Specimen Collection, Transport, and Processing
There are no special requirements for the collection, transport, or processing of stool or blood specimens for H. cinaedi and H. fennelliae. Tissue biopsy material of the stomach for detection of H. pylori should be placed directly into transport media such as Stuart’s transport medium to prevent drying. Specimens for biopsy may be refrigerated up to 24 hours before processing; tissues should be minced and gently homogenized.
Direct Detection
Pathologists use the Warthin-Starry or other silver stains and Giemsa stains to examine biopsy specimens. Squash preparations of biopsy material can be Gram-stained with good results; the 0.1% basic fuchsin counterstain enhances recognition of the bacteria’s typical morphology. Sampling error may occur during processing, therefore resulting in no identification of the organisms.
Presumptive evidence of the presence of H. pylori in biopsy material may be obtained by placing a portion of crushed tissue biopsy material directly into urease broth or onto commercially available urease agar kits. A positive test is considered indicative of the organism’s presence. Another noninvasive indirect test to detect H. pylori is the urea breath test. This test relies on the presence of H. pylori urease. The patient ingests radioactively labeled (13° C) urea, and if the organism is present, the urease produced by H. pylori hydrolyzes the urea to form ammonia and labeled bicarbonate that is exhaled as CO2; the labeled CO2 is detected by either a scintillation counter or a special spectrometer. This test has excellent sensitivity and specificity. Two enzyme immunoassays H. pylori stool antigen tests (Premier Platinum HpSA, Meridian Diagnostics, Inc., Cincinnati, Ohio; FemtoLab H. pylori, Connex, Martinsried, Germany) and a one-step immunochromatographic assay using monoclonal anti bodies (Immunocard STAT! HpSA, Meridian Bioscience Europe) have been introduced to directly detect H. pylori. Finally, a variety of molecular methods have been developed to directly detect H. pylori in clinical specimens and to identify bacterial strains and host genotype characteristics, bacterial density in the stomach, as well as antimicrobial resistance patterns.
Cultivation
Stool specimens submitted for culture of H. cinaedi and H. fennelliae are inoculated onto selective media used for Campylobacter isolation but without cephalothin such as Campy-CVA. For the recovery of H. pylori from tissue biopsy specimens including gastric antral biopsies, nonselective agar media, including chocolate agar and Brucella agar with 5% sheep blood, have resulted in successful recovery of the organisms. Selective agar such as Skirrow’s and modified Thayer-Martin agar also support growth. Recently, the combination of a selective agar (Columbia agar with an egg yolk emulsion, supplements, and antibiotics) and a nonselective agar (modified chocolate agar with Columbia agar, 1% Vitox, and 5% sheep blood) was reported as the optimal combination for recovering H. pylori from antral biopsies. Incubation up to 1 week in a humidified, 5% to 10% O2 environment, at 35° to 37° C may be required before growth is visible.
Approach to Identification
Colonies of Helicobacter spp. may require 4 to 7 days of incubation before small, translucent, circular colonies are observed. Organisms are identified presumptively as Helicobacter pylori by the typical cellular morphology and positive results for oxidase, catalase, and rapid urease tests. H. pylori, H. cinaedi, and H. fennelliae are definitively identified by using a similar approach to Campylobacter spp. (see Table1).
Table1. Differential Characteristics of Clinically Relevant Campylobacter, Arcobacter, and Helicobacter spp.
Serodiagnosis
Serologic diagnosis is also available for H. pylori. Numerous serologic enzyme-linked immunoassays (EIAs) designed to detect immunoglobulin G (IgG) and immunoglobulin A (IgA) antibodies to H. pylori are commercially available. Reported performance of these assays varies as a result of the reference method used to confirm H. pylori infection, antigen source for the assay, and the population studied. In addition to variability in assay performance, the clinical utility of these assays has not been determined. It is uncertain as to whether or not these assays are capable of differentiation of active versus past H. pylori infections. However, a single study has confirmed the role of seroconversion in determining a cure of H. pylori infection.
