ORIGINAL ARTICLE

Helicobacter pylori -induced chronic active gastritis in Saudipatients with special reference to the ultrastructural pathology

Mubarak Al-Shraim & Khaled Radad & Refaat A. Eid &

Fahmy El-Sayed

Received: 29 August 2013 /Accepted: 28 November 2013# Springer-Verlag London 2013

Abstract The present study was designed to address themost characteristic features of Helicobacter pylori(H. pylori )-induced chronic active gastritis in Saudipatients and to use transmission electron microscopy(TEM) to shed some light on the nature of the bacillaryand coccoid forms of H. pylori that still carries somedebate. In which, 40 antral biopsies were obtained from40 patients presented for dyspeptic disorders at AseerCentral Hospital, Abha, Saudi Arabia. Each specimenwas divided into two parts for light and TEM examina-tions. Thirty-three (82.5 %) out of the 40 specimenswere positive for H. pylori as shown by Giemsa stain.The presence of H. pylori was confirmed by immuno-staining with a polyclonal rabbit anti-H. pylori primaryantibody in 36 cases of the examined 40 antral biopsies(90 %) including all Giemsa-positive cases. Histologi-cally, 30 of the Giemsa-positive cases (90 %) showed

chronic active gastritis characterized by infiltration ofgastric mucosa with neutrophils, eosinophils, lympho-cytes and plasma cells. In 18 H. pylori positive patients(54.5 %), gastric glands appeared destructed and infil-trated with neutrophils. There was also intestinal gobletcell metaplasia of gastric glands in 2 (6 %) ofH. pylori -affected patients. Examination of 10 randomlyselected H. pylori positive specimens with TEM showedtwo forms of H. pylori , bacillary and coccoid forms,infecting gastric mucosa. Both forms were seen extra-cellularly in mucus layer and intercellular spaces andintracellularly in gastric epithelial cells. Interestingly,bacillary forms were seen in endocytic vesicletransforming into coccoid forms inside gastric epithelialcells. In conclusions, most of the presented patientsshowed histopathological features of chronic active gas-tritis. Ultrastructurally, bacillary form represents themain extracellular form of Helicobacter pylori and coc-coid form might be formed in the gastric epithelial cellsas a kind of bacillary transformation.

Keywords Chronic activegastritis .H.pylori .Histopathology .

Stomach .Ultrastructure

Introduction

Helicobacter pylori is a gram-negative and microaerophilicbacterium. Since its isolation byWarren andMarshall in 1983,H. pylori has been considered one of the most commonbacterial infections worldwide. It infects approximately50 % of the world’s population with a prevalence of up to90 % in developing countries (Brown 2000; Calvino-Fernández and Parra-Cid 2010). H. pylori infection was re-ported to occur from person to person through contaminatedwater and food. Risk factors for transmission of H. pylori

Mubarak Al-Shraim and Khaled Radad contributed equally to thepublication.

M. Al-ShraimPathology Department, College of Medicine,King Khalid University, Abha, Saudi Arabiae-mail: malshraim@yahoo.com

K. Radad (*)Pathology Department, Faculty of Veterinary Medicine,Assiut University, Assiut, Egypte-mail: khaledradad@hotmail.com

R. A. EidElectron Microscopy Unit, College of Medicine,King Khalid University, Abha, Saudi Arabiae-mail: refaat_eid@yahoo.com

F. El-SayedClinical Pathology Department, Faculty of Medicine, Al‐AzharUniversity, New Damietta, Egypte-mail: fahmmymohammed45@yahoo.com

Comp Clin PatholDOI 10.1007/s00580-013-1863-9

include socioeconomic status, household crowding, ethnicity,migration from high prevalence regions and infection status offamily members (Torres 2000).

Once H. pylori is acquired, it can colonize gastric mucosafor decades as the organism survives the gastric acid pH (Celliet al. 2009). Although the majority of H. pylori infections areasymptomatic, it has been reported to play an important role inthe pathogenesis of chronic active gastritis, duodenal andgastric ulcer, gastric cancer, and mucosa-associated lymphoidtissue (MALT)-lymphoma (Arnold et al. 2012). Among thesedisease presentations, chronic gastritis was reported to occurin everyone with H. pylori infection. On the other hand, asmall proportion of infected individuals experienced one ofother clinical forms (Marshall 1995). The development ofthese diseases was reported to depend upon the virulence ofH. pylori strain, host genetic susceptibility, and environmentalcofactors (Atherton 2006).

Our current study was designed to address the most char-acteristic histopathological features of H. pylori -inducedchronic active gastritis in 30 Saudi patients with dyspepticdisorder. Moreover, it tried to use TEM examination to shedsome light on the nature of bacillary and coccoid forms ofH. pylori , which still carries some debate.

Materials and methods

Study population

Forty male patients (40–55 years old) were admitted withdyspeptic disorders at Aseer Central Hospital, Abha, SaudiArabia during the year 2012. None of the patients has beenreceived treatment. During gastroscopic examination, 40 an-tral biopsies were obtained from the patients, and each spec-imen was divided into two parts for light and electron micro-scopic examinations.

Light microscopic examination

Specimens for light microscopy were collected in 10 % neu-tral buffered formalin. After 48 h of fixation, specimens wereroutinely processed for histopathological examination includ-ing embedding and sectioning. For each specimen, two slideswere made, each containing 3–4 tissue sections. The first slidewas stained with hematoxylin and eosin (HE), and the secondslide was stained with the modified Giemsa stain (rapidRomanowsky method) (Bancroft and Stevens 1996). Light

Fig. 1 Giemsa-stained andimmunostained antral specimens.a Giemsa-stained sectionshowing no evidence forH. pylori in the lumen of a gastricgland (asterisks). b Giemsa-stained section showing thepresence of numerous deep blue-stained bacilli of H. pylori in thelumen of a gastric gland(asterisks). c Immunostainedsection showing numerousH. pylori particles in gastricglands (arrow heads)

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microscopic examination of the stained sections was per-formed by investigators who were blinded to the work.

Immunohistochemistry staining

Tissue sections were deparafinized, rehydrated, and pretreatedwith citrate buffer (pH 6.0) for antigen retrieval. Then, sec-tions were kept in 3 % of hydrogen peroxide for 10 min toblock endogenous peroxidase activity. After that, sectionswere incubated with a polyclonal rabbit anti-H. pylori primaryantibody (B0471, Dako Corporation, Denmark) at a dilutionof 1:50 at room temperature for 1 h. After washing three timeswith phosphate buffered saline, the Dako EnVision Dual LinkSystem-HRP (K40561, Dako Corporation, Denmark) wasapplied for 30 min. Eventually, sections were incubated indiaminobenzidine (DAB) for 10min followed by hematoxylincounterstaining and mounting. H. pylori-infected gastric mu-cosa from chronic gastritis patients served as positive controls.

Negative controls were obtained by replacing primary anti-body with phosphate buffered saline (Rotimi et al., 2000).

Transmission electron microscopy

Transmission electron microscopy examination was selec-tively performed in 10 specimens of H. pylori activegastritis. Each specimen was trimmed, fixed in glutaralde-hyde solution in 0.1 M sodium cacodylate buffer, pH 7.2,and placed in a thermal box cooled to 4 °C for 2 h. Theywere post-fixed in 1 % osmium tetroxide in a sodiumcacodylate buffer and then dehydrated in an ascendingseries of ethyl alcohol and embedded in Spurr’s resin.Ultrathin sections stained with uranyl acetate and leadcitrate were examined by TEM (Jeol 100 CXII, Japan)operated at 80 kV in the Electron Microscopy Unit, Pa-thology Department, College of Medicine, King KhalidUniversity (Bancroft and Stevens 1996).

Fig. 2 Histopathologicalexamination of HE-stained antralsections obtained from H. pyloripositive patients. a Gastricmucosa showing desquamatedtissues in the lumen of somegastric glands (arrows) andinflammatory cellular infiltrationsin the lamina propria (asterisks).b Gastric mucosa showingneutrophilic infiltration in thelamina propria (arrows). Therewere also two gastric glands thatappeared degenerated andinfiltrated with neutrophils (G). cLamina propria showingeosinophilic infiltration (arrowheads). d Lamina propriashowing lymphoplasmocyticinfiltration (arrow heads). eLymphocytic infiltration andaggregation in the lamina propria(asterisks). There were alsodegeneration and desquamationof gastric epithelium (smallasterisks). f Gastric glandsshowing intestinal goblet cellmetaplasia (asterisks)

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Results

Histopathology

Light microscopic examination of Giemsa-stained sectionsdemonstrated that 33 out of 40 (82.5 %) patients werepositive for H. pylori infection, and seven (17 %) ofpatients were negative of the infection. H. pylori appearedas numerous deep blue-stained bacilli in the lumens ofgastric glands (Fig. 1a, b). Immunostaining of antral spec-imens with a polyclonal rabbit anti-H. pylori antibodyshowed that 36 out of 40 cases (90 %) including allGiemsa-positive cases were positive for H. pylori infection(Fig. 1c). Histopathological examination of HE-stainedsections prepared from antral biopsies showed that 30(90 %) out of 33 of the Giemsa-positive cases showedevidence of chronic active gastritis. In which, there wereneutrophilic (Fig. 2a, b), eosinophilic (Fig. 2c), andplasmolymphocytic (Fig. 2d, e) cell infiltrations of gastricmucosa. In 18 H. pylori positive patients (54.5 %), gastricglands appeared destructed and infiltrated with neutrophils(Fig. 2a, b). Intestinal goblet cell metaplasia of gastricglands was seen only in two (6 %) of H. pylori positivepatients (Fig. 2f).

Ultrastructural pathology

Transmission electron microscopy demonstrated a number ofH. pylori infecting gastric mucosa in all examined biopsies.Two forms of H. pylori , bacillary and coccoid, were seen extra-cellularly in the mucus layer (Fig. 3a) and in between gastricepithelial cells (Fig. 3b) and intracellularly in gastric epithelialcells (Fig. 3c, d). The presence ofH. pylori in between secretorygranules indicated their intracellular location inside gastric epi-thelial cells. Extracellularly,H. pylori with long flagella appearedattached to the gastric epithelial cells either by adhesion pedestals(Fig. 4a) or through membrane fusion (Fig. 4b). Entering ofH. pylori into the gastric epithelial cells occurred through phago-cytic mechanism as attached bacteria to the gastric epithelial cellssurrounded by membrane pseudopodia followed by engulfment(Fig. 4a). Interestingly, bacillary forms of H. pylori appearedtransforming into coccoid forms inside gastric epithelial cells, theprocess perhaps by which the organism protects itself fromunfavorable conditions. In which, bacillary forms blended firstforming U-shaped forms with an increase in the protoplasmiccylinder and then after, transformed into coccoid form(Fig. 3c, d). Some gastric epithelial cells showed vacuolation,myelin figures, and increased lysosomal activities (Fig. 4c). Ul-trastructural examination also revealed destruction of gastric

Fig. 3 Ultrastructuralmicrographs for gastric mucosa inH. pylori positive patients. a Anumber ofH. pylori present in themucus layer (asterisk), coccoidform (thin arrows) and bacillaryform (thick arrows). b Both formsof H. pylori appear in betweengastric epithelial cells (arrows). cThe presence of H. pylori(bacillary form) inside a gastricepithelial cell (arrows). d Somebacillary forms ofH. pylori (thickarrows) appear transforming intococcoid forms (thin arrows) insidea gastric epithelial cell. Thepresence of H. pylori in betweensecretory granules indicated theirintracellular location insidegastric epithelial cells (whiteasterisks)

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epithelium with the presence of some H. pylori in betweendestructed tissues (Fig. 4d).

Discussion

Among various diagnostic tests, histopathological examina-tion of gastric biopsies is mandatory at the initial presentationof patients because in addition to its accuracy in diagnosingH. pylori infection, it sheds some light on the status of thegastric mucosa (Burette 1998). In our current study, H. pyloriwas identified in 33 (82.5 %) and 36 (90 %) cases usingGiemsa and immunostaining, respectively. In consistent,Hartman and Owens (2012) diagnosed 83 % of H. pyloripositive cases using Giemsa-stained antral biopsies. Rotimiet al. (2000) reported that immunostaining was the highestsensitive invasive method for detection of H. pylori infectionamong other histological staining protocols. Histopathologicalexamination of antral biopsies was reported to provide aneasier and more cost-effective alternative mean for diagnosingH. pylori infection (Wabinga 2002).

Examination of HE-stained antral specimens revealed thatmost of H. pylori positive patients (90 %) had chronic activegastritis characterized by neutrophilic, eosinophilic, andlymphoplasmocytic infiltrations of gastric mucosa,

destruction of gastric glands, and intestinal goblet cell meta-plasia. Matsukawa et al. (2010) reported that both neutrophilicand lymphoplasmocytic infiltrations into the gastric mucosa isa well-known phenomenon that characterizes H. pylori-in-duced chronic active gastritis. Although no confirmed associ-ation between eosinophilic infiltration and H. pylori infection(Papadopoulos et al. 2005), some studies reported that eosin-ophils increased in H. pylori-induced gastritis (Piazuelo et al.2008). Varying degree of gastric gland destruction was seen in18 ofH. pylori positive patients. Destruction of gastric glandswas reported to be associated with chronic inflammatoryprocess induced by H. pylori infection (Dîrnu et al. 2012).No mucosal atrophy was seen as a result of glandular destruc-tion in examined specimens. Intestinal goblet cell metaplasiaof gastric glands was observed in only two cases of H. pyloripositive patients. Similarly, Fikret et al. (2001) found thatthree cases out of 42H. pylori positive cases showed intestinalgoblet cell metaplasia. Intestinal metaplasia is usuallyregarded as a precancerous lesion (Dîrnu et al. 2012).

Transmission electron microscopy showed two morpho-logical forms ofH. pylori , bacillary, and coccoid in the gastricmucosa of all examined specimens. The nature of these twoforms still carries some debate in the literature. Therefore,TEM examination was done in our study to shed some lighton the nature of bacillary and coccoid forms of H. pylori . It

Fig. 4 Ultrastructuralmicrographs for gastric mucosa inH. pylori positive patients. a Bothcoccoid (thin arrow) and bacillary(thick arrow) forms of H. pyloriattach to a gastric epithelial cell byadhesion pedestals (arrow heads).b A coccoid form with longflagellum (arrow) attaches to agastric epithelial cell throughmembrane fusion (white arrowhead). The bacterium appears in aprocess of phagocytosis (blackarrow heads). c A gastricepithelial cells showingvacuolation (V), myelin figures(thick arrow), and lysosomalactivity (thin arrow). dDestruction of gastric epitheliumwith the presence of H. pylori inbetween the destroyed tissues(arrows)

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was found that both morphological forms were seen extracel-lularly in the mucus layer and intercellular spaces betweengastric epithelial cells and intarcellularly inside gastric epithe-lial cells. Bacillary form appeared to be the main extracellularH. pylori bacteria infecting our examined specimens. In con-sistent, Agustí et al. (2010) stated thatH. pylori usually existsin bacillary form in both the natural habitat and the humanhost. On the other hand, coccoid bacteria found extracellularlyin between bacillary ones could be resulting either from sec-tional direction on bacillary form or a kind of bacillary trans-formation (Chun et al. 2002).

Some extracellular organisms appeared attaching to thegastric epithelial cells by the mean of adhesion pedestals orthrough membrane fusion followed by entering of the organ-ism into gastric epithelial cells. In this context, Ozbek et al(2010) reported that H. pylori , either cocoid or ellipsoid,attached to the epithelial cells through egg-cup-like pedestals.Also, they concluded the presence of the organism within theendocytic vesicle inside the epithelial cells indicating theirinternalization by phagocytosis. Intracellularly, both forms ofH. pylori were detected in the gastric epithelial cells. In which,bacillary form was seen in autophagic vacuoles where theytransformed into coccoid forms. Transforming process ofbacillary forms began with ingrowth in the periplasm on oneside forming U-shaped forms which eventually converted tococcoid forms. Similarly, Citterio et al (2004) observed thatbacillary form converted into coccoid form via intermediateU-shaped and V-shaped forms in prolonged culture ofH. pylori . It was reported that such conversion might be aprocess by which the organism protects itself from unfavor-able conditions such as lack of nutrients, increase of gastricpH and exposure to antibiotics (Chun et al. 2002, Agustí et al.2010, Poursina et al. 2013). Some specimens showed vacuo-lation, myelin figures, and increased lysosomal activities ingastric epithelial cells and destruction of gastric epitheliumwith the presence of some H. pylori in between destructedtissues. Degenerative changes as the result of H. pylori infec-tion was similarly reported in the form of vacuolar degenera-tion, dilated endoplasmic reticulum, reduction of organellesand destruction of cell junctions, and construction in gastricepithelial cells SGC-7901 (Wang et al. 2012). Bai et al. (2010)returned these alterative changes to the vacuolating toxin(VacA) of H. pylori .

In conclusions, most of the presented patients showedhistopathological features of chronic active gastritis. Giemsaand immunostaining confirmed H. pylori infection in 83.5and 90 % of the examined cases. Ultrastructurally, bacillaryform represents the main extracellular form of H. pylori , andcoccoid form might be formed in the gastric epithelial cells asa kind of bacillary transformation.

Acknowledgments The authors wish to thank Prof. Wolf-DieterRausch, Institute of Medical Chemistry, Department of Natural Sciences,

University of Veterinary Medicine Vienna, Austria, for proof reading thismanuscript.

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