CITATION: Winterbach, R. et al. 1994. Incidence of heat-labile enterotoxin-producing Escherichia coli detected by means of polymerase chain reaction amplification. South African Medical Journal, 84:85-87.The original publication is available at http://www.samj.org.zaDiarrhoea can be caused by many different organisms, some of which are notoriously difficult to identify. One of these is enterotoxin-producing Escherichia coli. Recently a new diagnostic technique that uses polymerase chain reaction DNA amplification was developed for detection of the 'A' subunit of the labile enterotoxin-producing E. coli gene. This technique was used to evaluate the incidence of heat-labile (LT+) enterotoxin-producing E. coli in the causation of diarrhoea. The results from this study showed that LT+ E. coli is a cause of diarrhoea in the western Cape and that 5,3% of non-diagnosed diarrhoea patients in Tygerberg Hospital were infected with this pathogen. This represented less than 1% of the total number of cases of diarrhoea investigated in this hospital. The peak coincides with the wetter months in this locality and the infection rate is lower than that reported in most other countries. Given the low incidence of occurrence of this organism we do not recommend routine implementation of the diagnostic procedure. However, this test may be useful at times, e.g. to ascertain the source of a diarrhoea epidemic.Publisher’s versio

Janse Van Rensburg, J.

Van Helden, P. D.

Victor, T.

Winterbach, R.

Stellenbosch University SUNScholar Repository

elderly woman had left colon diverticula and right colon angiodysplasia bur which was the culprit could not be established. A blind subtotal colectomy which could take care of both lesions was deemed unwise in her frail physical state. Angiodysplasia is still very rare among blacks. In summary, our study confirms the previous report from Johannesburg that diverticular disease is emerging among urban blacks in South Africa and further draws attention to haemorrhage as a predominant presenting feature. These findings might equally apply to other newly urbanised com munities who have historically been free of this disease. The increasing global mobility of patients and clinicians requires the latter to be fami-liar with different patterns of disease and their presenta-tion in different races and communities; this will help prevent mortality caused by missed diagnoses. We wish to thank Ms Eleanor Gouws of the MRC Biostatistics Division, Durban, for help with statistical ana-lysis. REFERENCES I. Burkitt DP, Clements JL jun, Earon BS. Prevalence of diverticular disease, hiatus hernia and pelvic phleboliths in black and white Americans. Lancer 1985; 2: 880-881. 2. Segal I, Solomon A, Hunt JA. Emergence of diverticular disease in urban South African blacks . Ga.stroemerology 1977; 72 : 215-219. 3. Luvuno FM. Role of intra-operative colonic lavage and a decom-pressive ileostomy in management of rransmural amoebic colitis. Br J SlIrg 1990; 77:1 56-159. 4 . Golligher J. D iverticulosis and diverticulitis of the colon . In: Golligher J, ed. SlIrgery of (he Anus, Rectllm and Cololl . 5th ed. London: Bailliere Tindall, 1984: 1083- 1116. 5. Archampong EQ, Christian F , Badoe EA. Diverticular disease in an indigenous African community. AmI R Coli Surg Engl 1978; 60: 464-470. 6. Arrington P, Judd CS jun. Cecal diverticulitis. Am J Surg 1981; 142: 56-59. 7. Markham ~'1, Li AKC. Diverticulitis of the right colon - experi-ence from H ong Kong. Gill 1992; 33: 547-549. 8. Miangolarra CJ. Diverticulitis of the right colon: an important sur-gical problem. Ann Surg 1961; 153: 861-870. 9. Potter GO, Sellin JH. Lower gastrointestinal bleeding. GastraemerolClin N orthAm 1988; 17: 341-355 . 10 . C ussons PO, Berry AR. Comparison of the value of emergency mesenteric angiography and intraoperative colonoscopy with ante-gtade colonic irrigation in massive haemorrhage. J R Call Surg Edinb 1989; 34: 91 -93. Incidence of heat-labile enterotoxin-producing Escherichia coli detected by means of polymerase chain reaction amplification R. WINTERBACH, P. D. VAN HELDEN, J. JANSE VAN RENSBURG, T. VICTOR Abstract Diarrhoea can be caused by many different organ-isms, some of which are notoriously difficult to identify. One of these is enterotoxin-producing Escherichia coli. Recently a new diagnostic tech-nique that uses polymerase chain reaction DNA amplification was developed for detection of the 'A' subunit of the labile enterotoxin-producing . E. coli gene. This technique was us~d to evaluate the incidence of heat-labile (L T +) enterotoxin-pro-ducing E. coli in the causation of diarrhoea. The results from this study showed that L T + E. coli is a cause of diarrhoea in the western Cape and that 5,3% of non-diagnosed diarrhoea patients in T ygerberg Hospital were infected with this pathogen. This represented less than 1% of the total number of cases of diarrqgea investigated in this hospital. The peak coincides with the wetter months in this locality and the infection rate is lower than that reported in most other countries. Given the low incidence of occurrence of this organism we do not recommend routine imple-mentation of the diagnostic procedure. However, this test may be useful at times, e.g. to ascertain the source of a diarrhoea epidemic. S Atr Med J 1994; 84: 85-87. Ml{c Centre for Molecular and Cellular Biology and De\lartment of Medical Physiology and Biochemistry, University of Stellenbosch, Tygerberg, CP R. WINTERBACH , DIP. MED. TECH. P. D . VAN HELDEN, PH.D. J. JANSE VAN RENSBURG, 4th-year Medical Student T . VICTOR, PH.D. Accepted 4 Jan 1993. ~ure diarrhoea has a significant impact on public health' (particularly among children in developing countries) . Previous studies have shown that rotavirus and Salmonella spp. are among the common causes of diarrhoea in urban areas, whereas enterotoxi-genic Escherichia coli, Shigella spp. and other bacterial agents are a more important cause . of diarrhoea in rural areas, where the water supply and public health mea-sures are often poor.' E. coli is reported to be one of the most important causes of diarrhoeal illness among child-ren and adults in developing countries and in the indus-trialised world.' Numerous techniques are available for the identifica-tion of these aetiological organisms - including E. coli. Most often, MacConkey and blood agar plates together with serological typing are used for the diagnosis of E. coli.' Techniques fo r the direct identification of enterotoxigenic E. coli or gene products include the fol-lowing: (i) tissue culture and skin permeability factor assay;' (iZl Yl -adrenal cells in tissue minicultures;6 (iiz) direct serological assay with passive immune haemo-lysis;' and (iv) identification by means of enzyme-linked irnmunosorbent assay with antisera against labile toxin after bonding to specific antibodies or gangliosides.' Techniques aimed at the detection of specific genes such as D A colony hybridisation," direct detection of enterotoxin-producing genes with radio-active DNA probes' and non-radioactive polynucleotide gene probe assay for identification of enterotoxigenic E. coli, 'o were also used for identification . However, serological typing and tissue culture tech-niques have been found to be labour-intensive as well as nonspecific and insensitive. '0 The use of radiolabelled 85 cloned DNA probes and oligonucleotides in colony hybridisation for heat-labile (L T +) and stable (ST+) toxin genes is not very sensitive. These methods are fairly sophisticated and, although specific,"''' 'z are not suitable for routine laboratory diagnosis. More recently a new technique, polymerase chain reaction (PCR) amplification of DNA, was developed for the amplifica-tion of the 'A' subunit of the L T + toxin gene, without prior DNA extraction. 11 Detection of the amplified product is possible with polyacrylamide gel electrophoresis, which is sensitive enough to detect the L T + enterotoxin gene in stool-derived samples. The PCR technique described is rela-tively sensitive, specific, simple and inexpensive. Since diarrhoea is such an important disease (parti-cularly among children), this study was undertaken to evaluate the role of L r enterotoxin-producing E. coli in the causation of diarrhoea in patients at T ygerberg Hospital. Materials and methods Stool specitnens For the amplification of the L T + toxin gene, stool speci-mens from patients with severe diarrhoea were obtained. These were collected over a period of 12 months Qanuary - December 1990). The specimens used for this study were selected according to the following crite-ria: (i) the stool specimens were all from patients with severe diarrhoea; (ii ) no Shigella spp., Salmonella spp., Campylobacter spp., Klebsiella spp., Scaphylococcus spp. or any parasites were identified during routine screening for the abovementioned organisms by the microbiologi-cal laboratory at T ygerberg Hospital; and (zii) there had to be overgrowth of E. coli after overnight incubation on MacConkey plates. After the MacConkey plates had been selected, they were directly tested for the presence of LT+ enterotoxin-producing E. coli by means of the PCR amplification technique described below. Patient data Information was obtained from the hospital files of those patients whose stool specimens proved to be L T + E. coli-positive after the PCR analysis. Detection ofLT+ toxin-producing E. coli by means of peR amplification PCR amplification with gene-specific primers was essen-tially done as described in earlier studies. " A scraping of E. coli from the MacConkey plates was diluted in 400 !ll of sterile saline and boiled for 20 minutes. Ten micro-litres of this lysed and denatured suspension of bacteria were combined in a total volume of 99 !ll with a premix-ture of 1 x PCR buffer (10 mM Tris-HCL [PH 9,0 at 25°C] , 1,5 mM MgClz, 50 mM KCI, 0,01 % gelatin (w/v), 0,01 % Triton X-100), a 200 ~ final concentra-tion (each) of dATP, dCTP, dGTP and dTTP and 0,4 ~ of each primer. After the addition of 0,5 U of Taq DNA polymerase (Promega), the mixture was overlaid with 40 !ll of mineral oil and the heating cycle of 93°C for 1 minute, 55°C for 1 minute and 72°C for 2 minutes was repeated 35 times in a thermal cycler. The ampli-fied product was analysed by means of electrophoresis on ethidium bromide containing 12% polyacrylamide gels, and the DNA was visualised by means of transillu-mination. LT+ enterotoxin-producing E. coli (ATCC 43886) and sterile saline were used as positive and nega-tive controls respectively. Results Detection ofLT+ E. coli by m.eans of peR analysis Clinical isolates were analysed over a period of 1 year by means of culture, PCR and the electrophoresis system. Fig. 1 is a sample result and indicates that L r E. colt could be a cause of diarrhoea in the western Cape. Some negative samples were spiked with a few L T + bac-teria and analysed again. Positive results were obtained thereafter, which indicated that a negative result was not due to the presence of polymerase inhibitors in the sam-ples tested. A negative result was obtained in each batch of samples for the control tube containing saline instead of template (Fig. 1, lane 10). Seventeen out of 323 sam-ples were positive for Lr enterotoxin plasmid (5,3%) (Table I) . 1 2 3 4 5 6 7 8 9 10 110 FIG. 1. Detection of L T+ enterotoxin-positive E. coli in the clinical isolates. The stool samples where no enteropathogen could be identified after routine screening were inocu-lated on MacConkey plates. A scraping from this colony growth was directly amplified with primers L T31 and L T51 . Twenty-microlitre samples of the amplified product were used for gel electrophoresis and detection with ethidium bromide. A 12% polyacrylamide gel was used. The sample in lane 10 contains no template DNA, only sterile saline. The numbers on tl:1e left indicate molecular sizes in base pairs. Lanes 2, 4 and 9 are the only lanes which contain L T+-positive samples. Patient data Patient data are presented in Table 1. All the patients, although admitted for different reasons, had severe diar-rhoea. Most of these patients were under 5 years old. Thirteen of the admissions were during the months March to June. More female patients (13) had diarrhoea than male patients (4); most of these patients were coloured (N = 14). Only 3 black and no white patients were LT+ E. coli-positiv e . On admission most of the patients were diagnosed as having gastro-enteritis, mal-nutrition (including marasmus and kwashiorkor) and necrotising enterocolitis. The patients spent between 2 days and 28 days (average 11 days) in hospital before either being discharged or before the diarrhoea dis-appeared. These patients were not treated for E. coli infection specifically, since the results obtained were generally not available to the clinicians treating the patients at the time of hospitalisation. TABLE !. Incidence of L T- enterotoxin-producing E. coli over a period of 1 year PCR- Diagnosis Month of positive Age made on admission cases Race Sex . (yrs) admission Jan 0 Feb 0 Mar C M 4 Gastro-enteritis C F 2 Gastro-enteritis 5 C F Gastro-enteritis, malnutrition, anaemia C M 12 Lymphoma C F < 1 Necrotising entero-olitis, septicaemia Apr 2 B F Gastro-enteritis, malnutrition, C F 2 Gastro-enteritis May B F Gastro-enteritis, malnutrition, 4 B F Gastro-enteritis C F Gastro-enteritis, malnutrition C F 52 Diverticulitis Jun 2 C F 1 Gastro-enteritis C M 1 Gastro-enteritis Jul C F 24 Chronic diarrhoea Aug C F < 1 Jaundice Sep C F Gastro-enteritis Oct 1 C M < 1 Gastro-enteritis Nov 0 Dec 0 A total of 323 selected samples was analysed, of which 5,3% tested positive for L T- enterotoxin-producing E. coli. The race group classification refers to the so-called coloured (C) and black (8 ) races respectively. Discussion Some diagnostic laboratories have reponed that there are many cases of diarrhoea (e.g. 40 %) where the pathogen could not be identified.13 The aim of this study was to determine the incidence of L T + E. coli and the role it played in the causation of diarrhoea at Tygerberg Hospital where the pathogenic organism was not diag-nosed. The results presented in this study show that 5,3% of non-diagnosed diarrhoea patients were infected with this pathogen . This represents less than 1 % of all cases (vari-ous causes) of diarrhoea investigated in this hospitaL The predominance of female patients is in contrast to the male dominance found in Ibadan, Nigeria. " The incidence is higher than the 2,6% L r E. coli found in Seoul, South Korea, ' and the 2,5% E. coli found on the island of H ong Kong/ bur is lower than the 16,6% LT+ E. coli found at Bandar-Abbas, Iran," the 24,3% previ-ously reponed for both L r and ST+ toxins in a differ-ent region in South Africa '· and the 45,3% Lr E. coli found at Lugar Sobre la Tierra Blanca, Mexico.I7 The reason for this difference could be the differences in the sampling of the study population or the differences in the methods used to identify the L r E. coli. Pathogenic E. coli is found mainly where water supply and public health services are poor and in densely populated areas. Variation in health serving and urban behaviour could also explain differences in infection rates. The high incidence of admissions during the months of March to June coincides with the wetter autumn and winter months in this locality. An annual seasonal peak of infection with this enteropathogen in Mexico also occurs in April to July; " however, in Seoul, South Korea, the seasonal peak is during the dry months of October and November. ' The low incidence of this organ ism at Tygerberg Hospital does not justify the expense of routinely assay-ing all diarrhoea patients; this method may nevenbeless be useful at times, e.g. to determine the cause of a diarrhoea epidemic. We thank the Deparonem of Microbiology, Tygerberg Hospiral, for supplying the clinical material. REFERENCES 1. Kyung-Hee K, Inn-Sao S, Jung Mogg K, Choon Won K, Yang-Ja C. Eriology of childhood diarrhoea in Korea. J Clin Microbiol 1989; 27: 11 92- 11 96. 2. Wing Ceong Y, Li Lung M, Yeung CY, Johns T , Mun H on NG. Escherichia coli associared wirh childhood diarrhoeas. J CIi" Microbiol 1987; 25: 2 145-2149 . 3. Andreoli TT, Carpenrer CCJ, Plum F, Smith LH. Essentials of Medicine. Philadelphia: WB Saunders, 1986. 4 . Edwards PR, Ewing WH. identificacion of EnrelObacreriaceae. 3rd ed. Minneapolis: Burgess Publishing, 1972. 5. Mosely SL, Huq I, Alim ARMA, Samad-pour-Moralebi M, So M, Falkow S. Enreroroxigenic Escherichia coli: identification and char-acrerization. J Infecl D is 1980; 142: 279-285. 6. Sack DA, Sack RB. Tesr for enreroroxigenic Escherichia coli using Yl adrenal cells in miniculrure. Infecc lmmun 1975; 11: 334-336. 7. Speirs ]I, Sravric S, Konowalchuk J. Direcr serological assay for rhe hear-labile enre roroxin of E scherichia coli, using pass ive immune hemolysis. Infecc Immun 1977; 16: 604-609. 8. Mosely SL, H uq I, Alim ARMA, Samad-pour-Moralebi M , So M, Falkow S. D erection of enreroroxigenic Eschendzia coli by DNA colony hybridization. J Infecc Dis 1980; 142: 892-898. 9. G omes TAT, Toledo MRF, Trabulsi LR, Wood PK, Glenn M o rri s J . DNA pro b es fo r ident ificarion of ente roinvasive Escherichia coli. J Clin Microbiol 1987; 25: 2025-2027. 10. Sommerfelr HS, Grewe! H.~S, Bhan MK. Simplified and accurare nonradioactive polynucleotide genprobe assay for identification of enreroroxigenic Escherichia coli. J Clin Microbiol 1990; 28: 49-54. II. Victor T, Du Toir R, Van Zyl ], Besrer AJ, Van H eIden PD. Improved merhod fo r rhe rourine iden rificarion of rox igen ic Escherichia coli by DNA amplification of a conserved region of the hear-labile roxin 'A' subunit. J elin Microbiol 199 1; 29: 158-1 61. 12 . M oseley SL, H ardy lW, Huq I , Echeverria P, Falkow S. Isolation and nucleotide sequence derermination of a gene encoding a hear-labile enterotoxin of Escherichia coli. Infecc Immun 1983; 39: 11 67-1174. 13. Coovadia H M , Loening WEK. Pediatrics and Child Health . 2nd ed. Cape Town: Oxford Universiry Press, 1988. 14. Ako-Nai AK, Lamikanra A, Ola 0 , F adero FF. A srudy of the incidence of enreroroxigenic Escherichia coli (ETEC) secre ring heat-labile toxin in twO communities in south-western Nigeria. JTrapMed Hyg I990; 93: 116-11 8. 15. Karouli M , Jaafari A, Kerabi GR. The role of diarrhoeagenic Escherichia coli in acure diarrhoeal diseases in Bandar-Abbas, Iran. J Med Microbiol 1988; 27: 71 -74. 16. Scoub BD, Greeff AS, Lecarsas G. A microbiological investigation of acure summer gastroenteriris in black South African infants. J Hyg Camb 1977; 78: 377-385. 17 . Cravioto A, Reyes RE, Trujillo F, el al. Risk of diarrhea during the firsr year of life associared with initial and subsequent colonization by specific enreropathogens. AmJ Epidemiol 1990; 131: 886-904. 18. L opez-Vidal Y, Calva 11, Trujillo A, el al. Enteroroxins and adhesins of enreroroxigenic Escherichia coli: are they risk facrors fo r acure diarrhoea in the communiry? J Infecc Dis 1990; 162: 442-447. 87 

Incidence of heat-labile enterotoxin-producing Escherichia coli detected by means of polymerase chain reaction amplification

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Incidence of heat-labile enterotoxin-producing Escherichia coli detected by means of polymerase chain reaction amplification

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