Value of Serology as a Noninvasive Method for Evaluating the Efficacy of Treatment of Helicobacter pylori Infection

Transcription

1 1038 Value of Serology as a Noninvasive Method for Evaluating the Efficacy of Treatment of Helicobacter pylori Infection Guillermo I. Pérez-Pérez, Alan F. Cutler, and Martin J. Blaser From the Division of Infectious Diseases, Vanderbilt University School of Medicine, and the Infectious Disease Section, Department of Veterans Affairs Medical Center, Nashville, Tennessee, and the Section of Gastroenterology, Sinai Hospital, Detroit, Michigan The systemic humoral response to Helicobacter pylori was studied in 86 infected adult patients before antimicrobial therapy and at intervals following therapy. Endoscopy with collection of biopsy specimens was performed immediately before treatment; a 13 C-labeled urea breath test was performed, and blood specimens were collected before treatment and at 1, 3, 6, 9, and 12 months after treatment. Serum samples from three patient groups (eradication success [n Å 50], eradication failure [n Å 16], and no treatment [n Å 20]) were assayed for IgA and IgG antibodies to H. pylori by enzyme-linked immunosorbent assay. Levels of antibody to H. pylori before treatment were similar in all three groups. As expected, the no treatment and eradication failure groups had no significant changes in antibody levels during the study period. In contrast, for the eradication success group, the specific IgA and IgG antibody levels decreased progressively and significantly. We conclude that serology is a potentially useful way to monitor the success of treatment of H. pylori infection without using invasive or more expensive methods. clinically useful for other chronic infectious diseases, including syphilis, brucellosis, and coccidioidomycosis, although the de- cline in antibody level is slow (often requiring 6 12 months for optimal accuracy). The aim of this study was to use a highly sensitive and specific serological assay to monitor the antibody response before and after antimicrobial therapy for eradication of H. pylori infection. We sought to determine the accuracy of serology for determining bacterial eradication in relation to time after therapy. It now is well established that gastric colonization with Helicobacter pylori induces chronic gastritis, which may lead to peptic ulcer disease [1 3]. Once acquired, H. pylori infection persists in the gastric mucosa for years or decades in the absence of treatment [4, 5], thus eliciting a stable systemic antibody response [6]. Eradication of H. pylori infection results in reduction of gastritis and in diminished recurrence of duodenal ulcers [7]. Until now, the monitoring of treatment usually involved either repeated gastroscopy or the use of urea breath tests (UBTs) [8]. Endoscopy is invasive and relatively expensive [9]. Although the UBT is noninvasive, less expensive, and quite accurate, it requires special equipment, trained personnel, and relative expense (especially for developing countries [8]); may involve radioisotopes; and is still relatively unavailable in most geographic areas. Moreover, for both techniques, sensi- tivity for detecting treatment failure is not 100% [10]. Serology has proven to be an accurate noninvasive test for detecting H. pylori infection [4, 11, 12]. Several studies have suggested that a decline in antibody levels might be useful for evaluation of eradication of H. pylori infection [13 15]. Previous studies [16 18] showing declining IgG antibody levels after confirmed eradication did not include an untreated control group. Serological assessment of treatment efficacy is This article is part of a series of papers presented at the Second International Workshop on Helicobacter pylori Infections in the Developing World held in Lima, Peru, in January Financial support: This study was supported in part by the Iris and Homer Akers Fellowship in Infectious Diseases. Reprints or correspondence: Dr. Guillermo I. Pérez-Pérez, Vanderbilt University School of Medicine, Division of Infectious Diseases, A-3310 Medical Center North, st Avenue South, Nashville, Tennessee Clinical Infectious Diseases 1997;25: This article is in the public domain. Materials and Methods Patients and specimens. Eighty-six patients with H. pylori infection who received care in the Division of Gastroenterology at Henry Ford Hospital (Detroit) between 1990 and 1992 were included in this study. Of these patients, 66 (76.7%) had partici- pated in an investigation evaluating specific treatments for the eradication of H. pylori infection. For 63 of the 66 patients, the treatment regimen was based on triple-antimicrobial therapy (2- week course of bismuth subsalicylate [525 mg q.i.d.], tetracy- cline [500 mg q.i.d.], and metronidazole [500 mg t.i.d.]), and the three other patients were treated with only colloidal bismuth subcitrate (Denol, Gist-Brocades, West Byfleet, England). There were 20 patients involved in other protocols who were not treated for their H. pylori infection. For all patients, baseline studies included endoscopy with collection of two antral biopsy specimens for histological examination with Warthin-Starry silver stain and one biopsy specimen for Campylobacter-like organism (CLO) (rapid urease) testing as previously reported [19]. Serum samples for examination of antibodies to H. pylori and for the 13 C-labeled UBT (Cambridge Isotope Laboratories, Boston) were obtained before treatment, 1 month after the

2 CID 1997;25 (November) Serological Evaluation of Treatment of H. pylori Infection 1039 termination of treatment, and then at 3-month intervals. One, Table 1. Sensitivity of diagnostic methods in the pretreatment eval- two, three, or four follow-up specimens were obtained from uation of 86 Helicobacter pylori infected patients. 100%, 84%, 50%, and 27% of the patients, respectively. No. of No. of Study definitions. For all patients, confirmation of H. pylori patients positive Sensitivity infection by the 13 C-labeled UBT was required for entry into Test studied tests (%) the study; the criteria used were previously reported [19]. In addition, H. pylori infection was diagnosed by positive War- CLO (rapid urease) test* Histological staining thin-starry silver staining of gastric mucosa specimens ob- ELISA for IgA tained by antral biopsy. In cases in which Warthin-Starry silver ELISA for IgG staining was negative, diagnosis of H. pylori infection was established on the basis of the CLO test. Status of H. pylori NOTE. All 86 patients had positive 13 C-labeled urea breath tests, as re- quired for entry into the study. CLO Å Campylobacter-like organism. infection during follow-up visits was established by the 13 C- * A positive test was defined as a change of color within the first 24 hours. labeled UBT. Eradication of H. pylori infection was defined A positive test was defined as the demonstration of an organism with the as a negative 13 C-labeled UBT 1 month after cessation of ther- typical appearance of H. pylori. The patient was considered seropositive when an optical density ratio of apy, as is now considered to be standard [20]. 13 C-labeled 1.0 was obtained. UBTs repeated 3 months after cessation of therapy, which are more accurate and eliminate false-negative results due to suppression but not elimination of infection [21], verified no the three patients treated solely with colloidal bismuth subcitrate. further changes in status of H. pylori infection. None of those patients whose infection was eradicated relapsed, and none of The enrollment characteristics of the three patient groups those patients who were not treated or for whom initial treatment (eradication success, eradication failure, and no treatment) are failed to eradicate the infection had spontaneous clearance summarized in table 2. The control (no treatment) group had of the organism. a higher average age and proportion of other pathologies associ- Serological analysis. Serological assays for IgA and IgG ated with the upper gastrointestinal tract (55% vs. 1.5%, respectively) antibodies to H. pylori were performed by ELISAs according than did the treatment groups. These patients repreantibodies to previously reported methods, and values were expressed as sented less severe clinical cases. This clear disproportion optical density ratios (ODRs) [4, 5, 11, 12]. The antigen was between the groups was due to specific recommendations of a pool of five H. pylori strains, and the sensitivity and specific- the Human Subjects Committee who approved the study in ity of testing with this antigen were ú90% [11, 12]. All followup The proportions of males and females were similar be- serum specimens from one patient were tested in the same tween the groups. None of the enrollment values could be used run with the original serum sample to evaluate the percentage to predict the results of treatment. change from baseline. By using the 13 C-labeled UBT to establish the presence of Statistical analysis. The results were expressed as means H. pylori infection, none of the treated patients in whom { SEM, and statistical analyses included comparison of means H. pylori infection was eradicated relapsed during the observation between groups by the t test. x 2 and Fisher s exact tests were period. Conversely, and as expected, in none of the patients also performed for two-by-two tables. A P value of.05 was who were not treated or for whom treatment failed was there considered statistically significant. spontaneous elimination of H. pylori infection. Since antibody kinetics were very similar between persons who were never treated or for whom treatment failed, we combined Results these two groups in all subsequent analyses. Levels of IgG and IgA antibodies specific to H. pylori decreased significantly We determined the sensitivity of each of the methods used only in the eradication success group (figure 1). In that for the diagnosis of H. pylori infection (table 1) and compared group, by the third month of follow-up, the mean levels of these sensitivities with that of the 13 C-labeled UBT, which was specific antibodies decreased ú30% from the original values. used for study entry. Although the 13 C-labeled UBT and CLO A persistent drop in IgG and IgA antibody values was observed test are based on the same enzymatic principle (urease activity), until the end of the study at 12 months of follow-up. In the the sensitivity of the CLO test was only 82.7%. As expected, eradication success group, the mean reduction in IgG antibody the sensitivity of the ELISAs was similar to that of histological ODR was greater than 40% of the pretreatment values by 6 examination. months. H. pylori infection was found to be eradicated in 50 (75.8%) Although the mean changes in antibody levels between of the 66 treated patients at 1 month after cessation of therapy. pretreatment and follow-up samples are of interest, the observations Sixteen of the treated patients remained infected with H. pylori; in figure 1 represent changes for all groups studied. these patients were included in the eradication failure group. A more important analysis for clinical purposes is to compare As expected, H. pylori infection was not eliminated in any of the change in antibody level for an individual patient in rela-

3 1040 Pérez-Pérez, Cutler, and Blaser CID 1997;25 (November) Table 2. Enrollment characteristics of Helicobacter pylori infected adult patients. Treatment group Characteristic Eradication success Eradication failure No treatment group No. of patients Mean age { SEM in y 58.2 { { { 2.9 Male patients (%) No. of patients in diagnostic category Duodenal ulcer Gastric ulcer NUD Other* Mean IgA ODR at baseline { SEM 2.48 { { { 0.3 Mean IgG ODR at baseline { SEM 3.85 { { { 0.7 Mean 13 C-labeled UBT value (%) at baseline { SEM 24.7 { { { 5.5 NOTE. NUD Å nonulcer dyspepsia; ODR Å optical density ratio [11]; UBT Å urea breath test. * Including 10 patients with NUD associated with nonsteroidal antiinflammatory drug use and one patient with atrophic gastritis. The increase in the mole fraction of tracer 13 CO 2 at 60 minutes compared with that at baseline, with ú6% considered positive. tion to the pretreatment serum sample. A summary of this analysisisshownintable3,inwhichthecriterionofchanging from seropositive to seronegative was used to define serological evidence of treatment success. Although seroreversion to IgG antibodies was not found in any sample from a patient for whom treatment failed, the sensitivity for ascer- taining treatment success was low (only 25.6% at 6 months and 47% at 12 months). We next sought to determine whether comparison between pretreatment and posttreatment antibody levels can more quickly and accurately reflect treatment success. As working guidelines, we defined seroreversion in a patient as a decrease Figure 1. Effect of specific antimicrobial therapy on the mean level { SEM of IgA antibody (A) and IgG antibody (B) tohelicobacter pylori antigens in 50 patients for whom therapy was successful ( ), 16 patients for whom therapy failed ( ), and 20 controls who received no specific treatment ( ). * Å P value of õ.005; /ÅP value of õ.05.

4 CID 1997;25 (November) Serological Evaluation of Treatment of H. pylori Infection 1041 Table 3. Seroreversion rates among patients after specific treatment observed as early as 3 months after cessation of treatment for Helicobacter pylori infection. (table 4). Percent of patients seroreverting* No. of patients studied at Discussion specified time Eradication Eradication For optimal posttreatment follow-up of H. pylori infections, failure success accurate, rapid, and inexpensive assays are needed. Because Mo. of Eradication Eradication techniques based on either endoscopy or UBTs have important follow-up failure success IgA IgG IgA IgG limitations [10, 22], we addressed whether serological techniques may be useful. Our study is limited in that we were not able to obtain specimens from each patient at each follow-up, and thus, comparisons of the different times involved different patients. We are not aware of any systematic selection that would bias the results obtained. To address the issue of bias * Seroreversion was defined as an optical density ratio of õ1.0. from follow-up differences, we divided the patients into those Includes patients in eradication failure and no treatment groups. who had peptic ulcer disease (n Å 58) and those with other milder conditions (n Å 28). There was no substantial difference in the mean total number of visits for each group (2.7 { 0.12 in IgG and IgA antibody levels from the pretreatment serum vs. 2.4 { 0.22, respectively; P Å.22). However, because the value of ú30% or ú50%. We also used a decrease of 50% therapy used was not as effective as treatments presently being in either IgA or IgG antibody value from the pretreatment advocated [23], we were able to follow a reasonable number serum sample as a criterion of seroreversion. In the eradication of patients for whom therapy failed as well as a control group failure group, the seroreversions that occurred were present of patients who did not receive any treatment. mainly for IgA antibodies and during the early months after All studies of follow-up after treatment are limited by the therapy (table 4). Three patients with positive 13 C-labeled lack of an absolute gold standard because both biopsy-based UBTs had a decrease of ú30% in IgG antibody values 6 and breath test based assays may falsely indicate treatment months after the cessation of therapy. Unfortunately, there success [10]. Despite these limitations, the results of our studies was not another follow-up sample from these patients, and are consistent with previous observations that serological techniques whether this drop in IgG antibody levels was an artifact or can be used to determine treatment efficacy [14 16]. the 13 C-labeled UBT results were false-positive due to the One limitation of the serological techniques is that they have sensitivity of the test could not be confirmed. In contrast, not been subjected to standardization involving different labo- among the patients for whom treatment was successful, rates ratories. of seroreversion to both IgG and IgA antibodies increased Biopsy-based tests examine samples from only a small progressively during the entire study period. Statistically sig- (õ1%) portion of the stomach, whereas the noninvasive UBT nificant differences in seroreversion rates between patients in and serology are global (in effect testing samples from the the eradication success and eradication failure groups were entire stomach). This phenomenon may help explain why the Table 4. Seroreversion rates among patients after specific treatment for Helicobacter pylori infection. Percent of patients seroreverting* No. of patients IgG antibody IgA antibody studied at specified time ú30% ú50% ú30% ú50% Mo. of follow-up F S F S F S F S F S NOTE. F Å eradication failure group includes patients who were not treated; S Å eradication success group. * Criteria for seroreversion are a ú30% or ú50% decrease in antibody level from the pretreatment value at the specific time during follow-up. P õ.05 compared with rate among persons in eradication failure group.

5 1042 Pérez-Pérez, Cutler, and Blaser CID 1997;25 (November) UBT was more sensitive than the CLO test in the initial diagno- obtain follow-up specimens as early as 3 months after the end sis of H. pylori infection. The presence of other urease-positive of therapy. In contrast, the criterion of using a ú30% decrease organisms (e.g., in the mouth) has little impact on UBT results in IgG antibody level is more sensitive for detecting treatment when tests are performed according to now standard criteria. success, and if used at an appropriate time (e.g., 9 months), If appropriate diagnostic criteria can be selected, serology and it is highly specific. For persons in whom there is no particular UBT have the double advantage of being both noninvasive and urgency to detect treatment efficacy, follow-up at 9 12 months globally based [24, 25]. A 13 C-labeled UBT recently has been might be ideal. On occasion, patients for whom therapy ultimately licensed by the U.S. Food and Drug Administration, and such fails have a transient and small decrease in antibody tests should become widely available in the near future. levels [16, 18, 26], presumably due to suppression of the infection. Because levels of antibody specific to H. pylori are often Examining specimens obtained beyond 6 months after the very high before treatment, the definition used for seroreversion cessation of therapy should overcome this problem. is critical to its clinical utility. For example, as shown in table In conclusion, we confirm that serology can be used for 3, the standard criterion of changing from seropositive to sero- the individual patient to noninvasively determine efficacy of negative is relatively insensitive and slow to develop for detecting therapy for H. pylori infection. Further investigations should treatment success. In contrast, the data in figure 1 sug- help better define optimal diagnostic criteria and timing of gest that using a comparison between pretreatment and follow-up examinations. posttreatment antibody levels in a particular patient will be more useful. The results of using several different definitions of seroreversion that are shown in table 4 confirm that this is a superior approach to that shown in table 3. However, even Acknowledgments so, this comparative method has several limitations. The authors thank T. T. Schubert and A. B. Schubert for their First, the comparative method requires that the pretreatment contributions during the study. serum sample be saved and then assayed simultaneously with the posttreatment serum sample. Although these are logistic handicaps, they can be easily overcome in a dedicated labora- References tory in which pretreatment samples are stored and are easily 1. Warren JR, Marshall B. Unidentified curved bacilli on gastric epithelium accessible. 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