634
n e i t h e r serological, b i o c h e m i c a l n o r genetic m a r k e r s , except for t h e v e r o t o x i n genes, are consistently p r e s e n t in n o n - O 1 5 7 : H 7 v e r o t o x i n p r o d u c i n g E s c h e r i c h i a coll. T h e m e t h o d s available at p r e s e n t for detecting v e r o t o x i n genes o r v e r o t o x i n s will reliably d e t e c t all such isolates however.
References 1. Riley LW, Remis RS, Heigerson SD, McGee HB, Wells JG, Davis BR, Heberl R J, Olcott ES, Johnson LM, Hargrett NT, Blake PA, Cohen L: Hemorrhagic colitis associated with a rare Escherichia coil serotype. New England Journal of Medicine 1983, 308: 681--686. 2. Karmali MA: Infection by verocytotoxin-producing Escherichia coil Clinical Microbiology Reviews 1989, 2: 15-38. 3. Bryant HE, Athar MA, Pal CH: Risk factors for Escherichia coli O157:H7 infection in an urban community. Journal of Infectious Diseases 1989, 160: 858-864. 4. Martin DL, MacDonald KL, White KE, Soler JT, Osterholm MT: The epidemiology and clinical aspects of the hemolytic uremic syndrome in Minnesota. New England Journal of Medicine 1990, 323: 1161-1167. 5. Gasser C, Gautier E, Steck A, Siebenmann RE, Oechslin R: H~imolytisch-ur~imische Syndrome: bilaterale Nierenrindennekrosen bei akuten erworbenen h~imolytischen Anamien. Schweizerische Medizinische Wochenschrift 1955, 85: 905-909. 6. Bopp CA, Greene KD, Downes FP, Sowers EG, Wells JG, Wachsmnth IK: Unusual verotoxin-producing Escherichia coli associated with hemorrhagic colitis. Journal of Clinical Microbiology 1987, 25: 1486--1489. 7. Kleanthous H, Smilh HR, Scotland SM, Gross R J, Rowe B, Taylor CM, Milford DV: Haemolytic uraemic syndromes in the British Isles, 1985-8: association with verocytotoxin producing Escherichia coll. 2: Microbiological aspects. Archives of Disease in Childhood 1990, 65: 722-727. 8. Smith HR, Scotland SM, Chart H, Rowe B: Vero cytotoxin production and presence of VT genes in strains of Escherichia coli and Shigella. FEMS Microbiology Letters 1987, 42: 173-177. 9. Bitzan M, Karch H, Maas MG, Meyer T, Riissmann H, Aleksic S, Bockemiihl J: Clinical and genetic aspects of Shiga-like toxin production in traditional enteropathogenic Escherichia coil International Journal of Medical Microbiology 1991, 274: 496-506. 10. Tzlpori S, Karch H, Waehsmuth IK, Robins-Browne RM, O'Brien AD, Lior H, Cohen ML, Smithers J, Levine MM: Role of a 60-megadalton plasmid and Shiga-tike toxins in the pathogenesis of infection caused by enterohemorrhagic Esct, erichia coil O157:H7 in gnotobiotic piglets. Infection and Immunity 1987, 55: 3117-3125. 11. Levine MM, Xu JG, Kaper JB, Lior H, Prado V, Tall B, Nataro J, Karch H, Waehsmuth K: A DNA probe to identify enterohemorrhagic Escherichia coli of O157:H7 and other serotypes that cause hemorrhagic colitis and hemolytic uremic syndrome. Journal of Infectious Diseases 1987, 156: 175-182.
Eur. J. Clin. Microbiol. Infect. Dis.
12. Jerse AE, Yu J, Tall BT, Kaper JB: A genetic locus of enteropathogenic Escherichia coli necessary for the production of attaching and effacing lesions on tissue culture cells. Proceedings of the National Academy of Science of the USA 1990, 87: 7839-7843. 13. Ewing H: Edwards and Ewing's identification of Enterobacteriaceae. Elsevier, New York, 1986. 14. Scotland SM, Rowe B, Smith HR, Wiilshaw GA, Gross RJ: Veto cytotoxin-producing strains of Escherichia coli from children with haemolytic uraemic syndrome and their detection by specific DNA probes. Journal of Medical Microbiology 1988, 25: 237-243. 15. t01rskov F, Orskov I: Serotyping of Escherichia coll. Methods in Microbiology 1984, 14: 43-112. 16. Beutin L, Montenegro MA, Orskov I, Orskov F, Prada J, Zimmermann S, Stephan R: Close association of verotoxin (Shiga-like toxin) production with enterohemolysin production in strains of Escherichia coil Journal of Clinical Microbiology 1989, 27: 2559-2564. 17. Thompson JS, Hodge DS, Borczyk AA: Rapid biochemical test to identify verocytotoxin-positive strains of Escherichia coli serotype O157. Journal of Clinical Microbiology 1990, 28: 2165-2168. 18. Boss P, Monckton RP, Nieolet J, Bumens AP: Nachweis von Toxingenen verschiedener E. coil Pathotypen beim Schwein mit nichtradioaktiv markierten Sonden. Schweizer Archiv ftir Tierheilkunde 1992, 134: 31-37. 19. Ostroff SM, Tarr PI, Neili MA, Lewis JH, HargrettBean N, Kobayashi JM: Toxin genotypes and ptasmid profiles as determinants of systemic sequelae in Escherichia coli O157:H7 infections. Journal of Infectious Diseases 1989, 160: 994-998.
Evaluation of Two Commercial Enzyme Immunoassays for the Diagnosis of Helicobacterpylori Infection E M . A g u i r r e 1, C.Y. P a s c u a l 2, E J . M e r i n o 3., A.C. Velasco 4
Sera from 65 patients with upper gastrointestinal tract s y m p t o m s and proven Helicobacter pylori infection, and from 42 negative controls were tested with two commercial E I A s ( G A P test, BioRad; and E C P test, Biometra) and two n o n - c o m mercial EIAs, o n e performed with w h o l e sonicated cells and the other with acid extract of 1Department of Microbiology and 2Department of Allergy, "La Paz" Hospital, Madrid, Spain. 3 Department of Microbiology, Insatud General Hospital, Paseo de Santa B~irbara s/n, 42002 Soria, Spain. 4Department of Microbiology, "Prfncipe de Asturias" Hospital, Alcal,'i de Hcnares, Madrid, Spain.
Vol. 11, 1992
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Helicobacter pylori as antigen. The G A P assay showed a sensitivity of 83.1% and a specificity of 47.6 %. The ECP assay showed a sensitivity of 87.7 % and a specificity of 61.9 %. For both nonCOmmercial EIAs these figures were 87.7 % and 88.1%, respectively. Independent of the interpretive criteria established by the manufacturers, receiver operating characteristic curves were plotted for better evaluation of the four methods. Both commercial tests showed a lower probability of yielding a correct diagnosis than the non-commercial tests (p < 0.05). Although commercial EIAs are convenient for the diagnosis of Helicobatter pylorI infection, the accuracy of the two COmmercial tests evaluated in this study was lower Compared to that of the two non-commercial EIAs.
Helicobacter pylori has been recognized as a major cause of gastritis worldwide (1, 2). Histological examination and culture of gastric biopsy specimens are the methods usually employed for the diagnosis of Helicobacterpylori infection, aIthough new non-invasive techniques such as the Serological detection of anti-Helicobacter pylori antibodies could also be a good alternative for diagnosis, follow-up and epidemiological study (3). Several commercial EIA tests (4) are now available. In this study we compared the performance of two non-commercial EIAs with that of two commercial EIAs for detecting specific antibodies in sera from dyspeptic patients and from healthy individuals. Materials and Methods. Sixty-five sera tested Were from patients (9 children and 56 adults) with Upper gastrointestinal tract symptoms and proven Helicobacter pylori infection diagnosed by at least one of the following methods: isolation of the microorganism in 58 patients, histological detection of Helicobacterpylori in antral biopsies by Giemsa staining (5) in 46 patients or detection of Helicobacterpylori in smears of antral biopsies by Gram staining (6) in 39 patients. Forty-two sera were from children (aged 6 months to 3 Years) seen in our hospitals for other diseases not related to the gastrointestinal tract. Sera were tested with two commercial EIAs (GAP test, BioRad Laboratories, Italy, and ECP test, Biometra, Germany), and two non-commercial EIAs, one performed with whole sonicated cells (WSC) and the other with acid extract (AE) of Helicobacter pylori as antigen. A pilot experiment was carried out in order to choose the negative controls: 15
635
sera from patients in whom Helicobacter pylori was not detected on histological or microbiological examination (7 patients with atrophic gastritis and 8 individuals with normal mucosa in the antral biopsy) were tested with the four EIAs. The results obtained in all four EIAs were higher in samples with abnormal histological findings than in those with normal histological findings, this difference being statistically significant for the WSC and AE non-commercial EIAs and for the ECP test (p < 0.01, p < 0.03 and p < 0.01, respectively; Mann-Whitney test). For this reason, instead of using sera from Helicobacter pylori-negative patients, we chose sera from children (aged 6 months to 3 years) as negative controls. For the EIA using whole sonicated cells (WSC), five Helicobacterpylori strains isolated from different patients with upper gastrointestinal symptoms were cultured at 37 °C on a selective medium described by Dent and McNutty (6) under microaerophilic conditions (10 % CO2 and 5 % O2). After 6 to 8 days of incubation, the cells were harvested in sterile distilled water, washed twice in sterile distilled water, suspended in 0.1 M carbonated buffer (pH 9.6) and sonicated (13 jam, peak-to-peak amplitude) on ice 10 times for 30 sec each time, with 30 sec rest periods between each sonication (MSE MK2, UK). After sonication the cells were centrifuged at 23,000 x g for 20 min at 4 °C and the supernatant was retained. The protein concentration was determined by transfer to nitrocellulose membranes (7), and the suspension was stored at a final concentration of 10 lug/ ml at -20 °C until used. Optimal concentrations of antigen solution, dilutions of sera and peroxidaseconjugated goat immunoglobulins to human IgG (Nordic, Holland) were determined by checkerboard titration using a pool of sera from 15 Helicobacterpylori-positive patients and another pool from 14 children under five years of age as the positive and negative standards, respectively, One day before the assay 100 lal of the antigen suspension were pipetted into the wells of flat bottom high binding plates (Costar, USA), covered and let stand overnight at 4 °C. Prior to the assay the plates were washed three times with a washing solution composed of PBS (pH 7.2) containing 0.05 % Tween 20 (Merck-Schuchard, Germany). For the assay 100 pl of sera, diluted 1:200 in the washing solution and 2 % human albumin (Behring, Germany), were pipetted in triplicate into the coated wells and incubated at 37 °C for 90 min. The washing step was repeated after incubation, and 100 pl of a 1:5000 dilution of
636
peroxidase-conjugated goat immunoglobulins to human IgG in the washing solution plus 2 % human albumin and 10 % horse serum (Flow Laboratories, Germany) were added. The plates were incubated at 37 °C for 90 min and washed again. For the color reaction 100 lal of a solution of 30 mg of ortho-phenylenediamine (MerckSchuchard) in 50 ml of 0.1 M phosphate buffer (pH 6.0) was used to which 250 ~1 of 10 % hydrogen peroxide were added. The reaction was stopped after 15 min with 50 ~tl of 5 M sulfuric acid, and read after 15 min at 492 nm with a Titertek Multiskan Plus reader (Flow Laboratories). Sample scores were calculated as a ratio obtained by dividing the optical density (OD) of each patient serum by the OD of a pool of positive sera, which showed reactivity up to a dilution of 1:12,800, included in every run. The preparation of acid extract antigen (AE) for the second non-commercial EIA was based on the procedure described for Campylobacterjejuni by Blaser and Duncan (8). The same five Helicobacter pylori strains mentioned above were used to obtain the antigen by acid extraction. Strains were cultured and washed in the same way as described for the WSC test. The purified bacteria were suspended in 0.2 M glycine-hydrochloride buffer (pH 2.2) at a concentration of approximately 0.1 g (wet weight) of cells to 2.5 ml of buffer. The suspensions were stirred at 25 °C for 15 min and centrifuged at 11,000 x g for 15 min at 4 °C. The supernatant was retained, and the pH neutralized with 1 M sodium hydroxide. The supernatant was dialyzed with sterile distilled water for 24 hours at 4 °C through a dialysis membrane (Visking Tubing, UK) with a molecular weight cut-off of 10,000. T h e protein concentration of acid extract was determined, diluted in 0.1 M carbonated buffer (pH 9.6) to a final concentration of 15/ag/ml and stored at a temperature of -20 °C until used. Optimal concentrations of all reagents were also determined by checkerboard titration. One day before the assay the antigen preparation was diluted in 0.1 M carbonated buffer (pH 9.6) to give a final concentration of 10 lag protein/ml; 100 ~tl were added to each well and the plates covered and incubated overnight at 4 °C. The procedure for the AE assay was identical to that described for the WSC assay. The cut-off value in both the WSC and AE assay was determined using receiver operating characteristic (ROC) curves. The GAP assay is a semi-quantitative test for the detection of specific IgG antibodies to Helicobac-
Eur. J. Clin. Microbiol. Infect. Dis.
ter pylori using purified antigens (9) of this bacterium (strain not stated by the manufacturer). The results can be classified into five different categories (negative, indeterminate and three positive scores [1+, 2+, and 3+]). The ECP is a quantitative assay for determination of Helicobacter pylori specific IgG antibody levels using Helicobacter pylori purified antigen (specifications not stated by the manufacturer). The results are obtained by comparing the ODs of the samples with a standard curve of ODs constructed from known antibody concentrations included in the commercial kit. Both commercial tests were carried out according to the manufacturers' instructions. In order to evaluate within-run precision, all sera were tested twice in the commercial assays and three times in the non-commercial assay. The average of the different ODs (expressed as a percentage of the mean OD) obtained with every sera was calculated. For better evaluation of the four methods independent of the interpretive criteria for OD results established by the manufacturers, ROC curves were also determined for all four procedures by dividing the average OD of the patient sera by the average OD of positive controls.
Results and Discussion. Table 1 shows the results obtained with the four methods. The GAP test showed a sensitivity of 83.1%, a specificity of 47.6 %, a predictive positive value (PPV) of 80.6 % and a predictive negative value (PNV) of Results obtained with four EIA procedures using sera fromHelicobacterpylori-positivepatients and negative controls.
Table 1:
Patients (n = 65)
Controls (n = 42)
GAP Positive Negative Indeterminate
54 5 6
13 20 9
ECP Positive Negative
57 8
16 26
WCS Positive Negative
57 8
5 37
AE Positive Negative
57 8
5 37
Vol.
11, 1992
637
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80 %. The E C P test showed a sensitivity of 87.7 %, a specificity of 61.9 %, a PPV of 78.1% and a PNV of 76.5 %. For both non-commercial E/As these figures were 87.7 %, 88.1%, 91.9 % and 82.2 %, respectively. No statistically significant differences were found between the age of patients with negative results obtained in at least one test (mean ± SD 42 _+24 years) and the age of patients with positive results obtained in all tests (47 ± 19 years). The ability to correctly determine the presence or absence of Helicobacter pylori was significantly higher for the two non-commerciaI EIAs than for the commercial tests (p < 0.05; McNemar statistic With continuity correction). There were no statistically significant differences between the G A P and E C P tests.
The R O C plots obtained for every test are shown in Figure 1. The probability of a correct diagnosis with the G A P test was 0.872, with the ECP test 0.864, with the WCS test 0.930, and with the A E test 0.926 (Wilcoxon statistic). The WSC test performed significantly (10) better than the G A P method (z = 2.24; p < 0.05), and than the ECP method (z = 2.23; p < 0.05). The A E also performed significantly better than the G A P test (z = 2.01; p < 0.05), and than the E C P test (z = 2.32; p < 0.05), but no statistically significant differences were found between the two commercial methods. The intra-assay variability was 6.3 % for the WSC test, 5.7 % for the A E test, 6.9 % for the ECP test, and 9.8 % for the G A P test. The intra-assay variability of the G A P test was significantly
638
Eur. J. Clin. Microbiol. Infect. Dis.
higher than that of the other three methods (p < 0.02; paired-sample Student's t-test).
100"
A "gold standard" for the evaluation of the methods currently available for the diagnosis of Heticobacterpylori infection has not yet been es, tablished. Some authors suggest that histological examination of multiple biopsies is the most accurate method for detecting the infection (11). In our patients, both histological examination and Gram staining and culture of biopsies were performed.
,--90'
The scores in seven sera from patients with atrophic gastritis demonstrated histologically, in whom Helicobacter pylori could not be found, were significantly higher than in those from patients with normal histological findings, strongly suggesting the possibility of false negative culture/histology results in some patients. Due to patchy distribution of Helicobacterpylori in the gastric mucosa, there is a potential for error in the histological or microbiological assessment of this infection (11, 12). Furthermore, it is obvious that the accuracy of some diagnostic methods such as histological examination and culture may vary when performed by different investigators. This indicates that the accuracy of available methods should be assured before they are considered as a valid reference method for the evaluation of other new techniques. Although children under four years old are not very representative of the clinical population in whom the tests were applied, there is an almost negligible prevalence of Helicobacterpylori infection at this very young age in the Western world, as stated in previous reports (13, 14). Serological tests may have several different applications (screening, confirmation and follow-up of the infection, epidemiological studies, etc.) and can be used for the study of populations with very different prevalences of infection. For this reason we evaluated the performance of these four EIA tests by comparing the ROC curves (15) of all tests, in order to obtain a more comprehensive evaluation. The employment of ROC curves allows an overall evaluation of the assays, overcoming possible errors in determination of the cut-off value due to inappropriate selection of the reference population (4). ROC curves are generated by plotting the corresponding true-positive rate (sensitivity) and false-positive rate (100 % minus the specificity) obtained for each serum sample when its particular result is considered as the hypothetical cut-off of the test, and are useful for the evaluation of diagnostic accuracy not only for
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the performance of the four enzyme immunoassays evalutatcd. GAP 12. ECP tl,; WCS x; AE i. a specific cut-off value, but also for every possible decision level (16). Figure 2 shows that GAP and EPC tests render a very similar performance, and that the non-commercial tests have a higher specificity (a lower false-positive rate) than commercial tests at any useful sensitivity level (true-positive rate). Contradictory conclusions on the performance of the GAP test have been reported previously (4, 9). Our results agree with those of van den Oever et al. (4). Following the manufacturer's instructions, 15 sera evaluated in the GAP assay showed an indeterminate result. In 11 of these sera tests were repeated, but 6 remained indeterminate• The manufacturer alerts to the possibility that some healthy individuals over 50 years of age may exhibit false positive 1+ results. In our study six patients above 50 years of age showed this result, increasing the rate of indeterminate results. The GAP assay thus showed a substantial proportion of results with little usefulness as indicators of infection. This test also showed a higher intra-assay variability than the ECP test, which could explain why the manufacturer of the former recommends testing every sample in duplicate. The manufacturer of the ECP EIA does not provide the blocking solution or hydrogen peroxide needed for carrying out the procedure, which is inconvenient in a commercial test. Providing that the whole plate is used in one single run, the cost is about $ 7 per serum sample for the ECP and about $14 for the GAP, which is
Vo1.11,1992
USually p e r f o r m e d in duplicate following the r e c o m m e n d a t i o n of the manufacturer. In conclusion, E I A s are useful, reasonably fast, non-invasive m e t h o d s for the diagnosis and epidemiological study of Helicobacter pylori infection. C o m m e r c i a l E I A s m a k e these procedures available to a wide n u m b e r of investigators, although the cost p e r s a m p l e may still be too high for routine purposes. However, the p e r f o r m a n c e of the two c o m m e r c i a l E I A s evaluated is still far below that of the two n o n - c o m m e r c i a l E I A s and the standard normally required for use in the clinical laboratory.
References 1. Blaser MJ: Epidemiology and pathophysiology of Campylobacterpylori infections. Reviews of Infectious Diseases 1990, 12, Supplement 1: 99-106. 2. Tytgat GNJ: Campylobacter pylori: epidemio|ogica[ considerations. Scandinavian Journal of Gastroenterology 1989, 24, Supplement 160: 1-2. 3. Wyatt J J, Rathhone BJ: The role of serology in the diagnosis of Campylobacter pylori infection. Scandinavian Journal of Gastroenterology 1989, 24, Supplement 160: 27-34. 4. van den Oever HLA, Loffeld RJLF, Stobberingh EE: Usefulness of a new serological test (Bio-Rad) to diagnose Helicobacter pylori-associated gastritis. Journal of Clinical Microbiology 1991, 29: 283-286. 5. Gray SF, Wyatt Jl, Rathbone B J: Simplified techniques for identifying gastric Campylobacter pylori on tissue sections. Journal of Clinical Pathology 1986, 39: 12791280. 6. Dent JC, MeNutty CAM: Evaluation of a new selective medium for Campylobacterpylori. European Journal of Clinical Microbiology & Infectious Diseases 1988, 7: 555-558. 7. Sehaffner W, Weissmann C: A rapid, sensitive and specific method for the determination of protein in dilute solution. Analytical Biochemistry 1973, 56: 502514. 8. Blaser MJ, Duncan D J: Human serum antibody response to Campylobacter ]e/uni infection as measured in an enzyme-linked immunosorbent assay. Infection and Immunity 1984, 44: 292-298. 9. Crabtree JE, Shalicross TM, Heatley RV, Wyatt Jl: Evaluation of a commercial EIA for serodiagnosis of Helicobacter pylori infection. Journal of Clinical Pathology 1991, 44: 326-328. 10. Hanley JA, McNeil B J: A method of comparing the areas under receiver operating characteristic curves derived from the same cases. Radiology 1983,148: 839843. 11. Barthel JS, Everett ED: Diagnosis of Campylobacter pytori infections: the "gold standard" and the alternatives. Reviews of Infectious Diseases 1990, 12, Supplement 1: 107-114. 12. Morris A, All MR, Brown P, Lane M, Patton K: Campylobacter pylori infection in biopsy specimens of gastric antrum: laboratory diagnosis and estimation of sampling error. Journal of Clinical Pathology I989, 42: 727-732.
639
13. Megraud F, Brassens-Rabbe MP, Denis F, Belbouri A, Hoa DQ: Seroepidemiology of Campylobacter pylori infection in various populations. Journal of Clinical Microbiology 1989, 27: 1870-1873. 14. Drumm B: Helicobacter pylori. Archives of Disease in Childhood 1990, 55: 1278--1282. 15. Robertson EA, Zweig MH, van Steirteghem AC: Evaluating the clinical efficacy of laboratory tests. American Journal of Clinical Pathology 1983, 19: 7887. 16. Hanley JA, McNeil BJ: The meaning and use of the area under a receiver operating characteristic curve. Radiology 1982, 143: 29-36.
Sensitivity of Seven Commercial Assays in the Detection of Hepatitis B Virus Type 2-Like Infection E Le6n*, J.A. L6pez, J.M. Echevarrfa
Two hundred serum samples taken from patients with hepatitis B virus type 2-like infections were tested with seven commercial enzyme immunoassay methods for detection of HBsAg. Positive results were obtained with all methods in some samples, the rate of detection of HBsAg ranging from 9 % to 90 % for the different methods. A direct correlation was found between the analytical sensitivity of a method and its ability to yield positive results. It is suggested that these findings should be taken into consideration when selecting HBsAg detection methods in blood banks in order to avoid transmission of the HBV2 agent to recipients of blood or blood products.
T h e presence of isolated reactivity for hepatitis B surface antigen ( H B s A g ) in h u m a n serum has been interpreted by several authors as resulting f r o m infection with a hepatitis B virus ( H B V ) variant strain (1), which has b e e n designated H B V type 2 ( H B V 2 ) (2). Although data have been published suggesting that the H B V 2 agent is transmitted mainly by the oral route a m o n g the general population (3), characterization of HBV2-1ike infections a m o n g blood donors (4, 5) and recipients of b l o o d transfusion (4), as well as Scrvicio de Microbiologfa Diagn6stica, Centro Nacional de Microbiologfa, Virologia e lnmunologfa Sanitarias, Instituto de Salud Carlos III, 28220 Majadahonda, Madrid, Spain.
n e i t h e r serological, b i o c h e m i c a l n o r genetic m a r k e r s , except for t h e v e r o t o x i n genes, are consistently p r e s e n t in n o n - O 1 5 7 : H 7 v e r o t o x i n p r o d u c i n g E s c h e r i c h i a coll. T h e m e t h o d s available at p r e s e n t for detecting v e r o t o x i n genes o r v e r o t o x i n s will reliably d e t e c t all such isolates however.
References 1. Riley LW, Remis RS, Heigerson SD, McGee HB, Wells JG, Davis BR, Heberl R J, Olcott ES, Johnson LM, Hargrett NT, Blake PA, Cohen L: Hemorrhagic colitis associated with a rare Escherichia coil serotype. New England Journal of Medicine 1983, 308: 681--686. 2. Karmali MA: Infection by verocytotoxin-producing Escherichia coil Clinical Microbiology Reviews 1989, 2: 15-38. 3. Bryant HE, Athar MA, Pal CH: Risk factors for Escherichia coli O157:H7 infection in an urban community. Journal of Infectious Diseases 1989, 160: 858-864. 4. Martin DL, MacDonald KL, White KE, Soler JT, Osterholm MT: The epidemiology and clinical aspects of the hemolytic uremic syndrome in Minnesota. New England Journal of Medicine 1990, 323: 1161-1167. 5. Gasser C, Gautier E, Steck A, Siebenmann RE, Oechslin R: H~imolytisch-ur~imische Syndrome: bilaterale Nierenrindennekrosen bei akuten erworbenen h~imolytischen Anamien. Schweizerische Medizinische Wochenschrift 1955, 85: 905-909. 6. Bopp CA, Greene KD, Downes FP, Sowers EG, Wells JG, Wachsmnth IK: Unusual verotoxin-producing Escherichia coli associated with hemorrhagic colitis. Journal of Clinical Microbiology 1987, 25: 1486--1489. 7. Kleanthous H, Smilh HR, Scotland SM, Gross R J, Rowe B, Taylor CM, Milford DV: Haemolytic uraemic syndromes in the British Isles, 1985-8: association with verocytotoxin producing Escherichia coll. 2: Microbiological aspects. Archives of Disease in Childhood 1990, 65: 722-727. 8. Smith HR, Scotland SM, Chart H, Rowe B: Vero cytotoxin production and presence of VT genes in strains of Escherichia coli and Shigella. FEMS Microbiology Letters 1987, 42: 173-177. 9. Bitzan M, Karch H, Maas MG, Meyer T, Riissmann H, Aleksic S, Bockemiihl J: Clinical and genetic aspects of Shiga-like toxin production in traditional enteropathogenic Escherichia coil International Journal of Medical Microbiology 1991, 274: 496-506. 10. Tzlpori S, Karch H, Waehsmuth IK, Robins-Browne RM, O'Brien AD, Lior H, Cohen ML, Smithers J, Levine MM: Role of a 60-megadalton plasmid and Shiga-tike toxins in the pathogenesis of infection caused by enterohemorrhagic Esct, erichia coil O157:H7 in gnotobiotic piglets. Infection and Immunity 1987, 55: 3117-3125. 11. Levine MM, Xu JG, Kaper JB, Lior H, Prado V, Tall B, Nataro J, Karch H, Waehsmuth K: A DNA probe to identify enterohemorrhagic Escherichia coli of O157:H7 and other serotypes that cause hemorrhagic colitis and hemolytic uremic syndrome. Journal of Infectious Diseases 1987, 156: 175-182.
Eur. J. Clin. Microbiol. Infect. Dis.
12. Jerse AE, Yu J, Tall BT, Kaper JB: A genetic locus of enteropathogenic Escherichia coli necessary for the production of attaching and effacing lesions on tissue culture cells. Proceedings of the National Academy of Science of the USA 1990, 87: 7839-7843. 13. Ewing H: Edwards and Ewing's identification of Enterobacteriaceae. Elsevier, New York, 1986. 14. Scotland SM, Rowe B, Smith HR, Wiilshaw GA, Gross RJ: Veto cytotoxin-producing strains of Escherichia coli from children with haemolytic uraemic syndrome and their detection by specific DNA probes. Journal of Medical Microbiology 1988, 25: 237-243. 15. t01rskov F, Orskov I: Serotyping of Escherichia coll. Methods in Microbiology 1984, 14: 43-112. 16. Beutin L, Montenegro MA, Orskov I, Orskov F, Prada J, Zimmermann S, Stephan R: Close association of verotoxin (Shiga-like toxin) production with enterohemolysin production in strains of Escherichia coil Journal of Clinical Microbiology 1989, 27: 2559-2564. 17. Thompson JS, Hodge DS, Borczyk AA: Rapid biochemical test to identify verocytotoxin-positive strains of Escherichia coli serotype O157. Journal of Clinical Microbiology 1990, 28: 2165-2168. 18. Boss P, Monckton RP, Nieolet J, Bumens AP: Nachweis von Toxingenen verschiedener E. coil Pathotypen beim Schwein mit nichtradioaktiv markierten Sonden. Schweizer Archiv ftir Tierheilkunde 1992, 134: 31-37. 19. Ostroff SM, Tarr PI, Neili MA, Lewis JH, HargrettBean N, Kobayashi JM: Toxin genotypes and ptasmid profiles as determinants of systemic sequelae in Escherichia coli O157:H7 infections. Journal of Infectious Diseases 1989, 160: 994-998.
Evaluation of Two Commercial Enzyme Immunoassays for the Diagnosis of Helicobacterpylori Infection E M . A g u i r r e 1, C.Y. P a s c u a l 2, E J . M e r i n o 3., A.C. Velasco 4
Sera from 65 patients with upper gastrointestinal tract s y m p t o m s and proven Helicobacter pylori infection, and from 42 negative controls were tested with two commercial E I A s ( G A P test, BioRad; and E C P test, Biometra) and two n o n - c o m mercial EIAs, o n e performed with w h o l e sonicated cells and the other with acid extract of 1Department of Microbiology and 2Department of Allergy, "La Paz" Hospital, Madrid, Spain. 3 Department of Microbiology, Insatud General Hospital, Paseo de Santa B~irbara s/n, 42002 Soria, Spain. 4Department of Microbiology, "Prfncipe de Asturias" Hospital, Alcal,'i de Hcnares, Madrid, Spain.
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Helicobacter pylori as antigen. The G A P assay showed a sensitivity of 83.1% and a specificity of 47.6 %. The ECP assay showed a sensitivity of 87.7 % and a specificity of 61.9 %. For both nonCOmmercial EIAs these figures were 87.7 % and 88.1%, respectively. Independent of the interpretive criteria established by the manufacturers, receiver operating characteristic curves were plotted for better evaluation of the four methods. Both commercial tests showed a lower probability of yielding a correct diagnosis than the non-commercial tests (p < 0.05). Although commercial EIAs are convenient for the diagnosis of Helicobatter pylorI infection, the accuracy of the two COmmercial tests evaluated in this study was lower Compared to that of the two non-commercial EIAs.
Helicobacter pylori has been recognized as a major cause of gastritis worldwide (1, 2). Histological examination and culture of gastric biopsy specimens are the methods usually employed for the diagnosis of Helicobacterpylori infection, aIthough new non-invasive techniques such as the Serological detection of anti-Helicobacter pylori antibodies could also be a good alternative for diagnosis, follow-up and epidemiological study (3). Several commercial EIA tests (4) are now available. In this study we compared the performance of two non-commercial EIAs with that of two commercial EIAs for detecting specific antibodies in sera from dyspeptic patients and from healthy individuals. Materials and Methods. Sixty-five sera tested Were from patients (9 children and 56 adults) with Upper gastrointestinal tract symptoms and proven Helicobacter pylori infection diagnosed by at least one of the following methods: isolation of the microorganism in 58 patients, histological detection of Helicobacterpylori in antral biopsies by Giemsa staining (5) in 46 patients or detection of Helicobacterpylori in smears of antral biopsies by Gram staining (6) in 39 patients. Forty-two sera were from children (aged 6 months to 3 Years) seen in our hospitals for other diseases not related to the gastrointestinal tract. Sera were tested with two commercial EIAs (GAP test, BioRad Laboratories, Italy, and ECP test, Biometra, Germany), and two non-commercial EIAs, one performed with whole sonicated cells (WSC) and the other with acid extract (AE) of Helicobacter pylori as antigen. A pilot experiment was carried out in order to choose the negative controls: 15
635
sera from patients in whom Helicobacter pylori was not detected on histological or microbiological examination (7 patients with atrophic gastritis and 8 individuals with normal mucosa in the antral biopsy) were tested with the four EIAs. The results obtained in all four EIAs were higher in samples with abnormal histological findings than in those with normal histological findings, this difference being statistically significant for the WSC and AE non-commercial EIAs and for the ECP test (p < 0.01, p < 0.03 and p < 0.01, respectively; Mann-Whitney test). For this reason, instead of using sera from Helicobacter pylori-negative patients, we chose sera from children (aged 6 months to 3 years) as negative controls. For the EIA using whole sonicated cells (WSC), five Helicobacterpylori strains isolated from different patients with upper gastrointestinal symptoms were cultured at 37 °C on a selective medium described by Dent and McNutty (6) under microaerophilic conditions (10 % CO2 and 5 % O2). After 6 to 8 days of incubation, the cells were harvested in sterile distilled water, washed twice in sterile distilled water, suspended in 0.1 M carbonated buffer (pH 9.6) and sonicated (13 jam, peak-to-peak amplitude) on ice 10 times for 30 sec each time, with 30 sec rest periods between each sonication (MSE MK2, UK). After sonication the cells were centrifuged at 23,000 x g for 20 min at 4 °C and the supernatant was retained. The protein concentration was determined by transfer to nitrocellulose membranes (7), and the suspension was stored at a final concentration of 10 lug/ ml at -20 °C until used. Optimal concentrations of antigen solution, dilutions of sera and peroxidaseconjugated goat immunoglobulins to human IgG (Nordic, Holland) were determined by checkerboard titration using a pool of sera from 15 Helicobacterpylori-positive patients and another pool from 14 children under five years of age as the positive and negative standards, respectively, One day before the assay 100 lal of the antigen suspension were pipetted into the wells of flat bottom high binding plates (Costar, USA), covered and let stand overnight at 4 °C. Prior to the assay the plates were washed three times with a washing solution composed of PBS (pH 7.2) containing 0.05 % Tween 20 (Merck-Schuchard, Germany). For the assay 100 pl of sera, diluted 1:200 in the washing solution and 2 % human albumin (Behring, Germany), were pipetted in triplicate into the coated wells and incubated at 37 °C for 90 min. The washing step was repeated after incubation, and 100 pl of a 1:5000 dilution of
636
peroxidase-conjugated goat immunoglobulins to human IgG in the washing solution plus 2 % human albumin and 10 % horse serum (Flow Laboratories, Germany) were added. The plates were incubated at 37 °C for 90 min and washed again. For the color reaction 100 lal of a solution of 30 mg of ortho-phenylenediamine (MerckSchuchard) in 50 ml of 0.1 M phosphate buffer (pH 6.0) was used to which 250 ~1 of 10 % hydrogen peroxide were added. The reaction was stopped after 15 min with 50 ~tl of 5 M sulfuric acid, and read after 15 min at 492 nm with a Titertek Multiskan Plus reader (Flow Laboratories). Sample scores were calculated as a ratio obtained by dividing the optical density (OD) of each patient serum by the OD of a pool of positive sera, which showed reactivity up to a dilution of 1:12,800, included in every run. The preparation of acid extract antigen (AE) for the second non-commercial EIA was based on the procedure described for Campylobacterjejuni by Blaser and Duncan (8). The same five Helicobacter pylori strains mentioned above were used to obtain the antigen by acid extraction. Strains were cultured and washed in the same way as described for the WSC test. The purified bacteria were suspended in 0.2 M glycine-hydrochloride buffer (pH 2.2) at a concentration of approximately 0.1 g (wet weight) of cells to 2.5 ml of buffer. The suspensions were stirred at 25 °C for 15 min and centrifuged at 11,000 x g for 15 min at 4 °C. The supernatant was retained, and the pH neutralized with 1 M sodium hydroxide. The supernatant was dialyzed with sterile distilled water for 24 hours at 4 °C through a dialysis membrane (Visking Tubing, UK) with a molecular weight cut-off of 10,000. T h e protein concentration of acid extract was determined, diluted in 0.1 M carbonated buffer (pH 9.6) to a final concentration of 15/ag/ml and stored at a temperature of -20 °C until used. Optimal concentrations of all reagents were also determined by checkerboard titration. One day before the assay the antigen preparation was diluted in 0.1 M carbonated buffer (pH 9.6) to give a final concentration of 10 lag protein/ml; 100 ~tl were added to each well and the plates covered and incubated overnight at 4 °C. The procedure for the AE assay was identical to that described for the WSC assay. The cut-off value in both the WSC and AE assay was determined using receiver operating characteristic (ROC) curves. The GAP assay is a semi-quantitative test for the detection of specific IgG antibodies to Helicobac-
Eur. J. Clin. Microbiol. Infect. Dis.
ter pylori using purified antigens (9) of this bacterium (strain not stated by the manufacturer). The results can be classified into five different categories (negative, indeterminate and three positive scores [1+, 2+, and 3+]). The ECP is a quantitative assay for determination of Helicobacter pylori specific IgG antibody levels using Helicobacter pylori purified antigen (specifications not stated by the manufacturer). The results are obtained by comparing the ODs of the samples with a standard curve of ODs constructed from known antibody concentrations included in the commercial kit. Both commercial tests were carried out according to the manufacturers' instructions. In order to evaluate within-run precision, all sera were tested twice in the commercial assays and three times in the non-commercial assay. The average of the different ODs (expressed as a percentage of the mean OD) obtained with every sera was calculated. For better evaluation of the four methods independent of the interpretive criteria for OD results established by the manufacturers, ROC curves were also determined for all four procedures by dividing the average OD of the patient sera by the average OD of positive controls.
Results and Discussion. Table 1 shows the results obtained with the four methods. The GAP test showed a sensitivity of 83.1%, a specificity of 47.6 %, a predictive positive value (PPV) of 80.6 % and a predictive negative value (PNV) of Results obtained with four EIA procedures using sera fromHelicobacterpylori-positivepatients and negative controls.
Table 1:
Patients (n = 65)
Controls (n = 42)
GAP Positive Negative Indeterminate
54 5 6
13 20 9
ECP Positive Negative
57 8
16 26
WCS Positive Negative
57 8
5 37
AE Positive Negative
57 8
5 37
Vol.
11, 1992
637
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80 %. The E C P test showed a sensitivity of 87.7 %, a specificity of 61.9 %, a PPV of 78.1% and a PNV of 76.5 %. For both non-commercial E/As these figures were 87.7 %, 88.1%, 91.9 % and 82.2 %, respectively. No statistically significant differences were found between the age of patients with negative results obtained in at least one test (mean ± SD 42 _+24 years) and the age of patients with positive results obtained in all tests (47 ± 19 years). The ability to correctly determine the presence or absence of Helicobacter pylori was significantly higher for the two non-commerciaI EIAs than for the commercial tests (p < 0.05; McNemar statistic With continuity correction). There were no statistically significant differences between the G A P and E C P tests.
The R O C plots obtained for every test are shown in Figure 1. The probability of a correct diagnosis with the G A P test was 0.872, with the ECP test 0.864, with the WCS test 0.930, and with the A E test 0.926 (Wilcoxon statistic). The WSC test performed significantly (10) better than the G A P method (z = 2.24; p < 0.05), and than the ECP method (z = 2.23; p < 0.05). The A E also performed significantly better than the G A P test (z = 2.01; p < 0.05), and than the E C P test (z = 2.32; p < 0.05), but no statistically significant differences were found between the two commercial methods. The intra-assay variability was 6.3 % for the WSC test, 5.7 % for the A E test, 6.9 % for the ECP test, and 9.8 % for the G A P test. The intra-assay variability of the G A P test was significantly
638
Eur. J. Clin. Microbiol. Infect. Dis.
higher than that of the other three methods (p < 0.02; paired-sample Student's t-test).
100"
A "gold standard" for the evaluation of the methods currently available for the diagnosis of Heticobacterpylori infection has not yet been es, tablished. Some authors suggest that histological examination of multiple biopsies is the most accurate method for detecting the infection (11). In our patients, both histological examination and Gram staining and culture of biopsies were performed.
,--90'
The scores in seven sera from patients with atrophic gastritis demonstrated histologically, in whom Helicobacter pylori could not be found, were significantly higher than in those from patients with normal histological findings, strongly suggesting the possibility of false negative culture/histology results in some patients. Due to patchy distribution of Helicobacterpylori in the gastric mucosa, there is a potential for error in the histological or microbiological assessment of this infection (11, 12). Furthermore, it is obvious that the accuracy of some diagnostic methods such as histological examination and culture may vary when performed by different investigators. This indicates that the accuracy of available methods should be assured before they are considered as a valid reference method for the evaluation of other new techniques. Although children under four years old are not very representative of the clinical population in whom the tests were applied, there is an almost negligible prevalence of Helicobacterpylori infection at this very young age in the Western world, as stated in previous reports (13, 14). Serological tests may have several different applications (screening, confirmation and follow-up of the infection, epidemiological studies, etc.) and can be used for the study of populations with very different prevalences of infection. For this reason we evaluated the performance of these four EIA tests by comparing the ROC curves (15) of all tests, in order to obtain a more comprehensive evaluation. The employment of ROC curves allows an overall evaluation of the assays, overcoming possible errors in determination of the cut-off value due to inappropriate selection of the reference population (4). ROC curves are generated by plotting the corresponding true-positive rate (sensitivity) and false-positive rate (100 % minus the specificity) obtained for each serum sample when its particular result is considered as the hypothetical cut-off of the test, and are useful for the evaluation of diagnostic accuracy not only for
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the performance of the four enzyme immunoassays evalutatcd. GAP 12. ECP tl,; WCS x; AE i. a specific cut-off value, but also for every possible decision level (16). Figure 2 shows that GAP and EPC tests render a very similar performance, and that the non-commercial tests have a higher specificity (a lower false-positive rate) than commercial tests at any useful sensitivity level (true-positive rate). Contradictory conclusions on the performance of the GAP test have been reported previously (4, 9). Our results agree with those of van den Oever et al. (4). Following the manufacturer's instructions, 15 sera evaluated in the GAP assay showed an indeterminate result. In 11 of these sera tests were repeated, but 6 remained indeterminate• The manufacturer alerts to the possibility that some healthy individuals over 50 years of age may exhibit false positive 1+ results. In our study six patients above 50 years of age showed this result, increasing the rate of indeterminate results. The GAP assay thus showed a substantial proportion of results with little usefulness as indicators of infection. This test also showed a higher intra-assay variability than the ECP test, which could explain why the manufacturer of the former recommends testing every sample in duplicate. The manufacturer of the ECP EIA does not provide the blocking solution or hydrogen peroxide needed for carrying out the procedure, which is inconvenient in a commercial test. Providing that the whole plate is used in one single run, the cost is about $ 7 per serum sample for the ECP and about $14 for the GAP, which is
Vo1.11,1992
USually p e r f o r m e d in duplicate following the r e c o m m e n d a t i o n of the manufacturer. In conclusion, E I A s are useful, reasonably fast, non-invasive m e t h o d s for the diagnosis and epidemiological study of Helicobacter pylori infection. C o m m e r c i a l E I A s m a k e these procedures available to a wide n u m b e r of investigators, although the cost p e r s a m p l e may still be too high for routine purposes. However, the p e r f o r m a n c e of the two c o m m e r c i a l E I A s evaluated is still far below that of the two n o n - c o m m e r c i a l E I A s and the standard normally required for use in the clinical laboratory.
References 1. Blaser MJ: Epidemiology and pathophysiology of Campylobacterpylori infections. Reviews of Infectious Diseases 1990, 12, Supplement 1: 99-106. 2. Tytgat GNJ: Campylobacter pylori: epidemio|ogica[ considerations. Scandinavian Journal of Gastroenterology 1989, 24, Supplement 160: 1-2. 3. Wyatt J J, Rathhone BJ: The role of serology in the diagnosis of Campylobacter pylori infection. Scandinavian Journal of Gastroenterology 1989, 24, Supplement 160: 27-34. 4. van den Oever HLA, Loffeld RJLF, Stobberingh EE: Usefulness of a new serological test (Bio-Rad) to diagnose Helicobacter pylori-associated gastritis. Journal of Clinical Microbiology 1991, 29: 283-286. 5. Gray SF, Wyatt Jl, Rathbone B J: Simplified techniques for identifying gastric Campylobacter pylori on tissue sections. Journal of Clinical Pathology 1986, 39: 12791280. 6. Dent JC, MeNutty CAM: Evaluation of a new selective medium for Campylobacterpylori. European Journal of Clinical Microbiology & Infectious Diseases 1988, 7: 555-558. 7. Sehaffner W, Weissmann C: A rapid, sensitive and specific method for the determination of protein in dilute solution. Analytical Biochemistry 1973, 56: 502514. 8. Blaser MJ, Duncan D J: Human serum antibody response to Campylobacter ]e/uni infection as measured in an enzyme-linked immunosorbent assay. Infection and Immunity 1984, 44: 292-298. 9. Crabtree JE, Shalicross TM, Heatley RV, Wyatt Jl: Evaluation of a commercial EIA for serodiagnosis of Helicobacter pylori infection. Journal of Clinical Pathology 1991, 44: 326-328. 10. Hanley JA, McNeil B J: A method of comparing the areas under receiver operating characteristic curves derived from the same cases. Radiology 1983,148: 839843. 11. Barthel JS, Everett ED: Diagnosis of Campylobacter pytori infections: the "gold standard" and the alternatives. Reviews of Infectious Diseases 1990, 12, Supplement 1: 107-114. 12. Morris A, All MR, Brown P, Lane M, Patton K: Campylobacter pylori infection in biopsy specimens of gastric antrum: laboratory diagnosis and estimation of sampling error. Journal of Clinical Pathology I989, 42: 727-732.
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13. Megraud F, Brassens-Rabbe MP, Denis F, Belbouri A, Hoa DQ: Seroepidemiology of Campylobacter pylori infection in various populations. Journal of Clinical Microbiology 1989, 27: 1870-1873. 14. Drumm B: Helicobacter pylori. Archives of Disease in Childhood 1990, 55: 1278--1282. 15. Robertson EA, Zweig MH, van Steirteghem AC: Evaluating the clinical efficacy of laboratory tests. American Journal of Clinical Pathology 1983, 19: 7887. 16. Hanley JA, McNeil BJ: The meaning and use of the area under a receiver operating characteristic curve. Radiology 1982, 143: 29-36.
Sensitivity of Seven Commercial Assays in the Detection of Hepatitis B Virus Type 2-Like Infection E Le6n*, J.A. L6pez, J.M. Echevarrfa
Two hundred serum samples taken from patients with hepatitis B virus type 2-like infections were tested with seven commercial enzyme immunoassay methods for detection of HBsAg. Positive results were obtained with all methods in some samples, the rate of detection of HBsAg ranging from 9 % to 90 % for the different methods. A direct correlation was found between the analytical sensitivity of a method and its ability to yield positive results. It is suggested that these findings should be taken into consideration when selecting HBsAg detection methods in blood banks in order to avoid transmission of the HBV2 agent to recipients of blood or blood products.
T h e presence of isolated reactivity for hepatitis B surface antigen ( H B s A g ) in h u m a n serum has been interpreted by several authors as resulting f r o m infection with a hepatitis B virus ( H B V ) variant strain (1), which has b e e n designated H B V type 2 ( H B V 2 ) (2). Although data have been published suggesting that the H B V 2 agent is transmitted mainly by the oral route a m o n g the general population (3), characterization of HBV2-1ike infections a m o n g blood donors (4, 5) and recipients of b l o o d transfusion (4), as well as Scrvicio de Microbiologfa Diagn6stica, Centro Nacional de Microbiologfa, Virologia e lnmunologfa Sanitarias, Instituto de Salud Carlos III, 28220 Majadahonda, Madrid, Spain.
