session and dried sufficiently to remove surface moisture only. After inoculation they were incubated at 37 ~ in a gas jar with an anaerobic

Helicobacter pylori in Malawi 27I

Gas Pak bu t wi thout a catalyst. All plates were read after 5 days' incubation. Helicobacter pylori was identified by Gram-sta in , urease and oxidase tests.

Ant imicrobial sensitivities were de termined by means of a disc diffusion method. Strains of organisms were inoculated on b lood agar containing 1% isovitalex and discs containing the following antibiotics were applied: amoxycill in 2 #g, amoxycill in IO #g, tetracycline IO #g, penicillin I #g and metronidazole 5 #g. T h e plates were incubated as descr ibed above.

Histology

Histological sections of the specimens were stained with haematoxyl in and eosin for grading of gastritis and with Giemsa-s ta in in order to demonst ra te the bacteria. Po lymorphonuclear and mononuc lear cell infiltration were each graded f rom o to 3 and the scores summed. T he densi ty of colonisation with H. pylori was graded I + , 2 + or 3 + .

Serology

A n t i - n . pylori I gG was measured by enzyme-l inked immunosorben t assay ( E L I S A ) and use of a Por ton Cambr idge H E L I C O - G kit. 1~ Samples were measured in duplicate and the mean result obtained. An ant ibody con- centrat ion of IO un i t s /ml or more was taken to indicate n . pylori infection.

Statistical analysis

Statistical comparisons were made by means of the X z test with Yates correct ion for small number s where appropriate.

Results

Demographic and clinical features

A total of 16o patients (93 men and 67 women) whose mean age (S.D.) was 37 (12) years was studied. O f the women, 73 % were housewives while the commones t occupations among the men were artisans (17 %), farmers (12 %) and s tudents (I 1%). Only 15 % patients were cigarette smokers ; 20 % drank alcohol. Med ian durat ion of symptoms was 24 months (range 3 weeks to 33 years).

ltelicobacter pylori

Brain-hear t infusion agar cultures related to 136 patients were examined for H. pylori. T h e organism was identified in cultures f rom IOO patients. All of 5 i isolates tested were sensitive to ampicillin and tetracycline. However , five of 5o ( l O % ) were resistant to penicillin and 37 of 5o ( 7 4 % ) were resistant to metronidazole. O f 22 isolates from women, 16 (73 %) were resistant to metronidazole , a finding similar to 21 (75 %) of 28 isolates tested f rom men. T h e mean age of patients with sensitive isolates (34"9 years) was similar to that o f those with resistant isolates (36"5 years).

Helicobacter pylori was identified in histological sections in 141 (88 %) of 160 patients. T h e density of organisms was I + in 40 %, 2 + in 38 % and 3 + in 22 % patients. The re was no correlation be tween the densi ty of organisms observed histologically and the amount of growth on culture.

272 A.D. H A R R I E S E T AL .

Table I Gastritis, polymorphonuclear and mononuclear cell infiltration in relation to Helicobacter pylori infection

Number Number (%) with H. pylori

Gastritis score o 3 o (o) I I2 3 (25) 2 29 26 (9O) 3 34 3I (9I) 4 47 47 (Ioo) 5 32 31 (97) 6 3 3 (IOO) Polymorphonuclear cell infiltration o 56 41 (73) i 63 60 (95) 2 36 35 (97) 3 5 5 (ioo) Mononuclear cell infiltration o 3 o (o) I I6 6 (37) 2 51 47 (92) 3 90 88 (98)

H. pylori infection diagnosed by positive histological results and/or positive culture.

A definitive diagnosis of infection with H. pylori was made on the basis of a positive culture and /o r positive histology. According to these criteria, histology was I o o % sensitive and IOO % specific; culture was 81% sensitive and lOO% specific (i.e. all biopsy specimens negative for H. pylori histologically were negative for H. pylori on culture). Of biopsy specimens which were positive for H. pylori histologically but negative on culture, the density of organisms was I + in 78%, 2 + in I 8 % and 3 + in 4%.

Endoscopic diagnosis and Helicobacter pylori

A total of IO4 (65 %) biopsies was considered normal endoscopically. In 9o (87 %) of these, H. pylori was seen microscopically. Forty-one (26 %) patients had duodenal ulcers and 39 (95 %) of these had evidence of H. pylori infection. Seven patients had macroscopic gastritis, two oesophageal carcinoma and one a gastric ulcer. Helicobacter pylori was present in all of them. The organism was seen also in two of five biopsies from patients with gastric carcinoma.

Gastritis and Helicobacter pylori

Gastritis scores as well as polymorphonuclear and mononuclear infiltration in relation to colonisation with H. pylori are shown in Table I. The infiltration contained mononuclear cells in x57 and polymorphonuclear cells in xo4 patients. Polymorphonuclear cells were present in Io I (7 2 %) patients with H. pylori compared with 3 (I7 %) patients negative for H. pylori (P < o.ooI). The grade of mononuclear cell infiltration and the gastritis score were significantly higher in specimens with H. pylori densities 2 + or 3 + compared with H.

Table II

Helicobacter pylori in Malawi

Value of urease tests in the diagnosis of Helicobacter pylori infection

273

Number of Sensitivity Specificity PPV* N P V t Test isolates (%) (%) (%) (%)

'One minute ' urease test I28 read at i5 min

'One minute ' urease test I28 read at 24 h

Christensen's urease test 63 read at 4 and 24 h

75 IOO IOO 30

85 IOO IOO 40

56 IO0 IO0 IO

* Positive predictive value. t Negative predictive value.

pylori densities I + (P < 0"05). There were no such significant differences between H. pylori densities 2 + and 3 + .

T h e u r e a s e t e s t a n d Helicobacter pylori The sensitivity, specificity and predictive values of urease tests on endoscopic biopsy specimens are shown in Table II. The 'one minute ' urease test, read at 15 rain and at 24 h, as well as Christensen's commercial urease test, gave no false positive results. The sensitivity of the 'one minute ' test was higher when read at 24 h compared with what it was at 15 min, but both were more sensitive than the commercial test. Of biopsy specimens which were positive for H. pylori histologically but negative b y ' one minute ' urease testing, the density of organisms was I + in 73 % of those read at I5 min and 83 % of those read at 24 h.

Helicobacter pylori serological tests

When an antibody concentration of IO units/ml or more was regarded as indicative of infection with H. pylori, the sensitivity of serological testing was 98"5 % in I45 samples tested. Of I4 patients who were otherwise negative for H. pylori, I I were positive serologically, so giving a specificity of 21"4 %. Positive and negative predictive values for serological testing were 92 % and 60 % respectively.

Discuss ion

This study confirms previous reports 3'5-s from sub-Saharan Africa that non- ulcerous dyspepsia and duodenal ulcer are the commonest causes of epigastric pain, and that H. pylori is strongly associated with both conditions. Many patients were young and had experienced epigastric pain for a long time, often in association with other symptoms such as vomiting and loss of weight. Type B chronic active gastritis was found in most of our patients and, not unexpectedly, was found closely associated with H. pylori infection. The relation between the number of organisms and the intensity of the cellular infiltration, particularly of the polymorphonuclear component, has been previously reported from more developed countries. 11 Serological evidence

274 A. D. H A R R I E S E T A L .

f rom the Gambia 12 and Ethiopia la suggests that infection is established early in life and is much more prevalent than it is in more developed countries.

T w o biopsy specimens from the antral mucosa are repor ted to be sufficient to detect colonisation with H. p y l o r i 14 and was the reason for our choosing this n u m b e r for histological analysis. His topa tho logy was more sensitive in the diagnosis o f H. pylori infection compared with culture---a finding similar to that repor ted f rom Rwanda. a Abou t 8o % of the colonised specimens which failed to p roduce growth of H. pylori on culture had few organisms in histological sections. T h e low densi ty of infection, combined with the fact that cul ture may be technically difficult may explain the differences in detect ion rates. Even so, the culture med ium which we used is repor ted to yield a heavier growth of H. pylori more rapidly than other media. 15

Cul ture of H. pylori made it possible to s tudy antibiotic sensitivities. Mos t strains isolated in Malawi were resistant to metronidazole, in contrast to those in a recent s tudy f rom Europe, 16 showing that only 6 % isolates were metronidazole-resistant . It has been established that during the course of t reatment resistance to nitroimidazoles develops rapidly, especially with monotherapy. 17 In the Nether lands , 16 resistance was more common in women. This was a t t r ibuted to previous therapy with the antibiotic for vaginal infections. In a few patients s tudied in Zaire, 18 the f requency of metronidazole resistance was similar to our findings, being also uninfluenced by age and sex. Mos t Zairian patients recognised having received metronidazole before investigation, either through self-medication or prescr ipt ion for amoebiasis or giardiasis. Al though we did not specifically enquire about previous antibiotic therapy, a similar explanation for the findings is likely in Malawi. Other antibiotics, such as penicillin and tetracycline, are widely used in Malawi and it is unclear why resistance has not developed to them. T h e observat ion of metronidazole resistance, however , has impor tant therapeut ic implications because patients infected with metronidazole-resis tant H. pylori will not respond to the usually effective combinat ion of a nitroimidazole with b ismuth or amoxycillin. 18

T h e 'one minu te ' urease tests read at r 5 rain and at 24 h were Ioo % specific wi thout any false-positive results. These findings accord with those o f other studies. 19 W e read this urease test at I5 rain because it was impossible under the condit ions of the s tudy to read the colour change at I min. Increasing the concentrat ion of urea from 2-6 %2o increases the speed of the results bu t too much substrate may inhibit urease activity and result in decreased sensitivity. T h e 'one minu t e ' urease tests in our patients, however , were more sensitive than those for which commercial ly obta ined Chris tensen 's urea bro th was used. Moreover , the urea broth was easy to make and is considerably cheaper than the commercial preparation. Urease tests depend on the n u m b e r of organisms present in biopsy specimens, z~ Mos t false-negative reactions, therefore, were related to specimens with H. pylori in low densities.

In our hands serological means of diagnosis proved less useful. Al though its sensitivity was high (98"5%), its specificity was low ( 2 i % ) . T h e high sensitivity and resultant positive predict ive value reflect the high prevalence of H. pylori infection in our patient populat ion. However , its low specificity, which arose because antibodies were detected in I I of I4 patients who did not

Helicobacter pylori in Malawi 275

have demonstrable H. pylori infection, makes serology less valuable diag- nostically in these circumstances. T h e low degree of specificity may have come about for several reasons. First ly, this test has been designed to provide optimal results in developed countries 21 where different strains of H. pylori may give different serological results. A recent survey in T h e Gambia, where antigen was derived from H. pylori strains isolated there, showed improved performance of the test. 12 Secondly, ant ibody may still be detectable in persons who have been infected with H. pylori but who have recovered either spontaneously or following treatment . Finally, there is a very high prevalence of diarrhoeal disease in children and adults in developing countries and it is known that there is antigenic cross-reactivity between Carnpylobacter jejuni and H. pylori. 22 In order to try to improve the specificity of serological means of diagnosis, we are current ly examining the use of Malawian isolates as antigen as well as the value of seeking H. pylori IgA in saliva.

T h e results of this s tudy in Malawi suggest that fur ther work should be done in sub-Saharan Africa on H. pylori. Several questions need to be answered. H o w is the bacter ium acquired? Can it be eradicated and is eradication associated with cure of duodenal ulcer and improvement in non-ulcer dyspepsia ? T rea tmen t with b ismuth and amoxycill in in Africa has had mixed results, 7'za but requires fur ther evaluation in properly controlled studies.

(We thank the Ministry of Health, Malawi, for kind permission to perform this study, the Senior Medical Superintendent, Kamuzu Central Hospital, for helpful co- operation as well as Dr M. Steele and Sister O. Kadzakumanja for their valuable assistance with endoscopy. The study was supported by a grant from the University of Liverpool, and the Jean Clayton Research Trust, Liverpool School of Tropical Medicine. Bismuth chelate was provided by Gist-brocades for treatment of duodenal ulcer, and Helico-G serology kits by Porton Cambridge.)

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