Evaluation of salivary antibodies to detect infection with

Evaluation of salivaryantibodies to detect infection

with Helicobacter pylori

Mark B Loeb MD, Robert H Riddell MD, Cindy James RN, Richard Hunt MD, Fiona M Smaill MD

Helicobacter pylori infection is an important cause ofpeptic ulcers and gastritis (1). Gastric carcinoma and

lymphoma of the mucosa-associated lymphoid tissue havealso been linked to the organism (2-4). Gastric biopsy, thestandard method for diagnosis of H pylori infection, is both

invasive and expensive. Infection with H pylori stimulatesthe production of immunoglobulin (Ig) G antibodies, whichcan be detected in the serum (5), urine (6) and saliva (7).Serological antibody detection assays have potential diag-nostic value (8,9). The measurement of salivary antibodies

Can J Gastroenterol Vol 11 No 5 July/August 1997 437

MB Loeb, RH Riddell, C James, R Hunt, FM Smaill. Evaluationof salivary antibodies to detect infection with Helicobacter py-lori. Can J Gastroenterol 1997;11(5):437-440. Helicobacter py-

lori infection is an important cause of peptic ulcer disease andchronic gastritis. Infection with this bacterium stimulates the pro-duction of immunoglobulin (Ig) G antibody. Salivary IgG anti-body tests to detect H pylori infection offer a convenient andnoninvasive method of diagnosis. To evaluate an IgG salivary an-tibody kit, saliva was collected from 157 out-patients with dyspep-sia referred for endoscopy to a tertiary centre. A salivary IgGELISA antibody assay was performed using the Helisal Helico-

bacter pylori (IgG) assay kit, and at least four gastric biopsies wereobtained. H pylori infection was confirmed by demonstration ofthe organism on Warthin-Starry silver stain (sensitivity 85%,specificity 55%). The prevalence of infection with H pylori was30%. When the analysis was redone, excluding those treated witheradication therapy, the results were similar (sensitivity 86%,specificity 58%). The positive predictive value of the assay was45% and the negative predictive value was 90%. Despite the easeof sampling, the assay used has limited diagnostic utility, lackingthe predictive value to indicate which patients referred with dys-peptic symptoms to a tertiary care setting are infected with H py-

lori.

Key Words: Diagnostic test, Helicobacter pylori, Salivary antibodies

Utilité des anticorps salivaires pour ledépistage de l’infection à Helicobacter pylori

RÉSUMÉ : L’infection à Helicobacter pylori est une importante causede l’ulcère gastro-duodénal et de la gastrite chronique. Cetteinfection bactérienne favorise la production de gammaglobulines(IgG). Les tests de dépistage des IgG salivaires appliqués au diagnosticde l’infection à H. pylori constituent une méthode non vulnérante etpratique. Des échantillons de salive de 157 patients atteints dedyspepsie, non hospitalisés mais adressés pour endoscopie dans uncentre de soins tertiaires, ont été recueillis dans le but d’évaluer unetrousse de dépistage des IgG. Un dosage des IgG par méthode ELISA aété effectué au moyen de la trousse Helisal Helicobacter pylori IgG etau moins quatre biopsies gastriques ont été obtenues. L’infection àH. pylori a été confirmée au moyen de la coloration de Warthin-Starry(sensibilité 85 % et spécificité 55 %). La prévalence de l’infection àH. pylori a été évaluée à 30 %. Lorsque l’analyse a été répétée enexcluant les patients ayant subi un traitement d’éradication, lesrésultats ont été semblables (sensibilité 86 % et spécificité 58 %). Lavaleur prédictive positive du dosage a été de 45 %, la valeur prédictivenégative, de 90 %. Malgré la facilité du prélèvement des échantillons,le dosage possède une utilité diagnostique limitée, puisqu’il ne permetpas de prédire quels patients adressés dans un centre de soins tertiairespour symptômes dyspeptiques risquent d’être infectés par H. pylori.

Departments of Laboratory Medicine, Pathology and Gastroenterology, McMaster University Medical Centre, Hamilton, OntarioCorrespondence: Dr FM Smaill, McMaster University Medical Centre, Room 2N29, 1200 Main Street West, Hamilton, Ontario L8N 3Z5.

Telephone 905-521-2100 ext 6307, fax 905-521-5099, e-mail [email protected] for publication December 16, 1996. Accepted April 1, 1997

ORIGINAL ARTICLE

has been observed to be a convenient, noninvasive method todetect infection. The objective of this study was to evaluatethe use of salivary IgG antibodies to detect H pylori infectionin patients referred with dyspepsia who underwent endoscopy.

PATIENTS AND METHODSA total of 157 consecutive out-patients with at least a one-month history of dyspepsia, defined by upper abdominal painor discomfort accompanied by fullness, burning, belching,nausea, vomiting, fatty food intolerance or difficulty complet-ing a meal, referred to McMaster University Medical Centre,Hamilton, Ontario for endoscopy, were studied. Before endo-scopy, saliva was collected from each patient using the Omni-SAL collection device (Cortecs Diagnostics, Isleworth,United Kingdom), stored at 4°C and processed within twomonths. Treatment with proton pump inhibitors, nonsteroi-dal anti-inflammatory drugs (NSAIDs) or H pylori eradicationtherapy was recorded. At least four gastric biopsy samples wereobtained from each patient, including two from the antrum,one from the angulus and one from the body of the fundus.H pylori infection was confirmed by demonstration of the or-ganism on Warthin-Starry silver stain of the antral biopsies bya pathologist who was blinded to the serological result. If thesilver stains were negative, additional stains were done on thatpatient’s remaining gastric biopsies and repeated in inflamedantral biopsies to reduce the chances of failing to detect scantnumbers of organisms.

The Helisal Helicobacter pylori (IgG) assay kit (Cortecs Di-agnostics), an ELISA, was used to measure salivary antibodies.The assay was used as specified by the manufacturer with nomodifications. First, 100 �L of saliva was added to each well ofa 96-microwell plate coated with H pylori antigen. Following30 mins of incubation at room temperature wells were washedfive times using an automated plate washer. Then 50 �L of an-

tihuman biotinylate (rabbit antihuman IgG antibody con-jugated to biotin) was added to each well and allowed to in-cubate at room temperature for 30 mins. After five washes,50 �L of avidin peroxidase conjugate (horseradish peroxi-dase conjugated streptavidin) was added to each well andallowed to incubate for 15 mins. Following another fivewashes, 100 �L of substrate solution (1:1 mixture of te-tramethylbenzidine and hydrogen peroxide) was added.After 30 mins, 50 �L of stop solution (1 M sulphuric acid)was added to each well. Absorbance was read at 450 nm bya spectrophotometer. A standard curve was constructedduring each assay using the supplied standard solution. Bor-derline, positive and negative controls supplied by themanufacturer were run with each assay. Samples withELISA units of at least 1.00 were considered positive forsalivary H pylori antibody, samples of less than 0.80 wereconsidered negative and those between 0.80 and 0.99 wereequivocal, as recommended by the manufacturer.

RESULTSOf the 157 patients (mean age 44 years, range 17 to 75; 68males), 47 (30%) were infected with H pylori, as diagnosedby a positive biopsy. The sensitivity of the HelisalHelicobacter pylori (IgG) assay was 85% (40 of 47) and thespecificity 55% (61 of 110). Eighty-eight patients (56%)had a pathological diagnosis of gastritis, six (4%) of gastriculcers and nine (6%) of duodenal ulcers (Table 1). Of thesix patients with gastric ulcers, three had H pylori detectedon biopsy; two others were on NSAIDs in the month beforeendoscopy and one received eradication therapy in the yearbefore endoscopy. Of the nine patients with duodenal ul-cers, three had H pylori detected on biopsy, while five whowere biopsy negative received eradication therapy beforeendoscopy.

438 Can J Gastroenterol Vol 11 No 5 July/August 1997

Loeb et al

TABLE 1Diagnostic groupings based on Helicobacter pylori histological status and salivary antibody results

H pylori status

Number (%) of patients with endoscopic diagnosis of # (%) of patients withhistologic gastritisGastric ulcer Duodenal ulcer Other* None

H pylori biopsy positive (n=47)Positive salivary antibody (n=40)

3 (6)3 (8)

3 (6)2 (5)

26 (55)22 (55)

15 (32)13 (33)

45 (96)40 (100)

H pylori negative (n=110)Positive salivary antibody (n=49)

3 (3)0 (0)

6 (5)3 (6)

50 (45)29 (59)

51 (46)17 (35)

43 (39)4 (8)

*Includes patients with esophagitis, duodenitis, gastritis, hiatus hernia and chemical gastropathy

TABLE 2Treatment characteristics of patients

Helicobacter pylori status

Number (%) of patients treated with

Eradication therapy within12 months before endoscopy

Hydrogen ion pump inhibitors withinone month of endoscopy

NSAIDs within onemonth of endoscopy

Histology + / Antibody + (n=40) 2 (5) 6 (15) 2 (5)

Histology + / Antibody – (n=7) 1 (3) 1 (3) 0 (0)

Histology – / Antibody + (n=49) 9 (18) 11 (22) 4 (8)

Histology – / Antibody – (n=61) 6 (10) 14(23) 5 (8)

NSAID Nonsteroidal anti-inflammatory drug

Forty-five of the 49 salivary positive-biopsy negative pa-tients had no or minimal chronic inflammation on biopsy.Four had inflammation present. Nine of the 49 patients(18%) versus two (5%) salivary positive-biopsy positive pa-tients had received eradication antibiotic therapy in the 12months before biopsy. Also, 11 of the 49 patients (22%),compared with six (15%) salivary positive-biopsy positivepatients, had been on hydrogen ion pump inhibitors in themonth before endoscopy (Table 2). H pylori was detected inthe gastric body of only four of 53 salivary positive-antral bi-opsy negative cases.

DISCUSSIONA number of published studies have reported the use of sali-vary antibodies to detect H pylori infection, with sensitivityranging from 66% to 89% and specificity from 71% to 94%(10-16). Patel et al (10) studied 119 consecutive dyspepticpatients referred for endoscopy and determined the sensitiv-ity and specificity of both to be 85% with a salivary IgGELISA (an adaptation of the Helico-G serum ELISA kit),compared with microscopy and the urease test. The HelisalHelicobacter pylori (IgG) assay was evaluated in 79 patientsundergoing routine endoscopy by Moayyedi and co-workers(11). Sensitivity of 82% and specificity of 92% was notedwhen the assay was compared with a ‘gold standard’ that in-cluded histology, culture and a rapid urease test. Similar re-sults were noted by Clancy et al (12) (sensitivity 89%, speci-ficity 94%) when the Helisal Helicobacter pylori (IgG) assaywas compared with either biopsy or urease testing. Using thesame reference method, Christie et al (13) noted both alower sensitivity (88%) and specificity (71%) in 86 pa-tients. Simor et al (14), evaluating 195 patients using theHelisal Helicobacter pylori (IgG) assay with culture, histopa-thology or both as the reference method, found 81% sensi-tivity and 75% specificity. Similarly, Bathe and colleagues(15) noted sensitivity of 80% and specificity of 80% usinghistopathology as the reference method. Sensitivity of 84%and specificity of 81% were found by Fallon et al (16) whenthe results from the Helisal Helicobacter pylori (IgG) assaywere compared with those from serum IgG. When these in-vestigators, however, used gastric biopsy as the referencemethod, both the sensitivity and the specificity were lower(66% and 74%, respectively).

Although the sensitivity of the salivary antibody assay inour study (84%) was similar to that in previous studies, thespecificity (54%) was unexpectedly low. Serum antibodylevels fall slowly following successful eradication therapywith antibiotics (17). Our study, unlike at least two of theprevious studies (14,15), included patients with a history oferadication therapy for H pylori. Of patients with salivary an-tibody detected, over three times as many biopsy negativeversus biopsy positive had received eradication therapy inthe 12 months before endoscopy (18% versus 5%) (Table 2).If the analysis is redone excluding previously treated indi-viduals, however, the sensitivity and specificity of the sali-vary antibody assay change very little (86% sensitivity [38 of44] and 58% specificity [55 of 95]). Prior antibiotic treat-

ment is therefore not a likely explanation for the lowspecificity of the assay.

Another explanation for the low specificity may be thatthe salivary positive-biopsy negative individuals achievedonly partial eradication, reducing demonstrable organismsbut maintaining seropositivity due to persistent infection.Because only four of the 49 salivary positive-biopsy negativepatients had inflammation on biopsy, we believe partialeradication is an unlikely explanation.

It has been suggested that proton pump inhibitors maycause H pylori to migrate from the antrum to the fundus (18),with subsequent reporting of a false negative if only the an-tral biopsy is examined. In our study, because the proportionof salivary positive-biopsy negative patients who were onproton pump inhibitors was comparable to that of salivarypositive-biopsy positive individuals on the drug (22% versus15%), this is unlikely. Detection of H pylori in the gastricbody of only four of 57 salivary antibody positive-antral bi-opsy negative patients is further evidence that migration wasnot a major contributing factor to our high false positiverate.

Using biopsy alone (without culture or rapid urease test)may have resulted in underestimation of the number of truepositives. The patchy nature of H pylori infection may haveaccounted for false positive serology despite that multiple bi-opsies were examined (19). However, the absence of chronicinflammation on biopsy in the majority of the false positivepatients (45 of 49, 92%) argues against this having a signifi-cant effect.

The Helisal Helicobacter pylori (IgG) assay used in thisstudy was compared with gastric biopsy alone, not with a se-rum antibody assay. Several studies have confirmed the ac-curacy of serum antibody assays to detect H pylori infection(8,9,20-22). A serological assay to evaluate patients whowere histology negative and had received eradication ther-apy would have been helpful in this study. The majority ofsuch individuals, however, in this study did not have histo-logical evidence of chronic inflammation, making it unlikelythat they were infected.

The positive predictive value of the assay in this popula-tion of patients referred for endoscopy, with a prevalence ofinfection of 30%, is only 45% (40 of 89); the negative pre-dictive value is 90% (61 of 68). Results of this study suggestthat, compared with the many accurate serological assaysavailable, the present form of the Helisal Helicobacter pylori

(IgG) ELISA antibody assay is limited. It should be notedthat these results apply in the setting of a referred tertiarycentre where patients are often treated for H pylori infectionbefore referral. In conclusion, despite the ease of sampling,the Helisal Helicobacter pylori (IgG) assay has limited diag-nostic utility, lacking the predictive value to indicate whichpatients referred with dyspeptic symptoms to a tertiary caresetting are infected with H pylori.

ACKNOWLEDGEMENTS: We thank Axcan Pharma Inc(Mount-Saint-Hilaire, Quebec) for providing the Omni-SAL collec-tion device and Helisal Helicobacter pylori (IgG) assay kits.

Can J Gastroenterol Vol 11 No 5 July/August 1997 439

Salivary antibodies to Helicobacter pylori

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Evaluation of salivary antibodies to detect infection with