Helicobacter

TAXONOMY Back to top

The genus Helicobacter is classified in the family “Helicobacteraceae” of the

class Epsilonproteobacteria, formerly known as the epsilon subclass of

the Proteobacteria, with Helicobacter pylori as the type species (101). The other genus in the

family is Wolinella, with the type species Wolinella succinogenes, and both genera are

phenotypically similar to the genus Campylobacter. Helicobacter is a genus of expanding

diversity. Since the genus name was formally proposed in 1989 (48) with two species (H.

pylori and Helicobacter mustelae) and revised in 1991 to include Helicobacter

cinaedi and Helicobacter fennelliae (132), it has grown to comprise some 32 species,

including two species with Candidatus status (Table 1). Species of Helicobacter have genomic

G+C base contents ranging from 30 (H. acinonychis) to 48 (H. canis) mol%, which is similar

to the G+C content range of Campylobacter species. In addition, there are a number of

unique Helicobacter 16S rRNA gene sequences listed in GenBank that represent sound taxa

that have not yet met the criteria for official recognition but could be the basis of future new

species. All species described in this chapter except“Helicobacter winghamensis” have

formally validated names by international rules of nomenclature (101).

DESCRIPTION OF THE AGENTS Back to top

Members of the genus Helicobacter typically are curved, helical or spiral, or fusiform rodshaped

bacteria with or without periplasmic fibers. The cells have sizes ranging from 0.3 to

0.60 μm in width and 1 to 10 μm in length. Cells may become spheroid or form coccoid

bodies if they are cultured for a prolonged period or if growth conditions are not optimal.

Such forms typically cannot be subcultured. Helicobacters are all gram negative, cytochrome

oxidase producing, and non-spore-forming. Cells are motile and possess either single or

multiple flagella. There is considerable diversity among species in flagellum morphology.

Flagella are typically sheathed; for example, H. pylori has multiple (four to eight per cell)

monopolar sheathed flagella with terminal knobs (Table 2). In contrast, Helicobacter

pullorum, H. canadensis, and five other species with unsheathed flagella form a distinct

phylogenetic group within the genus (Fig. 1). Gastric helicobacters found in animals, with the

exception of Helicobacter baculiformis, have distinctive, tightly spiraled morphologies and

can exhibit tufts of up to 20 multiple flagella per cell. The optimum temperature for growth is

37°C. Helicobacters are organotrophs, possess a respiratory type of metabolism, and are

microaerobic. The optimal atmosphere for growth varies, as some species, such

as Helicobacter ganmani, a rodent enteric organism (115), grow best in an anaerobic

cabinet, although strict anaerobiosis can be lethal. Successful cultivation of helicobacters

typically requires a humid atmosphere maintained at 37°C with reduced levels of oxygen (5

to 10%) and increased levels of carbon dioxide (5 to 12%). Helicobacter species grow

poorly, if at all, in routine aerobic atmospheres. Key biochemical characteristics, such as

urease hydrolysis, nitrate reduction, indoxyl acetate hydrolysis, and alkaline phosphatase

activity, vary among species of Helicobacter and so are utilized in species identification

(Table 2). However, there is no single common feature which reliably distinguishes all

species ofHelicobacter from those of Campylobacter. All helicobacters lack the carbohydrate

utilization pathways typically exploited in conventional laboratory biochemical tests. Genomic

analysis of H. pylori shows that it does not appear capable of using complex carbohydrates

as energy sources, and comparisons with the Campylobacter jejuni genome indicate

significant differences in energy metabolism and chemotaxis systems (1).

EPIDEMIOLOGY AND TRANSMISSION Back to top

Helicobacter species are isolated from the gastrointestinal and hepatobiliary tracts of a

variety of mammalian hosts that include humans, dogs, cats, cattle, sheep, swine, rodents,

nonhuman primates, cheetahs, ferrets, rabbits, dolphins, whales, and horses, as well as

chickens and wild birds (Table 1).

H. pylori

H. pylori, with its characteristic strong urealytic ability, is the gastric helicobacter of humans

and is found almost exclusively in the human stomach, which provides the reservoir of

infection. Exceptions are isolates from primates previously named Helicobacter

nemestrinae, which is now considered a synonym of H. pylori.There is no evidence of animalto-

human transmission. The organism colonizes the cardia, corpus, and antrum of the

stomach and may also be found in areas of gastric metaplasia of the proximal duodenum.

Sero-epidemiology shows a widespread distribution, with estimates that close to half the

human global population is colonized, with clinical disease being the exception rather than

the rule (85). In North America and in Europe, up to 15% of children and up to 60% of

adults are infected, although there is evidence that prevalence rates are declining in

developed societies with improvements in sanitation and living standard (8,127). The

prevalence of H. pylori infection differs markedly between developing and developed

countries (124). In developing countries, infection occurs early in life; most children are

infected by the age of 10, and prevalence remains high (up to 90%) for all adult age groups.

In contrast, in developed countries, a progressive increase in prevalence is observed, from a

low percentage of infection in children to 40 to 50% infection rates in the older age groups.

This is not the consequence of a progressive acquisition of the infection but rather the result

of a cohort effect (124). Reported incidences of culture confirm that infections vary

considerably from country to country depending on local treatment guidelines and culturing

practices.

The modes and routes of transmission of H. pylori from person to person remain to be

definitely proven. There is epidemiological evidence for both oral-oral and fecal-oral

transmission, with the latter being more likely in developing countries, where sanitation and

contaminated water supplies may pose a greater risk (8). The role of contaminated public

water supplies has never been convincingly proven because of the rarity of culture-positive

water samples (139). There is no evidence that viable cells of H. pylori can survive the

disinfection levels in properly maintained main supplies, although survival may be possible as

a viable nonculturable form (94). Biofilms within water distribution systems have been

suggested as possible sites of passive accumulation (9).The rationale for oral-oral

transmission relies on the presence of H. pylori in regurgitated gastric juice, thus allowing H.

pylori to temporarily colonize the oral cavity. Another possibility is via vomitus, in which H.

pylori can remain viable for hours (108). Person- to-person transmission appears to be most

frequent in intrafamilial settings during childhood, particularly between mothers and siblings,

as well as among siblings and between other household contacts (106). Family groups

provide the best opportunity to study person-to-person transmission, but interpretation of

evidence is complex. Patterns of frequent horizontal spread deduced from DNA sequence

types were found both within families and between unrelated individuals in rural South

Africa, which may be a situation representative of high-prevalence areas in large parts of the

developing world (119). In urban families, in contrast, clonal transmission of H. pylori was

more frequent between first-degree relatives.

“H. heilmannii”-Like Organisms

Human infections with HHLO are generally considered uncommon, with prevalence rates

detected by histological observation ranging from <0.3% in developed countries to about 6%

in other regions (102). A higher rate of 2% was indicated for some United Kingdom patients

by a direct biopsy PCR assay designed to detect multiple HHLO species (16). Now that

individual HHLO taxa are better defined, retrospective reassessment using species-specific

assays of past cases attributed to “H. heilmannii” provide evidence of infection with one or

more species of zoonotic origin, notably H. salomonis, H. felis, “H.

suis,” and “CandidatusHelicobacter bovis” (26, 52, 134). These findings indicate cats, dogs,

and swine as possible sources of infection, but modes of transmission are unknown.

Enterohepatic Helicobacters

Enterohepatic helicobacters inhabit the intestinal and hepatobiliary tracts of various mammal

and bird hosts, and several species, such as H. bilis, H. canadensis, H. canis, H. cinaedi, H.

fennelliae, H. pullorum, and “H. winghamensis,” infect humans with clinical symptoms (Table

1). Little is known about prevalence and routes of transmission of these species, but the

implications are that they are transmitted to humans from animals (102). H. pullorum is a

recognized zoonotic risk, as it has been identified in carcasses of broiler chickens and laying

hens (144) and on uncooked retail chicken (47).

CLINICAL SIGNIFICANCE Back to top

H. pylori

Warren and Marshall (138) first proposed the association of H. pylori with peptic ulcer

disease, and since then it has become established as the most clinically important species

of Helicobacter. It is recognized as the main cause of peptic ulcer disease and a major risk

factor for gastric cancer (124). H. pylori infection is also an independent risk factor for the

development of atrophic gastritis, gastric ulcer disease, gastric adenocarcinomas, and gastric

mucosa-associated lymphoid tissue (MALT) lymphomas (124). Individuals infected with H.

pylori may develop acute gastritis (abdominal pain, nausea, and vomiting) within 2 weeks

following infection. The species establishes a chronic infection in the majority of infected

people, represented by chronic gastritis. Prominent mucosal inflammation is often evident in

the antrum (antrum-predominant gastritis), predisposing to hyperacidity and duodenal ulcer

disease. Many patients infected with H. pylori have recurrent abdominal symptoms (nonulcer

dyspepsia) without ulcer disease, and there appears to be a clinical benefit in eradicating H.

pylori in these patients (92). Duodenitis often occurs with H. pylori infection, and duodenal

ulcers develop in as many as 16% of infected individuals (39). Gastric MALT lymphoma, a

rare stomach cancer, is caused by H. pylori infection and is the only cancer which can

possibly be cured by antibiotics (141). Eradication of H. pylori is also recommended in cases

of unexplained iron deficiency anemia and chronic idiopathic thrombocytopenic purpura (82).

The clinical significance of H. pylori infection remains speculative in a number of other

chronic conditions, notably ischemic heart disease, inflammatory bowel disease, and liver

and biliary tract diseases (27, 68, 97).

“H. heilmannii”-Like Organisms

HHLO infection has been associated with mild-to-moderate gastritis, peptic ulcer disease,

and gastric MALT lymphomas in adults, although it has not unequivocally been established as

a causative agent (52, 95). HHLO infection is a rare finding in children (114). The etiology of

HHLO infections is unclear because they are uncommon and organisms are unculturable in a

routine clinical laboratory.

Enterohepatic Helicobacters

Isolated cases of infections in adults and children with enterohepatic helicobacters have been

reported over the past 20 years, but their clinical significance is often not clearly established.

Isolates are mainly from blood and, to a lesser extent, from fecal samples (36, 102). The

bacteremia-associated helicobacters, although rare, are the most clinically significant, as

they occur more frequently in patients with underlying conditions. It is presumed that these

helicobacters are able to invade the bloodstream via colonization of the human lower

gastrointestinal tract, possibly from mucosal cells damaged by combined chemo- and

radiotherapy.

H. cinaedi was initially described in homosexual men with proctitis (129). Infections may

present in various clinical manifestations (proctocolitis, gastroenteritis, neonatal meningitis,

localized pain and rash, and bacteremia), particularly in individuals with underlying

immunosuppressive conditions, such as AIDS, malignant diseases, and chronic alcoholism

(98, 102, 131). H. fennelliae was also first described from rectal swabs of homosexual men

with symptoms of proctitis (129) and has subsequently been implicated as a cause of human

gastroenteritis and bacteremia, particularly in immunocompromised individuals (102). H.

pullorum has been associated with several cases of human gastroenteritis (11, 122, 123).

Furthermore, DNA of this species was detected by PCR in the hepatobiliary tracts of patients

with chronic cholecystitis (38) as well as in intestinal biopsy specimens of Crohn’s disease

patients (68). The clinical significance of the latter findings is unclear.

Other species of Helicobacter isolated occasionally from infected humans but of unclear

clinical significance include H. canadensis (37) and “H. winghamensis” (90) from cases of

gastroenteritis, H. canis from cases of bacteremia and multi-focal cellulitis (74, 111), and H.

bilis from cases of bacteremia (36, 102). The presence ofH. bilis in human gallbladder tissue

(38) and of H. ganmani in the liver tissue of children with chronic liver disease (128) was

indicated by PCR assays, but clinical relevance was not established.

UnspecifiedHelicobacter DNA has been detected in liver specimens from patients with various

diseases, including hepatocellular carcinoma and cholangiocarcinoma (4, 23, 99, 116).

COLLECTION, TRANSPORT, AND STORAGE OF

SPECIMENS Back to top

Gastric Biopsy Specimens

Gastric biopsy specimens for the direct diagnosis of H. pylori are routinely obtained from the

antrum and corpus by esophagogastroduodenoscopy. While sterile normal saline may be

sufficient for short-term (up to approximately 2 h) transport of gastric biopsy specimens, a

transport medium should be used if available to maintain the viability of the organisms for

culture. H. pylori is sensitive to desiccation and to ambient atmosphere and temperature. A

semisolid transport medium may be used (e.g., Portagerm pylori [bioMerieux Inc., Durham,

NC]) or an in-house transport medium that comprises brain heart infusion broth (3.5%),

yeast extract (2.5%), sterile horse serum (5%), and Helicobacter pylori selective supplement

(Dent’s; 10-μg/ml vancomycin, 5-μg/ml trimethoprim, 5-μg/ml cefsulodin, and 5-μg/ml

amphotericin B [Oxoid Ltd., Basingstoke, United Kingdom]). Alternative media include

Stuart’s transport medium or Brucella broth with 20% glycerol. If culture of H. pylori is not

possible within 24 h, it is recommended that biopsy specimens be stored overnight at 4°C in

a transport medium and then transported at ambient temperature. For longer-term storage,

biopsy specimens should be frozen at −70°C in a 10%-glycerol-containing medium.

Fecal Specimens

H. pylori and other gastric helicobacters cannot ordinarily be isolated from human fecal

specimens, so samples are not recommended for routine culture. Fecal samples are used

for H. pylori stool antigen testing and either should be tested immediately or should be

stored immediately at—20°C. Repeated thawing and freezing of samples should be avoided.

As enterohepatic helicobacters can cause enteric disease, fecal specimens may be required

for culture. However, campylobacters are more likely to be tested for in the first instance,

and relevant protocols for their collection, transport, and storage also can be used for

enterohepatic species ofHelicobacter (as for Campylobacter [see chapter 53]).

Blood Specimens

Blood specimens are required for serological diagnosis of an H. pylori infection and may be

collected, transported, and stored by standard protocols. Also, as the enterohepatic

helicobacters may translocate across the intestinal barrier and cause invasive infections,

peripheral venous blood from suspected cases may be required for microbiological testing.

Blood may be collected in commercially available aerobic and anaerobic blood culture bottles

and transported and stored according to the protocols used for campylobacters, which are

more likely be tested for in the first instance (for Campylobacter, see chapter 53).

Other Clinical Specimens

Laboratory tests requiring the collection of other types of specimen have been developed to

assist in the diagnosis of H. pylori infection and may be undertaken under some

circumstances (89).

Gastric Juice

Gastric juice, obtained from the patient either by aspiration after the introduction of a

nasogastric tube or by the so-called string test, has been used as a possible source of H.

pylori for culture and PCR (39). Gastric juice does not offer a satisfactory alternative to a

biopsy specimen as a routine specimen because of problems caused in culture by overgrowth

of nasopharyngeal microbiota unless preventive steps such as acid pretreatment are taken

(140). Specimens, if used, should be transported at 4°C and processed without delay.

Urine

Fresh urine samples required for serological tests should be collected and transported by

standard protocols. Urine samples cannot be frozen because any resultant protein

precipitation may interfere with the tests (39).

Saliva

Saliva samples required for serological tests can be collected easily by having the patient spit

into a tube. An alternative that may be preferable is use of a special swab device rubbed

over the gums that is designed to obtain gingival transudate enriched in immunoglobulin G

(IgG) (39, 81). Specimens should then be transported by routine protocols.

DIRECT EXAMINATION Back to top

Microscopic Examination of Gastric Biopsy Specimens

Histopathological examination of gastric biopsy specimen sections preserved in a fixative

(10% formaldehyde) and embedded in paraffin is widely used for diagnosis of H.

pylori infection. Standard hematoxylin and eosin tissue staining is not sufficient to detect H.

pylori (110), whereas the Warthin-Starry stain allows excellent visualization of bacteria if

performed by trained histology personnel. Although the specificity is usually adequate, the

presence of bacteria with atypical morphologies may result in misinterpretations. Under

optimal conditions, histological diagnosis has a sensitivity and specificity of 95% (39).

Immunohistological staining with specific H. pylori antibodies can improve specificity.

Histological methods and interpretation of histological findings are outside the scope of this

chapter, but from the microbiology laboratory perspective, microscopic examinations of a

smear prepared directly from a biopsy specimen or from imprint cytology provide rapid

bacteriological test results for observation of cells of H. pylori (39, 89). Staining can be

performed using Gram stain, rapid Giemsa stain, or the fluorescent acridine orange stain.

The less common gastric HHLO can also be Giemsa stained and, when observed

microscopically, can be distinguished from H. pylori by their distinct tightly spiral morphology

(59).

Microscopic Examination of Stool and Other Pathological

Specimens

Direct Gram stain analysis of stool smears and other clinical samples is not routinely

performed for the detection of H. pylori or other helicobacters. Direct identification of

helicobacters in positive blood cultures may require special stains, particularly if tests are

performed by personnel unaccustomed to looking for such organisms. Thin, gull-shaped

bacteria such as H. cinaedi can be difficult to observe by Gram staining and require acridine

orange staining, dark-field microscopy, or Giemsa staining (62). A modified Gram stain with

carbol (0.5%) or basic fuchsin (0.1%) as the counterstain is also recommended for detection

(39).

Urease Testing of Gastric Biopsy Specimens for H. pylori

H. pylori produces large amounts of extracellular urease, which can rapidly be detected

following introduction of gastric biopsy tissue into a urea-containing medium. Urease

catalyzes the hydrolysis of urea into ammonia and carbonate. The net effect of ammonia

production is to increase local pH. Detection of urease activity forms the basis of several

simple, inexpensive, and easy-to-perform tests that are usually performed in an endoscopy

unit by clinicians (39, 89). Biopsy specimens are placed either in an agar gel or on a paper

strip containing a pH indicator. If organisms are present in sufficient numbers, a color

change will occur as a result of urea breakdown and ammonia production. Commercial rapid

urease tests that include agar gel-based tests (e.g., CLOtest [Kimberly-Clark, Neenah, WI])

and paper-based strip tests (e.g., PyloriTek [BARD, Murray Hill, NJ]) have been critically

evaluated, and specificities are usually excellent (89, 143). Detection sensitivity also is high

but is dependent on the H. pylori density in mucosal biopsy specimens and the number of

biopsy specimens sampled. Agar gel-based tests have their optimal sensitivity after 24 h of

incubation, whereas strip tests are optimal within an hour, making them truly rapid tests

(39). Urease broth media commonly available in microbiology laboratories, such as modified

Christensen medium and urea-indole medium, can be used but are not optimized to have

sensitivities equivalent to those of commercially available kits.

Urea Breath Test

Another important clinically performed test, based on the ability of H. pylori to produce

urease and developed specifically for detection of an active infection, is the urea breath test

(UBT). The UBT test has the advantage of being noninvasive, as urea, labeled with either a

carbon radioactive isotope (14C) or a nonradioactive natural isotope (13C), is ingested by the

patient. The labeled CO2 is absorbed by the blood and exhaled in expired air. The testing

methodology and factors influencing the result, standardization, and application in different

clinical settings have been comprehensively reviewed (39, 89). The use of the UBT has high

diagnostic accuracy (>95%) (82) and, where available, is consistently recommended for the

diagnosis of H. pylori infections in adults in both pre- and posttreatment settings (82, 89). A

recent prospective multicenter study indicated that the 13C UBT was also simple and accurate

for diagnosis of H. pylori infections in children (32).

H. pylori Fecal Antigen Detection

Stool antigen tests using an enzyme-linked immunosorbent assay (ELISA) provide another

valuable aid in the diagnosis of an active H. pylori infection. The test is easy to perform and

has the advantage of being noninvasive. Since becoming commercially available, kits

consisting of a polyclonal antibody fixed on microwells (e.g., Premier Platinum HpSA

[Meridian Bioscience Inc., Cincinnati, OH]) have been extensively evaluated on samples from

adults and children (41) and have proved to be an excellent diagnostic tool. A systematic

review of published data up to 2004 confirmed the value of such kits for primary

pretreatment as well as for follow-up posttreatment diagnosis (41). The test was further

developed by using specific monoclonal antibodies (89), and reviews and meta-analysis

based on evaluations of kits (e.g., IDEIA HpStAR [Oxoid Ltd., United Kingdom] and Premier

Platinum HpSA PLUS [Meridian, Bioscience Inc., Cincinnati, OH]) indicated improved

sensitivity compared to those of polyclonal tests (6, 42, 89). For example, high sensitivity

(94%) and specificity (100%) were reported for tests on pretreatment adult stools in

England (18), and the performance of tests was reported to be excellent for young children

in Finland (67). If the UBT is not available, the laboratory-based stool antigen test is

recommended for confirmation of eradication at least 4 weeks after treatment (82). The

presence of some false positives has been noted in the use of stool antigen tests for

posteradication diagnosis, possibly attributable to the presence of antigen in stools from

degraded coccoid forms (6). Some stool samples that were transiently positive by ELISA also

have been reported for children and were thought to have transient infections with H.

pylori or Helicobacter spp. (53). Monoclonal antibodies are used also in immunoenzymatic

rapid point-of-care tests for diagnosis of H. pylori infection (e.g., the ImmunoCard STAT!

HpSA [Meridian Bioscience Inc., Cincinnati, OH] and RAPID Hp StAR [Oxoid Ltd.,

Basingstoke, United Kingdom]), and their performance in the clinical/near-patient setting has

been critically evaluated (6, 18, 24, 89).

Nucleic Acid Detection

Detection of H. pylori in Gastric Biopsy Specimens

Nucleic acid assays based on PCR amplification and on fluorescence in situ hybridization with

species-specific probes provide useful approaches for the detection of H. pylori in gastric

biopsy specimens, as they are significantly faster than culture. The commonest targets for

amplification are 16S rRNA, ureA, glmM (formerly named ureC), vacA, and cagA genes

(125), and in addition 23S rRNA genes have been targeted for both detection and antibiotic

susceptibility testing (21, 118). There is currently no”gold standard” method for use in the

clinical laboratory setting for PCR of gastric biopsy specimens, and so it is advised that

PCRbased assays should not be the sole basis of determining the H. pylori status of a patient

(125). Nevertheless, PCR assays can provide added value in investigating culture-negative

gastric biopsy specimens, particularly those from cases for which other clinical tests indicate

an H. pylori infection (21). A systematic study of primers for H. pyloridetection found that

the four best-performing assays each attained a detection limit of <100 CFU/ml from gastric

tissue (125). However, no assay had 100% specificity or sensitivity, and all produced false

positives. Two of the best all-around assays based on the HP64-f/HP64-r primers for

the ureA gene and HP1/HP2 primers for the 16S rRNA genes had sensitivities and

specificities of >90% with gastric biopsy specimens.

Detection of H. pylori in Feces

The PCR assays developed for biopsy specimen testing, in particular those using primers

targeting the 16S rRNA and ureA genes, have been applied to stools to detect H. pylori with

various success rates (89), and their value for routine laboratory use is questionable. Feces

is a complex material containing a number of PCR inhibitors (93), and complex DNA

purification methods are needed to either eliminate or reduce the levels of such compounds

(61). The performance of the assays is restricted by the low numbers of H. pylori cells in

feces and by degradation of DNA during transit through the intestinal tract. Another test uses

a biprobe 23S rRNA gene real-time PCR assay (118), and a modified version (ClariRes assay

[Ingenetix, Vienna, Austria]), as well as clarithromycin susceptibility testing of stool

specimens of symptomatic children (77), has been applied to detection (for further details,

see “Antimicrobial Susceptibility” below).

Detection of HHLO in Gastric Biopsy Specimens

Specialist assays have been developed for direct PCR detection of species of HHLO in gastric

biopsy specimens (134), and simultaneous testing for both H. pylori and HHLO can be

performed using a multiplex PCR assay (16). Fluorescence in situ hybridization tests with

species-specific probes can also be applied to detect HHLO in human gastric biopsy

specimens (130).

Detection of Other Helicobacters in Clinical Samples

With the exception of an unvalidated 16S rRNA gene PCR assay for detection of H.

pullorum in human fecal extracts (11), there are no assays suitable for fecal detection of

enterohepatic Helicobacter species of clinical relevance. Genus-level PCR-based assays

targeting mainly 16S rRNA genes have been developed and used for direct detection of other

helicobacters in a variety of clinical samples that include dental plaque and saliva (49, 126),

intestinal tissue biopsy specimens (68), and liver biopsy specimens and associated tissues

(bile and gallbladder) (4, 116). These assays are generally undertaken for specific

epidemiological and disease association investigations and so are unlikely to be used in the

routine laboratory. Results of such PCR-based assays performed in the absence of other

evidence therefore should be interpreted with caution (125).

ISOLATION PROCEDURES Back to top

Isolation of H. pylori

H. pylori is readily isolated by culture from gastric biopsy specimens. Tissue should be

streaked over the culture medium with a minimum of delay or first homogenized to facilitate

a higher yield of bacteria. Agar-based media such as brain heart infusion agar, brucella agar,

Wilkins Chalgren agar, and Trypticase soy agar, can be used for primary culture. In our

experience, Columbia agar base supplemented with 10% defibrinated horse blood gives

excellent results. A selective medium (e.g., Helicobacter pylori selective medium [Oxoid Ltd.,

Basingstoke, United Kingdom]) containing Dent’s antibiotic supplement (see “Collection,

Transport, and Storage of Specimens” above) also gives adequate isolation results. Plates

should be incubated at 35 to 37°C in a humid microaerobic atmosphere (4% O2, 5% CO2,

5% H2, and 86% N2) achieved using either a gas jar and gas-generating system or an

incubator (e.g., the MACS VA500 microaerophilic workstation [Microbiology International,

Frederick, MD]). The exact gas mixes used vary between laboratories, but the presence of

5% H2 in the atmosphere enhances growth. Culture plates should be observed daily for the

appearance of small, smooth, circular colonies, which should appear after 48 h of incubation.

Plates must be incubated for 10 days before a negative result is given. Colonies should be

subcultured on nonselective medium for further investigation. Isolates may be stored at

−80°C in cryovials (e.g., the Microbank bacterial preservation system [PRO-LAB Diagnostics,

Austin, TX]).

Isolation of Other Helicobacters

There are no recommended culture methods available currently for use in routine clinical

laboratories for isolation of “H. heilmannii” and HHLO from human gastric biopsy specimens.

The first and only reported successful isolation of “H. heilmannii” from a human, achieved

after 7 days with a nonselective medium (7% lysed horse blood) in a 5% O2 and 10%

CO2 atmosphere (2), was subsequently identified as Helicobacter bizzozeronii (57). A novel

isolation method using high acidity and modified gaseous conditions has now been developed

for isolation of H. suis from pig gastric tissue but has not yet been evaluated on human

gastric biopsy specimens (5).

Enterohepatic helicobacters such as H. bilis, H. canadensis, H. canis, H. cinaedi, H.

fennelliae, H. pullorum, and“H. winghamensis” are isolated typically during investigation for

campylobacters in feces from humans with gastroenteritis. These organisms grow at 37°C

but not uniformly at 42°C, the temperature most often used for isolation of C. jejuni. Fresh

stool specimens should be examined using a selective medium or the nonselective

membrane filter method (70) with incubation for a minimum of 7 days at 37°C in a

microaerobic atmosphere (36, 133). Strains of some species may require 5 to 10% H 2 for

optimum growth, and recovery may be hindered if they are susceptible to antibiotics present

in the selective isolation medium.

Some enterohepatic helicobacters, such as H. cinaedi, H. canis, and H. fennelliae, are

isolated occasionally from blood of patients with suspected bacteremia using commercial

blood culture systems (e.g., the Bactec system [BD, Sparks, MD]). Isolates are usually

detected in aerobic blood culture bottles only and may be problematic to recover as they are

difficult to see microscopically and will probably grow poorly on subculture if plates are not

incubated for an extended period (minimum of 6 days) in a microaerobic atmosphere. The

isolation ofHelicobacter species from other sterile body fluids is rare, but a notable example

is the isolation of H. cinaedifrom joint fluid using a nonselective blood medium (56, 133).

IDENTIFICATION Back to top

Identification of Helicobacter species is based on a limited range of morphological,

physiological, and biochemical characteristics (Table 2). Helicobacters have various colony

phenotypes on blood agar, ranging from the discrete, gray, and translucent colonies of H.

pylori to swarming phenotypes of some gastric helicobacters (e.g., H. felis). Most isolates are

motile and should be routinely tested for oxidase, catalase, and urease activities according to

recommended procedures (28).

H. pylori and Other Gastric Helicobacters

In stained gastric biopsy samples, H. pylori cells usually have a curved or helical

morphology. However, on subculture, this “classical” morphology is often lost, and in Gramstained

preparations, cells may appear curved, U shaped, or even as straight rods. HHLO

cells are larger in size and have a more pronounced helical morphology in histological

examinations of gastric biopsy specimens (59). Helicobacter cells may appear faint on

conventional Gram staining and require prolonged counterstaining with carbol fuchsin (0.5%)

for enhanced visualization. Urease-negative organisms may be present occasionally in gastric

biopsy specimens, as H. cinaedi, although not cultured, has been identified by DNA analysis

(109). It is important therefore to perform other key biochemical tests, such as indoxyl

acetate hydrolysis and hippurate hydrolysis, to identify isolates of any unexpected species.

Enterohepatic Helicobacters

Enterohepatic helicobacters may appear as a swarming thin film (e.g., H. cinaedi and H.

fennelliae) or as discrete single colonies (e.g., H. canadensis and H. pullorum). By light

microscopy, they morphologically resemble other gram-negative spiral or curved bacteria.

The enterohepatic species possess several distinguishing characteristics (Table 2), and

biochemical and tolerance tests should be carried out according to the recommended

procedures (28). Species lacking urease activity isolated from humans, such as H.

canadensis, H. canis, H. cinaedi, H. fennelliae, and H. pullorum, superficially resemble enteric

campylobacters, and definitive identification may not be possible from phenotype alone.

Useful distinguishing tests are growth at 42°C, as both H. cinaedi and H. fennelliae are

negative, and indoxyl acetate hydrolysis, for which C. jejuniand Campylobacter coli are

positive and H. pullorum is negative. A PCR assay is described for identification of H.

pullorum (122), but the assay does not distinguish H. pullorum from H. canadensis, which

characteristically hydrolyzes indoxyl acetate and is resistant to nalidixic acid (37). H. canis is

unlike most other helicobacters in being both catalase negative and urease negative,

features that may cause confusion with “H. winghamensis”and H. bilis. Growth at 42°C and

the nitrate reduction and indoxyl acetate hydrolysis tests may be useful to distinguish H.

canis from other catalase-negative campylobacters. It is important to be aware that fecal

specimens occasionally can be cocolonized with

multiple Helicobacter and Campylobacter species, so making a complete diagnostic

evaluation is challenging (70). Helicobacter genus-specific PCR assays may be useful (76),

and sequencing of 16S rRNA genes may be required for a definitive identification.

TYPING SYSTEMS Back to top

Typing of H. pylori

Typing isolates of H. pylori has no role in direct patient management (82). Even so, typing

data may be useful in monitoring the effects of therapy and to establish whether a persistent

infection is due to eradication failure or reinfection, in investigating associations between

strain type and disease severity, in epidemiological investigations of routes and modes of

transmission, and in investigating the ancestry of strains worldwide that might be relevant in

vaccine development. There is no generally agreed-upon system for typing isolates of H.

pylori, although many different methods have been applied and evaluated (104). While a

somatic antigen serotyping scheme was proposed (91), genotypic methods are the most

widely used means of characterizing individual isolates of H. pylori. A key feature of H.

pylori is its high genetic diversity, with almost every isolate having a unique genotype arising

from within-genome diversification and reassortment by natural homologous recombination

(119). This diversification is thought to aid H. pylori in persistence during chronic infection

and in adapting to new gastric environments.

The highly polymorphic vacuolating cytotoxin (vacA) gene provides the basis of a widely

adopted PCR-based genotyping scheme with recommended primers (3). The vacA allelic type

is determined by the presence or absence of short, conserved nucleotide inserts within the

signal and middle regions (107). Common vacA allelic types identified worldwide are s1/m1

(vacuolating), s1/m2 (selectively vacuolating), and s2/m2 (nonvacuolating). The signal

region alleles can be further divided into s1a, s1b, and s1c subfamilies, and likewise the

midregion is subdivided into m2a and m2b subfamilies (104). Genotyping can be performed

either by using individual PCR assays or multiplex PCR assays (14) or by using the reverse

hybridization line probe assay (104). Molecular fingerprinting methods applied to H.

pylori include electrophoretic protein profiling, ribotyping, restriction fragment length

polymorphism (RFLP) analysis, pulsed-field gel electrophoresis (PFGE), amplified fragment

length polymorphism analysis, and plasmid profiling (104). These methods have limited

discriminatory power and have been superseded by PCRRFLP analysis because of its relative

technical simplicity and versatility. The technique has been applied widely in genotyping H.

pylori, using in particular urease (ureA) and flagellin (flaA) gene polymorphisms, and also as

a primary typing technique to differentiate among H. pylori in gastric biopsy specimens

without the need for culture (75). Furthermore, analysis of stool samples based on two

species-specific biprobe real-time PCR assays targeting the glmM and recA genes offers

potential as a noninvasive genotyping method for H. pylori (113). Direct nucleotide

sequencing is now a feasible approach to typing H. pylori, with the availability of highthroughput

sequencing technology. Sequence data are readily comparable by access to

publically available curated databases of sequences of loci for individual species (79) that

include H. pylori (http://pubmlst.org/perl/mlstdbnet/mlstdbnet.pl?filepub-hp_profiles.xml).

This database contains 1,933 unique sequence types (November 2009) based on seven loci

and provides an invaluable reference resource for typing.

Typing of Other Helicobacters

The need to type species of Helicobacter other than H. pylori is unlikely, and the genotyping

schemes described are of questionable value for routine use. These include amplified

fragment length polymorphism analysis and PFGE of H. pullorum (12, 40), plasmid profiling

and ribotyping of H. cinaedi and H. fennelliae (62), and PFGE and random amplified

polymorphism DNA analysis of H. cinaedi (66).

SEROLOGIC TESTS Back to top

Detection of H. pylori Antibody in Blood

H. pylori infection induces a specific systemic immune response to multiple antigens, with

only 2% of patients failing to seroconvert (89). The immune response typically shows a

transient rise in specific IgM antibodies followed by a rise in IgG and IgA antibodies that

persists during infection. Serology is widely used in primary screening for H. pylori infection,

as it is a simple, noninvasive test. A number of in-house and commercial kits have been

developed over the past 20 years for antibody detection, with the essential laboratory

technique being the standard ELISA. The performance and diagnostic utility of laboratory

ELISA kits (e.g., Cobas Core enzyme immunoassay [Roche, Mannheim, Germany]) and rapid

near-patient immunochromatographic tests (e.g., FlexSure HP [Beckman Coulter Inc., Brea,

CA]) have been critically evaluated in several reviews and meta-analyses (69, 73, 89, 120).

Serology (ELISA) kits that measure IgG antibodies are recommended based on overall

performance as an accurate means of diagnosing infection (69). The relevance of IgA in

testing is more controversial (69, 120). Some investigators have observed IgA to be equal to

IgG in performance, but a recent evaluation concluded that IgA alone yielded poorer overall

sensitivity and specificity, although it performed better in samples from children than those

from adults (120). IgM has been found to have little diagnostic value, with an unacceptably

low sensitivity (120). The Maastricht III Consensus Report recommended serological test kits

with high accuracy (>90%) in validated settings (82).

Serology is not recommended for posteradication follow-up when tests detecting an active

infection are preferable (82, 89). Antibody titers decrease very slowly after eradication, so a

singe serum sample does not differentiate past and ongoing infections. Some 30% of

patients have elevated IgG antibodies even after 5 years of successful eradication therapy

(135). False-positives therefore could result in some patients being inappropriately treated

for presumed H. pylori infection, particularly in low-prevalence populations (87). Serology is

useful in epidemiological studies of H. pylori infections, but such analyses likewise need to

take into account that some asymptomatic individuals without an active infection may test

positive.

Immunoblot analysis may also be used for the diagnosis of H. pylori infections, and the

commercial Helico Blot 2.1 test (Genelabs Diagnostics, Singapore) has been evaluated in

studies of adults and children (73, 136). The test may not be commonly performed in a

routine clinical laboratory setting, but its high sensitivity and high specificity (96%) in

patients <50 years old indicate that it could be used as a confirmatory test in some

situations (136). It may have applications also in detecting a past infection, especially by

monitoring the persistence of antibodies to CagA, a product of the cagA gene within

the cag pathogenicity island (136). While it is recognized that CagA protein and also VacA

protein, a cytotoxin produced in various amounts, are important H. pylori pathogenicity

factors, they are of little relevance in the management of infections (82).

Detection of H. pylori Antibodies in Urine

Specific H. pylori IgG antibodies are present in urine at low concentrations. A review of 18

published studies over the period 1998 to 2004 using kits that included commercial ELISAs

and rapid immunoenzymatic tests, listed sensitivities and specificities ranging from 82 to

100% and from 68 to 100%, respectively (89). While the accuracy is not affected by the pH

or the presence of bacteriuria, it may be influenced by a large amount of total IgG. Detection

of H.pylori antibody in urine is attractive because it is noninvasive and it could be useful for

epidemiological studies (82).

Detection of H. pylori Antibodies in Saliva

Salivary antibodies are secreted during the immune response to H. pylori infections (78).

Several commercial kits and in-house ELISAs been developed to detect H. pylori-specific IgG

in saliva, and a review of 15 published studies between 1994 and 2002 listed sensitivities

and specificities ranging from 64 to 94% and from 58 to 95%, respectively (89). The

detection of H. pylori antibody in saliva can be helpful for epidemiological studies (82).

Detection of Other Helicobacter Antibodies in Clinical Samples

Serology has no application in the routine diagnosis of human infections with gastric HHLO

and enterohepatic helicobacters, as there are no validated IgG or IgA assays currently

available. Sustained immunoglobulin responses to multiple antigens of H. cinaedi and H.

fennelliae have been documented (35), and there is recent evidence that a 30-kDa putative

membrane protein, identified as a major antigen of H. cinaedi, could be useful for

immunological and serological testing for clinical diagnosis and epidemiology (58).

ANTIMICROBIAL SUSCEPTIBILITY Back to top

H. pylori Antibiotic Therapy and Relevance of Resistance

The first-choice standard triple therapy to eradicate H. pylori comprises a proton pump

inhibitor, clarithromycin, and either amoxicillin or metronidazole (82). Therapy should ideally

be based on pretreatment antibiotic susceptibility testing, although this is not always

practical (50). The main cause of failure to eradicate H. pyloriwith the standard antimicrobial

regimen is clarithromycin resistance (89). Prevalence rates are 10 to 15% in the United

States (31, 103) and about 10% in Europe, with distinct regional variations (20, 65, 89). The

clinical impact of resistance is marked, with an eradication rate for the standard therapy

decreased by 70% (from 88 to 18%) (89). The key risk factor for clarithromycin resistance is

previous consumption of macrolides, and prevalence of resistance after failure of treatment

is extremely high, with rates of resistance to clarithromycin of up to 63% (19, 65).

Monitoring local clarithromycin prevalence rates is important, as the recommended threshold

at which clarithromycin should not be used or susceptibility testing should be performed is

15 to 20% (82).

Resistance to metronidazole, a key component of the triple-therapy regimen, is also

widespread and is estimated to decrease treatment success rates by 25% (89). Resistance

rates are 20 to 40% in the United States, with similar levels in Europe (typically 27%) (89).

In some other countries, resistance rates may be as high as 60 to 90%. In vitro resistance to

metronidazole may not accurately reflect in vivo resistance (34), and for that reason, routine

susceptibility testing is not recommended in Maastricht III guidelines (82). Nevertheless,

laboratory testing is important for surveillance of resistance, as a threshold of resistance in

the population of 40% provides a guide in deciding choice of treatment (82).

Resistance of H. pylori to other antibiotics used in therapy, such as amoxicillin and

tetracycline (an antibiotic used in second-choice treatment), is rarely found (< 1%) in the

United States and Europe (89), although higher rates to both antibiotics have been reported

in some Asian populations (64). Two other classes of antibiotics have emerged as thirdchoice

(rescue therapy) in the treatment of H. pylori infection: a fluoroquinolone,

levofloxacin, and a rifamycin, rifabutin. Increasing consumption of fluoroquinolones may lead

to higher prevalence of resistance in H. pylori, as rates of resistance to levofloxacin are

currently about 9% in the United States (10) and even higher in some other countries, such

as Italy, where resistance rates are up to 22% (7, 22). Resistance to rifabutin is virtually

absent in H. pylori (22, 44), although its use in eradication therapy has been limited. The

efficacy of furazolidone has also been evaluated (117), but no data are available on

resistance rates.

Phenotypic Susceptibility Testing of H. pylori Cultures

Gastric biopsy isolates of H. pylori should be tested against the antibiotics commonly used in

eradication therapy, in particular, clarithromycin, as resistance in vitro is clinically relevant.

Phenotypic methods of susceptibility testing, such as broth microdilution, disk diffusion, the

Etest, and agar dilution, can be applied toH. pylori. The Clinical and Laboratory Standards

Institute (CLSI; formerly NCCLS) and a workgroup of the European Helicobacter Study Group

have made a similar recommendation of an agar dilution method and breakpoint for testing

susceptibility to clarithromycin (25, 45, 89). In this method, Mueller-Hinton agar base with

5% aged sheep blood is incubated for 72 h at 35°C, with an MIC breakpoint for resistance of

1 μg/ml. The Etest (bioMerieux Inc., Durham, NC) may also be used to determine MIC (20),

and its results correlate well with broth dilution results. The disc diffusion method is costeffective

for routine testing, and an inhibitory zone of less than 17 mm around a

clarithromycin disk indicates a resistant strain (51).

Metronidazole in vitro susceptibility testing is intrinsically less reliable in terms of inter- and

intralaboratory reproducibility and is more difficult to standardize, as results appear to be

highly dependent on atmospheric conditions (89). Elevated MICs (>8 μg/ml) have been

correlated with treatment failures, and 8 μg/ml is the threshold commonly used to define

metronidazole resistance (89). The Etest is also used to determine metronidazole MICs for

resistant isolates, but comparisons with broth dilution results may not correlate fully (89).

The agar diffusion method with disks can be used for testing susceptibility to other antibiotics

less commonly used in eradication, such as tetracycline, ciprofloxacin, and rifabutin. For

instance, isolates were recorded as resistant if the growth inhibition zone for tetracycline was

<30 mm (10-μg disk) (21) and if any inhibition zone was observed for ciprofloxacin (1-μg

disk) and rifampin (5-μg disk) (22). The present tentative agar dilution MIC interpretive

criteria for resistance to those antibiotics are >1 μg/ml for tetracycline, >0.5 μg/ml for

levofloxacin, and >1 μg/ml for rifabutin (22, 44, 89). For resistant isolates, the MICs can be

determined using the Etest.

Genotypic Susceptibility Testing of H. pylori Cultures and in

Biopsy Specimens

Resistance to clarithromycin in H. pylori is attributed to point mutations at sites (A2142G and

A2143G) in the peptidyltransferase region of domain V of the 23S rRNA gene which inhibit

macrolide binding (137). Several methods involving gene amplification, rapid sequencing by

pyrosequencing, or fluorescent in situ hybridization have been developed for the rapid

detection of mutations associated with clarithromycin resistance (89, 105). PCR-RFLP

analysis was initially used on isolates to detect relevant mutations, but real-time PCR now

provides a simpler and more rapid approach. Adaptations allow detection with excellent

sensitivity of both H. pylori and its resistance to clarithromycin directly from gastric biopsy

specimens (13, 21, 100, 118). Real-time PCR assays also are available to ascertain

resistance to tetracycline by rapid detection of 16S rRNA gene point mutations (72) and to

ciprofloxacin/levofloxacin by rapid detection of point mutations in the quinolone resistancedetermining

region of the gyrA gene (43). Likewise, a real-time PCR test has been developed

to ascertain resistance to rifabutin by rapid detection of point mutations in the rpoB gene

(142). In contrast, development of a DNA-based assay to detect H. pylori resistance to

metronidazole has proved more problematic, as the mechanisms of in vitro resistance have

yet to be fully elucidated. Multiple null mutations in the NADPH nitroreductase

gene (rdxA) and in the NAD(P) H flavin oxidoreductase gene (frxA) may contribute to the

induction of resistance, but neither provides consistent markers for in vitro resistance testing

(15, 17). The metronidazole resistance phenotype may involve more-complex metabolic

changes than inactivation of therdxA and frxA genes, as there is evidence of a role for

oxygen and the intracellular redox status (60). Detection of RdxA protein by immunoblotting

is possible but needs further development (71).

Genotypic Susceptibility Testing of H. pylori in Feces

A biprobe 23S rRNA gene real-time PCR assay has been developed for direct clarithromycin

susceptibility testing of H. pylori in stool specimens (118), and an evaluation of a modified

version, the Helicobacter pyloriClariRes assay (Ingenetix, Vienna, Austria), reported that it

was at least as sensitive and more specific than the stool antigen test (112). However,

another evaluation of the assay on stool specimens from symptomatic children reported a

sensitivity of only 63% (77), and the resultant discussion highlighted the importance of

appropriate laboratory practice, especially in handling of the stool sample, to ensure accurate

performance of the assay (80).

Susceptibility Testing of Gastric HHLO

Optimal treatment remains to be established for HHLO, although there is evidence that

eradication by antimicrobial therapy, such as that used in conventional H. pylori eradication,

results in the resolution of gastritis and peptic ulcer disease (46, 59) as well as “H.

heilmannii”-associated, primary, low-grade MALT lymphoma (95). Although susceptibilities in

vitro were described for multiple isolates (from one patient) of an HHLO subsequently

identified as H. bizzozeronii (2, 57), usually there are no cultures of HHLO available for

testing. Consequently, there is no information for HHLO on their frequency of resistance to

clarithromycin and other antibiotics. To ascertain possible treatment options for HHLO, triple

therapy was shown to significantly reduce burden in experimentally infected mouse stomachs

(83). However, no PCR assays, such as those used to determine H. pylori clarithromycin and

tetracycline resistance in gastric biopsy tissue, have been developed for direct testing of

resistance in HHLO.

Susceptibility Testing of Enterohepatic Helicobacters

No recommended guidelines are available for treatment of a diagnosed infection with the

enterohepatic helicobacters H. cinaedi, H. canis, H. fennelliae, and H. pullorum and intestinal

flexispira-like helicobacters. Various antibiotic agents alone or in combination have been

successfully used in treating such infections, but there is insufficient information to

determine resistance rates for individual species. For the more commonly reported H.

cinaedi, effective therapy for infection may require prolonged courses for at least 2 to 3

weeks of multiple antibiotics, such as erythromycin, ciprofloxacin, gentamicin, levofloxacin,

tetracycline, and beta-lactams (63, 84, 102). Susceptibility testing of H. cinaedi appears to

be meaningful, as resistance in vitro has been correlated with treatment failures (63).

However, there are no guidelines for antimicrobial susceptibility testing with interpretive

criteria currently recommended for enterohepatic helicobacters. As a guide, it may be noted

that in testing H. cinaedi from a recurrent-bacteremia case, the approach used was

interpretation of susceptibility for clarithromycin based on the CLSI guidelines for H.

pylori and on published reports for metronidazole and amoxicillin but that for other

antibiotics, interpretation was based on CLSI guidelines for gram-negative bacilli (131).

EVALUATION, INTERPRETATION, AND REPORTING OF

RESULTS Back to top

The principal noninvasive tests for diagnosis of an H. pylori infection before treatment are

the UBT, enzyme immunoassay-based stool antigen tests, and high-accuracy ELISA-based

IgG serology. According to the clinical setting, endoscopic investigation may be indicated,

and then rapid urease testing, histology, and culture of gastric biopsy specimens can be

used. To assess H. pylori status for posttreatment follow-up, the UBT and stool antigen tests

are the recommended noninvasive tests, but not IgG serology, as serum antibody

concentrations fall slowly after eradication. In addition to test performance, other factors,

such as cost-effectiveness and patient attitudes, need to be considered in test selection

(33, 88). To perform antimicrobial susceptibility testing, bacteriological culture of H.

pylori from gastric biopsy specimens is recommended, especially in cases of repeated

treatment failure. Successful culture may be reported if the organism is microaerobic, has a

gram-negative morphology, and is oxidase, catalase, and urease positive. If culture is not

positive after 10 days of incubation, it can be reported as negative, but if clinical tests

indicate an H. pyloriinfection, it may be informative to perform a species-specific PCR assay

directly on the gastric biopsy specimen. Because of the potential unreliability of PCR assays,

resulting in false positives, such tests should not be used as the sole basis for diagnosis.

Testing for clarithromycin susceptibility should be performed using either the CLSI reference

method or a substantially equivalent method. Direct PCR testing of cultures or biopsy

specimens provides a rapid alternative to phenotypic testing to detect the presence of

discrete mutations conferring macrolide resistance. Eradication therapies are also likely to

include other agents, such as amoxicillin, metronidazole, and tetracycline and in problem

cases possibly rifabutin, levofloxacin, and furazolidone, depending on local clinical practice.

Interpretive criteria for these antimicrobials, where available, may be “tentative” but should

be used in the absence of recommended guidelines.

Gastric infection with non-pylori Helicobacter species is less common and should be

diagnosed from bacterial morphology in gastric biopsy specimens. In the microbiology

laboratory, they may be detected by an HHLO-specific PCR assay. Because of the lack of

rapid diagnostic methods for the enterohepatic species, these must be cultured for a

definitive identification. Enteric species such as H. canadensis and H. pullorum may

occasionally be isolated by techniques employed for the isolation of Campylobacter species,

particularly if nonselective media and incubation at 37°C are employed.

As Campylobacter isolates are typically only cursorily identified routinely,

enteric Helicobacter species are likely to be missed. Although they may have a limited role in

human gastroenteritis, their significance remains unclear. Other species, such as H. cinaedi,

H. canis, and H. fennelliae, may be rarely encountered from blood culture and other sites of

infection. They are unlikely to grow well aerobically but may be apparent after prolonged

incubation in an atmosphere containing additional CO2or on plates incubated “anaerobically”

(conditions of strict anaerobiosis will not support growth). As these enterohepatic

helicobacters are typically urease negative and can be confused with campylobacters,

accurate identification is often difficult, and a reference laboratory should be consulted. The

clinical significance of isolates may be unclear and should be assessed on a case-by-case

basis. Determination of antibiotic susceptibilities should be performed if needed to guide

antibiotic therapy decisions.