Salivary Tests and Gastrointestinal Investigations: Cotinine, Alcohol, and H. pylori

Salivary Cotinine Test

• Specimen Collection: Saliva or Urine

  • Obtained and tested within $4$ hours of sample collection.

  • Can be stored for up to $3$ days if refrigerated at $4 \, ^\circ\text{C}$ immediately after collection.

  • Can be frozen immediately at $-20 \, ^\circ\text{C}$ if a longer period of storage is required.

  • Stored specimens must be warmed to room temperature before testing.

• Assay Procedure - Saliva

  1. Place funnel in tube. Deposit saliva into funnel, collecting enough sample to fill at least $1/3 \text{rd}$ of the tube.

  2. Discard funnel and apply tube top, pressing until it snaps into place.

  3. Open a pouch containing an strip by tearing at the slit on the side and lay the strip on a flat surface.

  4. Invert the closed tube. Slowly squeeze $8$ drops of saliva sample from the tube directly onto the exposed white padded end (the green end is at the opposite end).

  5. Leave the strip to rest on a flat surface. Wait for bands to develop. Read the strips once the blue band at the end of the strip substantially disappears.

  6. If the blue band has not disappeared after $20$ minutes, apply several more drops of sample and incubate for an additional $20$ minutes. This is necessary for some very viscous salivas that require a longer time to migrate upwards.

  7. A red color must appear in at least one of the zones (Levels $0-6$) for the test results to be valid.

  8. If the blue band does not disappear after additional incubation, or if the readout appears as a smear instead of discreet bands, the sample is too viscous. Do not retest this sample; collect a new sample and rerun it on a new strip.

• How to Read the Strip

  • Level 0: Lowest color at Level $0$ = Record as Level $0$.

  • Level 1: Lowest color in Level $1$ = Record as Level $1$.

  • Level 2: Lowest color in Level $2$ = Record as Level $2$, and so on for higher levels.

• Interpretation of the Test

  • Saliva: A result of Level $1$ ($10$ - $30 \, \text{ng/mL}$) or higher indicates the use of tobacco products.

  • Urine: A result of Level $3$ ($100$ - $200 \, \text{ng/mL}$) or higher indicates the use of tobacco products.

Alcohol Salivary Test (AST)

• Description and Principle

  • A qualitative screening test used to detect the presence of ethyl alcohol in human saliva.

  • Consists of a plastic strip with a test pad at the tip, treated to react with alcohol in saliva.

  • A distinct colored line develops across the test pad if alcohol is detected at a Blood Alcohol Concentration (BAC) greater than or equal to $0.02\%$.

    • The United States Department of Transportation established $0.02\%$ BAC as the cut-off level for a positive alcohol presence.

  • The most preferable method for quantitative measurement of alcohol is gas chromatography for whole blood.

  • Depending on individual factors and quantity consumed, alcohol can be detected in urine, saliva, and breath samples for up to $12$ hours.

  • The distinct color on the reactive pad can be observed in less than $20$ seconds after the tip contacts saliva samples with an ethyl alcohol concentration greater than $0.02\%$.

• Limitations of the AST Strip

  • Designed for use with human saliva only.

  • A positive result indicates only the presence of alcohol and does not indicate or measure intoxication.

  • Technical or procedural errors, as well as other substances in certain foods and medicines, may interfere with the test and cause false results.

• Test Pad Components

  • Tetramethylbenzidine (TMB): $0.12 \, \text{mg}$

  • Alcohol oxidase (ALOx): $0.5 \, \text{IU}$

  • Peroxidase: $0.35 \, \text{IU}$

  • Proteins: $0.15 \, \text{mg}$

• Chemical Principle

  • The AST is based on the high specificity of ALOx for ethyl alcohol in the presence of peroxidase and an enzyme substrate like TMB.

• Interpretation of Results

  • Positive: A distinct colored line appears on the test pad, indicating a BAC greater than or equal to $0.02\%$.

  • Negative: No colored line appears on the test pad, indicating a BAC below $0.02\%$.

Investigation of the Stomach: Introduction and Physiology

• Introduction to GI Investigation

  • The exploration of the digestive tract is essential for diagnosing gastrointestinal (GI) diseases.

  • Investigation should begin with anamnesis (patient history).

  • Most Common GI Symptoms: abdominal pain, heartburn, nausea and vomiting, altered bowel habits, GI bleeding, jaundice.

  • Nonspecific Symptoms: dysphagia (difficulty swallowing), anorexia, weight loss, fatigue – these can also reflect a GI disease.

• Stomach Anatomy and Cell Secretions

  • Key Regions: Fundus, Lower Esophageal Sphincter, Body, Caudad region, Orad region, Antrum, Pylorus, Duodenum.

  • Gastric Cell Types, Location, and Secretion: (Referencing page 9)

    • Parietal cells: Location: Body. Secretion: $\text{HCl}$, Intrinsic factor.

    • Chief cells: Location: Body. Secretion: Pepsinogen.

    • G cells: Location: Antrum. Secretion: Gastrin (released into circulation).

    • Mucous cells: Location: Antrum, Mucous neck cells (necks of gastric glands), surface epithelial cells. Secretion: Mucus (and Pepsinogen, specifically from antral mucous cells).

• Mucus Secretion and its Protective Role

  • Secretions containing mucins are viscous and sticky, collectively termed mucus.

  • Mucins are secreted by mucous neck cells and surface epithelial cells of the stomach.

  • Mucus is stored in large granules in the apical cytoplasm of these cells and released by exocytosis.

  • pH Gradient: The cell surface has a pH of $7$, while the gastric juice has a pH of $2$. A bicarbonate ($\text{HCO}_3^-$) layer exists beneath the mucus layer.

  • Mucus Degradation: The mucus gel is subject to proteolysis by pepsins, which cleave disulfide bonds near the center of the mucin tetramers.

    • Proteolysis releases fragments that do not form gels, thus dissolving the protective mucus layer.

    • Maintenance of this protective layer requires continuous synthesis of new tetrameric mucins to replace those cleaved by pepsins.

  • Gastric Mucosal Barrier: Mucus and bicarbonate ($\text{HCO}_3^-$) protect the stomach surface from the corrosive effects of $\text{H}^+$ and pepsins.

    • The protective mucus gel formed on the luminal surface, along with entrapped alkaline secretions, constitutes this barrier, preventing damage to the mucosa by gastric contents.

Helicobacter pylori Testing

• H. pylori Infection and Pathogenesis

  • H. pylori infection most often manifests as predominantly antral gastritis, characterized by high acid production despite hypogastrinemia.

  • This increases the risk of duodenal ulcers, with gastritis typically localized to the antrum.

  • H. pylori organisms are adapted to the unique ecological niche provided by gastric mucus.

  • While H. pylori may invade the gastric mucosa, the precise contribution of invasion to disease pathogenesis is unknown.

  • Four Key Virulence Factors of H. pylori: (As linked to pathogenesis)

    • Flagella: Allow the bacteria to be motile within the viscous mucus layer.

    • Urease: Generates ammonia from endogenous urea, thereby elevating the local gastric pH around the organisms, protecting the bacteria from the stomach's acidic environment.

    • Adhesins: Enhance bacterial adherence to surface foveolar cells.

    • Toxins (e.g., Cytotoxin-associated gene A - CagA): May be involved in ulcer or cancer development through poorly defined mechanisms.

  • Overall Impact: These factors enable H. pylori to create an imbalance between gastroduodenal mucosal defenses and damaging forces that overwhelm these defenses.

  • Progression of Chronic Antral H. pylori Gastritis: May progress to:

    • Pangastritis, leading to multifocal atrophic gastritis.

    • Reduced acid secretion.

    • Intestinal metaplasia.

    • Increased risk of gastric adenocarcinoma.

  • The underlying mechanisms for this progression are unclear, but interactions between the host immune system and the bacterium appear critical.

• Classification of H. pylori Tests

  1. Non-invasive Tests:

     • Urea breath tests

     • Stool/fecal antigen test

     • Blood tests

  2. Invasive Tests:

     • Endoscopy (with biopsy for further analysis)

Non-Invasive H. pylori Tests

• Urea Breath Test

  • Principle: Exploits H. pylori's production of urease, an enzyme that protects the organism by alkalizing its immediate environment.

  • Test Procedure:

    • Patient swallows carbon-labeled urea on an empty stomach.

    • If H. pylori is present, its urease enzyme metabolizes the labeled urea into carbon dioxide ($\text{CO}_2$) and ammonia.

    • The produced $\text{CO}<em>2$ diffuses into blood vessels, is transported as bicarbonate to the lungs, and expelled as $\text{CO}</em>2$ with exhaled air, where it is captured for sampling.

  • Result Interpretation:

    • A positive result conclusively indicates H. pylori infection.

    • In the absence of H. pylori, administered urea is absorbed from the GI tract and subsequently voided.

  • False Negatives: Can occur with recent therapy, including proton pump inhibitors (PPIs), antibiotics, or bismuth compounds.

• Stool/Fecal Antigen Test

  • Description: Detects the presence of H. pylori antigen in a stool sample.

  • Advantages: Inexpensive, convenient, can be used for initial diagnosis and to confirm cure after treatment.

  • Interference: PPIs and bismuth can affect test results.

  • Mechanism: A one-step in vitro diagnostic test based on an immunochromatographic assay.

  • Interpretation: (Visual reference used in source)

    • Positive: Control line (C) and Test line (T) both appear.

    • Negative: Only Control line (C) appears.

    • Invalid: No lines appear, or only the Test line (T) appears without the Control line (C).

• Blood Tests (Antibody Detection)

  • Method: Used to measure antibodies to H. pylori (IgA, IgG, IgM classes) using a quantitative ELISA method or rapid tests.

  • Advantages: Inexpensive, convenient.

  • Disadvantages: Generally not useful for early follow-up or distinguishing active infection from past exposure.

  • IgA H. pylori Test (Rapid Test Example):

    • This assay is a double antigen chromatographic lateral flow immunoassay.

    • The test strip contains a burgundy-colored conjugate pad with colloidal gold coupled with H. pylori antigens and a nitrocellulose membrane.

    • The nitrocellulose membrane has a test line (T line) coated with H. pylori antigens, and a control line (C line) coated with goat anti-H. pylori antibody.

    • If specific H. pylori antibodies are present in the sample, the T line will appear as a burgundy-colored band.

    • If H. pylori antibodies are absent or below the detectable level, no T line will develop.

    • The C line should always appear as a burgundy-colored band, regardless of antibody presence. It serves as an internal qualitative control, indicating sufficient specimen volume and proper flow.

    • Interpretation for Rapid Test: Positive if both C and T lines appear; negative if only the C line appears.

  • IgG H. pylori Test:

    • Patients with H. pylori infection almost always develop IgG-class antibodies.

    • These antibodies develop within $3$ weeks after infection but can persist for years even after an infection has resolved.

    • Limitation: The presence of IgG antibodies does not distinguish between previous exposure and acute, ongoing infection.

    • Clinical Utility: Provides no clinical information on whether an H. pylori infection is active and causing current symptoms, or if symptoms stem from another cause.

    • Accuracy: Not accurate for current infections and has limited utility in areas with a high prevalence rate or as a test-of-cure.

Invasive H. pylori Tests: Endoscopy and Biopsy Analysis

• Endoscopy

  • Allows direct investigation of various parts of the gastrointestinal tract:

    • Oesophagus

    • Stomach and Duodenum (Esophagogastroduodenoscopy or EGD)

    • Small intestine (Enteroscopy)

    • Large intestine/colon (Colonoscopy, Sigmoidoscopy)

    • Bile duct (Endoscopic Retrograde Cholangiopancreatography - ERCP)

    • Rectum (Rectoscopy)

    • Anus (Anoscopy)

  • During endoscopy, a tissue biopsy sample is typically obtained.

  • Some very effective tests are available from biopsy, but they are less frequently ordered due to the invasive nature of the procedure.

• General Indications for Endoscopy

  • When symptoms in the digestive system are present: nausea, vomiting, abdominal pain, difficulty swallowing, and gastrointestinal bleeding.

  • For confirmation of a diagnosis, most commonly by performing a biopsy to check for conditions such as anemia, bleeding, inflammation, and cancers of the digestive tract.

  • Used as a therapeutic intervention, such as cauterization of a bleeding vessel, widening a narrow esophagus, clipping off a polyp, or removing a foreign object.

• Esophagogastroduodenoscopy (EGD)

  • Diagnostic Capabilities: Can diagnose conditions like stomach ulcers or gastro-oesophageal reflux disease (GORD).

  • Therapeutic Capabilities: Can treat conditions such as bleeding ulcers, esophageal blockages, non-cancerous growths (polyps), or small cancerous tumors.

  • Accuracy: More accurate than X-rays or barium tests for detecting inflammation, ulcers, and tumors of the esophagus, stomach, and duodenum. It is an excellent test for finding and treating the cause of bleeding from the upper gastrointestinal tract.

  • Biopsy Reasons: Commonly performed to diagnose celiac disease, ulcerative colitis, Crohn's disease, or to test for H. pylori (a common cause of duodenal and gastric ulcers). Occasionally used to distinguish between benign and malignant (cancerous) tissues.

  • Visualization: Permits an excellent view of the mucosal surfaces of the esophagus, stomach, and proximal duodenum.

  • Biopsy Procedure: Biopsies are a critical part of the procedure. Samples must be taken from at least $3$ sites within the stomach and duodenum because inflamed regions are often patchy.

  • Uses of Biopsy Samples: Used for histology to establish the presence of bacteria in inflamed areas and for rapid urease testing to establish the viability of the bacteria.

  • False Negatives: Antibiotic treatment within $4$ weeks of the endoscopy (or stool antigen test) may suppress bacteria to below detectable levels without full eradication. Therefore, antibiotics must be avoided prior to these tests to prevent false negative results.

  • Subsequent Tests Performed on Biopsy Samples: (After collection)

    1. Rapid urease test

    2. Pathological examination/microscopic examination

    3. Culture

    4. PCR

Post-Biopsy Tests for H. pylori

• Rapid Urease Test (from Biopsy)

  • Performance: High sensitivity ($80-95\%$) and specificity ($95-100\%$).

  • Principle: Detects urease in the tissue sample. It's simple, but false negative results can occur with recent use of PPIs, antibiotics, or bismuth compounds.

  • Based on the activity of viable H. pylori's urease enzyme, which converts urea to ammonia. This is how the bacteria naturally moderate the pH of their locale in the acidic stomach environment.

  • Procedure: Performed at the time of gastroscopy. A biopsy of mucosa is taken from the antrum of the stomach and placed into a medium containing urea and an indicator (e.g., phenol red).

  • The urease produced by H. pylori hydrolyzes urea to ammonia, which raises the pH of the medium, changing the specimen's color from yellow (NEGATIVE) to red (POSITIVE).

• Culture (from Biopsy)

  • Disadvantages: Expensive, time-consuming (can take several weeks), and the result depends on experience.

  • Advantages: Allows for the determination of antibiotic susceptibility. This test is necessary if the health practitioner needs to evaluate which antibiotic will likely cure the infection.

  • Procedure: Cultures, the ultimate confirmation method that the bacteria are alive, can be made from biopsy specimens if antibiotic sensitivity testing is required. They necessitate special growth media and take $5-7$ days to grow.

• Pathological Examination (Microscopic Examination from Biopsy)

  • Requirements: Requires pathology processing and staining.

  • Advantage: Provides valuable histologic information. The pathologist looks for H. pylori bacteria and any other signs of disease that may explain a person's symptoms.

• PCR (Polymerase Chain Reaction) (from Biopsy)

  • Method: Molecular detection of H. pylori DNA is feasible from biopsy samples. Fragments of H. pylori DNA are amplified and used to detect the bacteria.

  • Usage: Primarily used in research studies.

  • Sensitivity: Quite sensitive.

  • Limitation: Cannot determine if the bacterial source of the DNA is viable (i.e., if the bacteria are alive).