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IgM antibody response to antigens prepared from vegetative and coccoid forms of Helicobacter pylori
Experimental and Molecular Pathology 81 (2006) 171 – 175 www.elsevier.com/locate/yexmp
IgM antibody response to antigens prepared from vegetative and coccoid forms of Helicobacter pylori Manoucher Shahamat a , Navid Alem b , Mandana Asalkhou b , Nina Hamedi b , Neda Alem b , Kasra Morshedizadeh c , Mehdi Alem b,⁎ a
c
University of Maryland, Biotechnology Institute, Baltimore, MD 21202, USA b Micro Detect, Inc., 2852 Walnut Ave., Suite H-1, Tustin, CA 92780, USA Department of Internal Medicine, University Medical Center, Fresno, CA 93702, USA Received 2 February 2006, and in revised form 18 April 2006 Available online 9 June 2006
Abstract This report describes the utility of antigens prepared from different coccoid and spiral forms of Helicobacter pylori in a serological method. The presence of IgM antibody to H. pylori was determined in 22 human sera on antigens prepared from 24 strains of H. pylori. Antigens prepared from spiral form of certain strains of H. pylori detected IgM in all confirmed positive sera. Antigens obtained from the coccoid cells of the same strains could not completely detect IgM in the same sera. Testing sera on boosted antigens of the coccoid cells showed reduction in the number of false negative, indicating that the coccoid cells do not have one or more antigenic fractions essential for accurate detection of antibody. Our data suggest that H. pylori may lose CagA during the coccoid conversion process and regain it in the spiral form. In conclusion, we suggest that the antigen used for the detection of antibodies to H. pylori in serological methods should contain a broad spectrum of antigenic fractions and should be prepared from certain strains and culturable cells of H. pylori. © 2006 Elsevier Inc. All rights reserved. Keywords: IgM antibody; Helicobacter pylori; Antigen; Spiral form; Coccoid form; ELISA; CagA
Introduction Results from a large number of studies indicate that H. pylori is an etiologic agent of type B gastritis. This bacterium is the most common cause of infection worldwide, infecting more than half of the world population. Although H. pylori infections are more common in the developing world than in industrialized countries, still nearly one-third of all people in industrialized countries are infected and 10–15% of all infections result in duodenal ulcer or gastric cancer diseases. It is estimated that H. pylori infection is the leading cause of gastric cancer worldwide, and in developing countries, this organism induces acute bleeding duodenal ulcers which result in many deaths (Blaser and Berg, 2001; De Pascalis et al., 1999; Del Giudice et al., 2001; Hassall, 2001; Owen, 1995; Chanto et al., 2002; Peterson et al., 2000). Clinical aspects of H. pylori infection have been extensively reviewed (Statt et al., 1996; ⁎ Corresponding author. Fax: +1 714 832 8231. E-mail address: [email protected] (M. Alem). URL: http://www.microdetect.com (M. Alem). 0014-4800/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.yexmp.2006.04.003
Suerbaum and Michetti, 2002; Taylor and Parsonnet, 1995). The primary transmission route of H. pylori to humans appears to be by the fecal–oral route. Recent studies carried out in Peru, Japan and the United States have identified waterborne transmission of H. pylori (Fox, 1995; Sasaki et al., 1999; Sheri et al., 2004). Both live H. pylori and its DNA were found in well water and groundwater (Brown, 2000). Epidemiological studies and DNA analyses of the isolated strains have supported the view that contaminated well water is the origin of H. pylori infections in many parts of the world (Cellini et al., 2005; Deltenre and de Koster, 2000; Velazquez and Feirtag, 1999; Megraud, 1993). Widespread dissemination of H. pylori infection in the United States (30–40%) results in 200,000– 400,000 of new cases of duodenal ulcers every year (Megraud, 1993). Control protocols for minimizing H. pylori spread do not exist, and gastrointestinal illnesses are the major contributor of escalating health care costs (Banatavla et al., 1994). Although the prevalence of H. pylori infection varies worldwide, higher infection rates are seen in developing countries (Cave, 1997; Dore et al., 1999; Klein et al., 1991). Serological evidence shows that 52% of the world population (ranging from 15% in Australia to 87% in
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Poland) is infected with this pathogen, and certain ethnic groups are at a higher risk (Horiuchi et al., 2001; Lindkvist et al., 1999). A variety of tests are available for detection of H. pylori infections. Many reports have shown that serological techniques are simple, reliable and convenient for detection of antibodies to H. pylori in infected individuals. The advantages of serological methods are that these methods are not complicated, time-consuming, expensive or invasive. Although detection of specific IgG and IgA antibodies to H. pylori has been used for diagnosis of infections, the titers of both IgG and IgA antibodies remain detectable for a long period of time after eradication of infection. Therefore, testing sera for IgM to H. pylori, in conjunction with IgG and IgA detection, may be used to differentiate between current and past infections (Alem et al., 2002). There are significant differences in the choice of H. pylori antigens used for antibody detection. H. pylori antigens contain proteins with molecular weight of 14, 19, 30, 44–66, 84, 110 and 120 kDa. Studies for detection of antibodies to H. pylori have shown inconsistent antibody response pattern to H. pylori antigenic fractions from patient to patient (Newell, 1989; Newell and Satcey, 1989). Using the Western blot method, Alem et al. have confirmed these findings for detection of IgG in serum (Alem et al., 1993a), IgG in urine (Alem et al., 1993b), IgA in serum (unpublished data) and IgM in serum (Alem et al., 2002). All these studies have shown that, in serological methods, all antigenic fractions of H. pylori should be used. Therefore, selection of antigen prepared from the right strain or a mixture of antigens prepared from more than one strain is essential for better performance. Detection of IgM antibodies to H. pylori and its diagnostic values has been previously reported. In 2002, Alem et al. (2002) evaluated the usefulness of the detection of IgM antibodies to H. pylori using 9043 human sera. In their study, they used ELISA method and an antigenic mixture of two H. pylori strains (ATCC 43504 and 43629). Antigens obtained from these two strains, in addition to urease, contain a significant amount of CagA and VacA protein fractions. It has been reported that CagA and VacA positive H. pylori strains predominate in patients with ulcers and peptic cancer. Published reports suggest that CagA and VacA proteins are better markers for detection of antibodies to those H. pylori strains which are associated with the greatest clinical impact (Telford et al., 1994; Cavacci et al., 1993). In their study (Alem et al., 2002), Alem et al. reported that 2.2% of randomly selected human sera are positive for IgM to H. pylori. In another unpublished study performed at Micro Detect, Inc., testing 546 randomly selected sera for IgM to H. pylori resulted in selecting only 12 IgM positive (2.19%) specimens. The primary purpose of the present study was to determine the IgM antibody response to antigens prepared from 24 different coccoid and vegetative (spiral) forms of H. pylori.
Table 1 General information related to different strains of H. pylori used in this study and results obtained by testing all positive and negative sera on all antigens prepared from spiral form of 24 strains of H. pylori #
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
H. pylori strains
Urease/ Source Catalase tests
Sera
HP-MS-52 a 26694 HP-MS-RA a 31604 a 52815 a 33098 A-314 11219 a HP-MS-61 a 33097 11639 HP-MS-186 43629 52815 a HP-MS-UM1a 12648 a 11673 20200 a TX30a a HP-AMS a HP-MS-AN-19 11616 a 95e 7546 a 43504 and 43629 b
+/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+
LH PHLS LH PHLS PHLS PHLS PHLS PHLS LH PHLS NCTC LH PHLS PHLS LH
11 12 11 10 11 12 12 10 11 12 12 12 12 9 9
1 0 1 2 1 0 0 2 1 0 0 0 0 3 3
10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
+/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+
PHLS NCTC PHLS PHLS LH LH PHLS PHLS PHLS ATCC and PHLS
11 12 10 11 11 12 10 12 10 12
1 0 2 1 1 0 2 0 2 0
10 10 10 10 10 10 10 10 10 10
0 0 0 0 0 0 0 0 0 0
Positive
Negative
True + False − True − False +
PHLC: Public Health Laboratory Service, London, UK. NCTC: National Collection of Type Culture, London, UK, LH: isolated from biopsy samples in local hospitals, Maryland. a Produced false negative. b Control antigen.
All strains were grown on Columbia agar (Oxoid) with 7% defibrinated horse blood and subsequently incubated under microaerophilic conditions using Oxoid 3.5 l jars with BBL Campy Pak Plus (Becton Dickinson, Spark, MD) at 37°C for 72 h. The cultures were sequentially transferred twice prior to testing. Cells were collected and suspended in PBS. For induction of cells into the coccoid form, a freshly prepared suspension of three strains of H. pylori (43629, 12648 and HPAMS) was inoculated into a pre-washed 2-l flask containing 1 l of sterile water. Initial concentration of the cells was 107–108 cells/mL. Flasks were incubated at 15°C and 4°C. At established time intervals, samples were removed aseptically from the microcosms, cultured on blood agar plates for viable count and simultaneously enumerated using Acridine Orange Direct Count (AODC) as described by Hobbie et al. (1977) with some modifications. Morphological and viability changes in the cells were monitored during the process. Cells were collected from the water by centrifugation, and the pellets were re-suspended in PBS.
Patient specimens Materials and methods Bacterial strains, growth conditions and sample preparation The H. pylori strains used in this study are listed in Table 1. Twenty four strains of H. pylori obtained from NCTC (National Collection of Type Cultures, UK), PHLS (Public Health Laboratory Service, UK), and isolates from patients in local Hospitals, Baltimore, MD were included in this study. All strains were preserved at −80°C in blood before culturing.
A total of 22 serum specimens selected from randomly tested sera were evaluated in this study. Of these specimens, 12 were confirmed positive for IgM to H. pylori and the rest were tested negative for IgM, IgG and IgA to H. pylori. Of 12 positive specimens, 9 of them were only IgM positive. All IgM positive and IgM negative sera were selected by testing 546 sera from individuals attending a clinical laboratory in Southern California for evaluation of various medical disorders. A significant number of these specimens (random specimens) were sent to the laboratory mainly for clinical chemistry tests.
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Table 2 Comparison between results obtained by testing all positive sera on antigens prepared from coccoid and spiral forms of three selective strains of H. pylori Antigen used in ELISA was prepared from 12648 HP-AMS 43629
Positive sera Spiral forms
Coccoid forms
Positive
False negative
Positive
False negative
11/12 11/12 12/12
1 1 0
10/12 10/12 9/12
2 2 3
Urease test Urease activity of all strains used in this study was determined by the method previously described (Alem et al., 1993b). A liquid urease–catalase test, which consisted of 1 mL of a 2% urea–hydrogen peroxide solution containing bromothymol blue as an indicator, was used for the detection of urease and catalase.
SDS-PAGE and Western blot analysis The protein components of H. pylori antigens prepared from vegetative form of strain 43629 (RSB6) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The Western blot analysis of the antigen was performed by the method previously described (Alem et al., 2002).
ELISA method The status of serum IgM to H. pylori for all patient specimens was determined using PYLORI DETECT IgM assay (Micro Detect Inc., Tustin, CA). The same specimens were also tested in ELISAs which contained specific antigens prepared from each one of the strains presented in Table 1. Each patient specimen was first diluted 1:10 in 1 mg/mL goat anti-human IgG before adjustment of the final dilution of 1:100. The reactivity of serum IgM to antigens prepared from all strains was determined by using ELISA reagents manufactured by Micro Detect Inc.
Results Table 1 shows the results of IgM detection in all 22 patients' sera using the antigens prepared from different strains. All 10 confirmed IgM negative sera were negative on antigens prepared from all 24 strains of H. pylori. Of the 24 different prepared antigens, only 10 of them were able to detect all 12 confirmed
Fig. 2. A typical Western blot pattern showing the location of CagA fraction. Specific H. pylori IgG antibody in the serum of patient number 8 reacted with CagA fraction present in the antigen prepared from vegetative form of H. pylori strain 43629.
IgM positive sera. The rest (fourteen) of antigen preparations were not able to detect IgM antibody in all tested sera, and a number of positive sera turned to false negative (Table 1). The titer of IgM antibodies in all false negative sera determined as EV (ELISA Value) was close to the borderline zone, indicating that the antigens prepared from these 14 strains did not contain all antigenic fractions of H. pylori necessary for the antibody measurements. The deficiency in antigens prepared from the 14 strains was confirmed by boosting these antigens with an antigen mixture of 43504 and 43629 strains (1:1) and retesting the positive sera. The majority of false negative sera turned positive on boosted antigens. Table 2 shows the results obtained by testing all positive sera on antigens prepared from coccoid forms of selected strains. Usingantigens prepared from coccoid forms of three selected H. pylori strains in ELISA resulted in obtaining a high number of false negatives. The number of specimens which turned to false negative on antigen prepared from spiral forms of the same strains was fewer, indicating that one or more antigenic fractions of tested strains were absent in antigens prepared from coccoid forms. SDSPAGE (Fig. 1) and Western blot studies (Fig. 2) performed in our laboratory showed that strain 43629 is a CagA and VacA positive strain of H. pylori. Since the number of false negatives on the antigen prepared from coccoid form of this strain was higher than the other two strains, we concluded that the lost fraction could be CagA. Discussion
Fig. 1. SDS-PAGE of antigen prepared from H. pylori strain 43629. Lane M represents the molecular mass markers. Lanes 1–7 contain different concentrations of SDS-treated antigen prepared from vegetative form of strain 43629. Location of fraction 120 kDa (CagA) can clearly be identified in lanes 1–3.
Results from a number of studies have shown a close correlation between the presence of H. pylori on the gastric mucosa of patients and histologically confirmed gastritis, peptic ulcer diseases and gastric carcinoma (Peterson et al., 2000; Suerbaum and Michetti, 2002). Reports have shown that detection of antibodies to H. pylori can be used for diagnostic purposes (Statt et al., 1996). Since the use of specific IgM serves a diagnostic tool for establishing active or current infection, in this study, we determined the IgM antibody response to antigens prepared from
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different coccoid and vegetative forms of H. pylori. Our data showed that antigen prepared from a number of vegetative strains used in our study did not posses all antigenic fractions and therefore a number of positive specimens turned to false negative when antigens from these strains were used in ELISA. Boosting the antigens prepared from these strains with an antigen mixture of 43504 and 43629 strains resulted in reduction in the number of false negatives. Sixteen of the total (23) false negatives (64%) turned positive after the boosting process, confirming deficiencies in the antigens of certain strains of H. pylori. This finding confirms our previous work (Alem et al., 2002; Alem et al., 1993a) and shows that, for a reliable detection of antibodies to H. pylori, selection of the right antigen in the assays is a major factor. H. pylori can exist in both spiral and coccoid forms. The coccoid form of H. pylori was described soon after its discovery. The coccoid forms of H. pylori may be viable but are not culturable (Boucher et al., 1994; Shahamat et al., 2004). The changes in the morphology of H. pylori, accompanied by physiological changes, make it very difficult to recover this bacterium by routine culturing methods (Shahamat et al., 2004). However, under special laboratory procedures, it is possible to resuscitate the coccoid cells (Shahamat et al., 1993). The morphological changes may represent a survival strategy of H. pylori and a number of other non-spore forming bacteria in the environment, something similar to the spores of spore forming bacteria. This may therefore be the method of transfer of H. pylori to the new hosts and may also be the reason that H. pylori survives in the environment (Hulter et al., 1996). Roe et al. (1999) studied the changes in the antigenic profiles and morphology of H. pylori during coccoid conversion and concluded that the pattern of antigenic fractions of H. pylori changes during coccoid conversion and protein bands at 120 kDa (CagA) and 35 kDa was not detected in coccoid forms. Figueroa et al. (2002) studied IgG response to the antigens of coccoid forms of H. pylori. They reported identical serum IgG response to the antigens from both coccoid and spiral forms. They also observed higher OD in ELISA on coccoid antigen preparation. These authors concluded that the higher OD obtained on the antigens from coccoid forms was related to the overexpression of one or more major epitopes in the coccoid forms. In this study, we analyzed IgM responses to the antigens prepared from coccoid and spiral forms of H. pylori cells. We induced conversion from spiral to coccoid form in three selective strains of H. pylori (Table. 2) and compared IgM responses to antigens preared from spiral and coccoid forms of H. pylori. The resulting data showed a higher number of false negatives on antigens prepared from coccoid cells. These findings clearly showed that during the conversion certain antigenic fractions of H. pylori disappear. Since the number of false negatives on antigens prepared from coccoid forms of 43629 strain (A CagA positive strain, Figs. 1 and 2) was higher (3/12), we concluded that the 120 kDa fraction is lost during coccoid conversion. This finding confirms the Roe et al. (1999) study. It also explains the reason for the higher OD obtained in ELISA on antigens prepared from coccoid forms in the Figueroa et al. (2002) study. The major antigen of H. pylori is urease and in the absence of CagA this protein has a higher binding chance to microwells in ELISAs. Since a significant number of sera obtained
from individuals infected by H. pylori do not have antibody to CagA, the higher OD in ELISA is related to the higher binding of antibodies to urease fractions. H. pylori, like other Gram-negative bacteria, may lose certain periplasmic proteins if subjected to osmotic shock. Our years of experience, working with H. pylori, has clearly shown that harvesting and subsequent suspension of H. pylori should be performed using buffer solutions which have the right pH and osmolarity to prevent leakage of certain antigenic fractions. Apel et al. (1988) were the first to analyze the 120 kDa (CagA) protein in H. pylori and found it to be a surface located protein. Odenbreit et al. (1999) suggested that CagA is a protein located in the periplasmic space. The result of our study suggests that, during the conversion to coccoid form, some strains of H. pylori either completely or partially lose CagA, which is the most important protein and has the greatest clinical and pathological impact on H. pylori infections. This phenomenon was also observed by Carbone et al. (2005). They were able to detect CagA from seven of thirty six environmental samples (19.4%). Since the coccoid form of H. pylori retains its urease activity, it is able to induce immune response, as previously suggested in many studies. This study confirms our previous works and suggests using antigens containing CagA for detection of antibodies to H. pylori in clinical specimens. It also shows that antigen prepared from coccoid form of H. pylori may not be a useful tool for detection of anti-H. pylori antibodies in patient sera. Furthermore, Weaver et al. (1999) reported the presence of coccoid form of H. pylori cells in stool. Hulter et al. (1996) suggested that H. pylori may be in the form of coccoid in stool and is exerted and survives in the environment in this form. As previously stated, in the coccoid form, H. pylori is viable but nonculturable. This may be the main reason associated with the difficulty of culturing the organism from stool. Presence of coccoid forms in stool supports fecal–oral transmission of H. pylori and the high prevalence of H. pylori infection in the developing world. It seems likely that many strains of H. pylori lose CagA protein in the coccoid stage and regain it after a period of time and conversion to spiral forms. As stated above, CagA positive strains of H. pylori are associated with the development of gastric cancer (Argen et al., 2005). It is shown that in animals coccoid forms of H. pylori can develop gastric inflammation with the same severity as infection with the spiral form (Nilsson et al., 2002). This ability of the coccoid cells indicates that H. pylori can persist in the environment in a dormant state for a long period of time, and when ingested, this bacterium may regain infectivity and produce the gastric disease. With fecally contaminated waters, the potential exists for fecal– oral transmission of H. pylori via water (Lu et al., 2002). Acknowledgment This research was partially supported by Micro Detect Inc. grant COMB05106. References Alem, M., Foley, T.J., Dooley, C.P., 1993a. Antibody response to H. pylori antigen fractions. “93rd General Meeting of ASM” (Abstract V-13). Atlanta, GA.
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IgM antibody response to antigens prepared from vegetative and coccoid forms of Helicobacter pylori Manoucher Shahamat a , Navid Alem b , Mandana Asalkhou b , Nina Hamedi b , Neda Alem b , Kasra Morshedizadeh c , Mehdi Alem b,⁎ a
c
University of Maryland, Biotechnology Institute, Baltimore, MD 21202, USA b Micro Detect, Inc., 2852 Walnut Ave., Suite H-1, Tustin, CA 92780, USA Department of Internal Medicine, University Medical Center, Fresno, CA 93702, USA Received 2 February 2006, and in revised form 18 April 2006 Available online 9 June 2006
Abstract This report describes the utility of antigens prepared from different coccoid and spiral forms of Helicobacter pylori in a serological method. The presence of IgM antibody to H. pylori was determined in 22 human sera on antigens prepared from 24 strains of H. pylori. Antigens prepared from spiral form of certain strains of H. pylori detected IgM in all confirmed positive sera. Antigens obtained from the coccoid cells of the same strains could not completely detect IgM in the same sera. Testing sera on boosted antigens of the coccoid cells showed reduction in the number of false negative, indicating that the coccoid cells do not have one or more antigenic fractions essential for accurate detection of antibody. Our data suggest that H. pylori may lose CagA during the coccoid conversion process and regain it in the spiral form. In conclusion, we suggest that the antigen used for the detection of antibodies to H. pylori in serological methods should contain a broad spectrum of antigenic fractions and should be prepared from certain strains and culturable cells of H. pylori. © 2006 Elsevier Inc. All rights reserved. Keywords: IgM antibody; Helicobacter pylori; Antigen; Spiral form; Coccoid form; ELISA; CagA
Introduction Results from a large number of studies indicate that H. pylori is an etiologic agent of type B gastritis. This bacterium is the most common cause of infection worldwide, infecting more than half of the world population. Although H. pylori infections are more common in the developing world than in industrialized countries, still nearly one-third of all people in industrialized countries are infected and 10–15% of all infections result in duodenal ulcer or gastric cancer diseases. It is estimated that H. pylori infection is the leading cause of gastric cancer worldwide, and in developing countries, this organism induces acute bleeding duodenal ulcers which result in many deaths (Blaser and Berg, 2001; De Pascalis et al., 1999; Del Giudice et al., 2001; Hassall, 2001; Owen, 1995; Chanto et al., 2002; Peterson et al., 2000). Clinical aspects of H. pylori infection have been extensively reviewed (Statt et al., 1996; ⁎ Corresponding author. Fax: +1 714 832 8231. E-mail address: [email protected] (M. Alem). URL: http://www.microdetect.com (M. Alem). 0014-4800/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.yexmp.2006.04.003
Suerbaum and Michetti, 2002; Taylor and Parsonnet, 1995). The primary transmission route of H. pylori to humans appears to be by the fecal–oral route. Recent studies carried out in Peru, Japan and the United States have identified waterborne transmission of H. pylori (Fox, 1995; Sasaki et al., 1999; Sheri et al., 2004). Both live H. pylori and its DNA were found in well water and groundwater (Brown, 2000). Epidemiological studies and DNA analyses of the isolated strains have supported the view that contaminated well water is the origin of H. pylori infections in many parts of the world (Cellini et al., 2005; Deltenre and de Koster, 2000; Velazquez and Feirtag, 1999; Megraud, 1993). Widespread dissemination of H. pylori infection in the United States (30–40%) results in 200,000– 400,000 of new cases of duodenal ulcers every year (Megraud, 1993). Control protocols for minimizing H. pylori spread do not exist, and gastrointestinal illnesses are the major contributor of escalating health care costs (Banatavla et al., 1994). Although the prevalence of H. pylori infection varies worldwide, higher infection rates are seen in developing countries (Cave, 1997; Dore et al., 1999; Klein et al., 1991). Serological evidence shows that 52% of the world population (ranging from 15% in Australia to 87% in
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Poland) is infected with this pathogen, and certain ethnic groups are at a higher risk (Horiuchi et al., 2001; Lindkvist et al., 1999). A variety of tests are available for detection of H. pylori infections. Many reports have shown that serological techniques are simple, reliable and convenient for detection of antibodies to H. pylori in infected individuals. The advantages of serological methods are that these methods are not complicated, time-consuming, expensive or invasive. Although detection of specific IgG and IgA antibodies to H. pylori has been used for diagnosis of infections, the titers of both IgG and IgA antibodies remain detectable for a long period of time after eradication of infection. Therefore, testing sera for IgM to H. pylori, in conjunction with IgG and IgA detection, may be used to differentiate between current and past infections (Alem et al., 2002). There are significant differences in the choice of H. pylori antigens used for antibody detection. H. pylori antigens contain proteins with molecular weight of 14, 19, 30, 44–66, 84, 110 and 120 kDa. Studies for detection of antibodies to H. pylori have shown inconsistent antibody response pattern to H. pylori antigenic fractions from patient to patient (Newell, 1989; Newell and Satcey, 1989). Using the Western blot method, Alem et al. have confirmed these findings for detection of IgG in serum (Alem et al., 1993a), IgG in urine (Alem et al., 1993b), IgA in serum (unpublished data) and IgM in serum (Alem et al., 2002). All these studies have shown that, in serological methods, all antigenic fractions of H. pylori should be used. Therefore, selection of antigen prepared from the right strain or a mixture of antigens prepared from more than one strain is essential for better performance. Detection of IgM antibodies to H. pylori and its diagnostic values has been previously reported. In 2002, Alem et al. (2002) evaluated the usefulness of the detection of IgM antibodies to H. pylori using 9043 human sera. In their study, they used ELISA method and an antigenic mixture of two H. pylori strains (ATCC 43504 and 43629). Antigens obtained from these two strains, in addition to urease, contain a significant amount of CagA and VacA protein fractions. It has been reported that CagA and VacA positive H. pylori strains predominate in patients with ulcers and peptic cancer. Published reports suggest that CagA and VacA proteins are better markers for detection of antibodies to those H. pylori strains which are associated with the greatest clinical impact (Telford et al., 1994; Cavacci et al., 1993). In their study (Alem et al., 2002), Alem et al. reported that 2.2% of randomly selected human sera are positive for IgM to H. pylori. In another unpublished study performed at Micro Detect, Inc., testing 546 randomly selected sera for IgM to H. pylori resulted in selecting only 12 IgM positive (2.19%) specimens. The primary purpose of the present study was to determine the IgM antibody response to antigens prepared from 24 different coccoid and vegetative (spiral) forms of H. pylori.
Table 1 General information related to different strains of H. pylori used in this study and results obtained by testing all positive and negative sera on all antigens prepared from spiral form of 24 strains of H. pylori #
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
H. pylori strains
Urease/ Source Catalase tests
Sera
HP-MS-52 a 26694 HP-MS-RA a 31604 a 52815 a 33098 A-314 11219 a HP-MS-61 a 33097 11639 HP-MS-186 43629 52815 a HP-MS-UM1a 12648 a 11673 20200 a TX30a a HP-AMS a HP-MS-AN-19 11616 a 95e 7546 a 43504 and 43629 b
+/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+
LH PHLS LH PHLS PHLS PHLS PHLS PHLS LH PHLS NCTC LH PHLS PHLS LH
11 12 11 10 11 12 12 10 11 12 12 12 12 9 9
1 0 1 2 1 0 0 2 1 0 0 0 0 3 3
10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
+/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+
PHLS NCTC PHLS PHLS LH LH PHLS PHLS PHLS ATCC and PHLS
11 12 10 11 11 12 10 12 10 12
1 0 2 1 1 0 2 0 2 0
10 10 10 10 10 10 10 10 10 10
0 0 0 0 0 0 0 0 0 0
Positive
Negative
True + False − True − False +
PHLC: Public Health Laboratory Service, London, UK. NCTC: National Collection of Type Culture, London, UK, LH: isolated from biopsy samples in local hospitals, Maryland. a Produced false negative. b Control antigen.
All strains were grown on Columbia agar (Oxoid) with 7% defibrinated horse blood and subsequently incubated under microaerophilic conditions using Oxoid 3.5 l jars with BBL Campy Pak Plus (Becton Dickinson, Spark, MD) at 37°C for 72 h. The cultures were sequentially transferred twice prior to testing. Cells were collected and suspended in PBS. For induction of cells into the coccoid form, a freshly prepared suspension of three strains of H. pylori (43629, 12648 and HPAMS) was inoculated into a pre-washed 2-l flask containing 1 l of sterile water. Initial concentration of the cells was 107–108 cells/mL. Flasks were incubated at 15°C and 4°C. At established time intervals, samples were removed aseptically from the microcosms, cultured on blood agar plates for viable count and simultaneously enumerated using Acridine Orange Direct Count (AODC) as described by Hobbie et al. (1977) with some modifications. Morphological and viability changes in the cells were monitored during the process. Cells were collected from the water by centrifugation, and the pellets were re-suspended in PBS.
Patient specimens Materials and methods Bacterial strains, growth conditions and sample preparation The H. pylori strains used in this study are listed in Table 1. Twenty four strains of H. pylori obtained from NCTC (National Collection of Type Cultures, UK), PHLS (Public Health Laboratory Service, UK), and isolates from patients in local Hospitals, Baltimore, MD were included in this study. All strains were preserved at −80°C in blood before culturing.
A total of 22 serum specimens selected from randomly tested sera were evaluated in this study. Of these specimens, 12 were confirmed positive for IgM to H. pylori and the rest were tested negative for IgM, IgG and IgA to H. pylori. Of 12 positive specimens, 9 of them were only IgM positive. All IgM positive and IgM negative sera were selected by testing 546 sera from individuals attending a clinical laboratory in Southern California for evaluation of various medical disorders. A significant number of these specimens (random specimens) were sent to the laboratory mainly for clinical chemistry tests.
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Table 2 Comparison between results obtained by testing all positive sera on antigens prepared from coccoid and spiral forms of three selective strains of H. pylori Antigen used in ELISA was prepared from 12648 HP-AMS 43629
Positive sera Spiral forms
Coccoid forms
Positive
False negative
Positive
False negative
11/12 11/12 12/12
1 1 0
10/12 10/12 9/12
2 2 3
Urease test Urease activity of all strains used in this study was determined by the method previously described (Alem et al., 1993b). A liquid urease–catalase test, which consisted of 1 mL of a 2% urea–hydrogen peroxide solution containing bromothymol blue as an indicator, was used for the detection of urease and catalase.
SDS-PAGE and Western blot analysis The protein components of H. pylori antigens prepared from vegetative form of strain 43629 (RSB6) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The Western blot analysis of the antigen was performed by the method previously described (Alem et al., 2002).
ELISA method The status of serum IgM to H. pylori for all patient specimens was determined using PYLORI DETECT IgM assay (Micro Detect Inc., Tustin, CA). The same specimens were also tested in ELISAs which contained specific antigens prepared from each one of the strains presented in Table 1. Each patient specimen was first diluted 1:10 in 1 mg/mL goat anti-human IgG before adjustment of the final dilution of 1:100. The reactivity of serum IgM to antigens prepared from all strains was determined by using ELISA reagents manufactured by Micro Detect Inc.
Results Table 1 shows the results of IgM detection in all 22 patients' sera using the antigens prepared from different strains. All 10 confirmed IgM negative sera were negative on antigens prepared from all 24 strains of H. pylori. Of the 24 different prepared antigens, only 10 of them were able to detect all 12 confirmed
Fig. 2. A typical Western blot pattern showing the location of CagA fraction. Specific H. pylori IgG antibody in the serum of patient number 8 reacted with CagA fraction present in the antigen prepared from vegetative form of H. pylori strain 43629.
IgM positive sera. The rest (fourteen) of antigen preparations were not able to detect IgM antibody in all tested sera, and a number of positive sera turned to false negative (Table 1). The titer of IgM antibodies in all false negative sera determined as EV (ELISA Value) was close to the borderline zone, indicating that the antigens prepared from these 14 strains did not contain all antigenic fractions of H. pylori necessary for the antibody measurements. The deficiency in antigens prepared from the 14 strains was confirmed by boosting these antigens with an antigen mixture of 43504 and 43629 strains (1:1) and retesting the positive sera. The majority of false negative sera turned positive on boosted antigens. Table 2 shows the results obtained by testing all positive sera on antigens prepared from coccoid forms of selected strains. Usingantigens prepared from coccoid forms of three selected H. pylori strains in ELISA resulted in obtaining a high number of false negatives. The number of specimens which turned to false negative on antigen prepared from spiral forms of the same strains was fewer, indicating that one or more antigenic fractions of tested strains were absent in antigens prepared from coccoid forms. SDSPAGE (Fig. 1) and Western blot studies (Fig. 2) performed in our laboratory showed that strain 43629 is a CagA and VacA positive strain of H. pylori. Since the number of false negatives on the antigen prepared from coccoid form of this strain was higher than the other two strains, we concluded that the lost fraction could be CagA. Discussion
Fig. 1. SDS-PAGE of antigen prepared from H. pylori strain 43629. Lane M represents the molecular mass markers. Lanes 1–7 contain different concentrations of SDS-treated antigen prepared from vegetative form of strain 43629. Location of fraction 120 kDa (CagA) can clearly be identified in lanes 1–3.
Results from a number of studies have shown a close correlation between the presence of H. pylori on the gastric mucosa of patients and histologically confirmed gastritis, peptic ulcer diseases and gastric carcinoma (Peterson et al., 2000; Suerbaum and Michetti, 2002). Reports have shown that detection of antibodies to H. pylori can be used for diagnostic purposes (Statt et al., 1996). Since the use of specific IgM serves a diagnostic tool for establishing active or current infection, in this study, we determined the IgM antibody response to antigens prepared from
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different coccoid and vegetative forms of H. pylori. Our data showed that antigen prepared from a number of vegetative strains used in our study did not posses all antigenic fractions and therefore a number of positive specimens turned to false negative when antigens from these strains were used in ELISA. Boosting the antigens prepared from these strains with an antigen mixture of 43504 and 43629 strains resulted in reduction in the number of false negatives. Sixteen of the total (23) false negatives (64%) turned positive after the boosting process, confirming deficiencies in the antigens of certain strains of H. pylori. This finding confirms our previous work (Alem et al., 2002; Alem et al., 1993a) and shows that, for a reliable detection of antibodies to H. pylori, selection of the right antigen in the assays is a major factor. H. pylori can exist in both spiral and coccoid forms. The coccoid form of H. pylori was described soon after its discovery. The coccoid forms of H. pylori may be viable but are not culturable (Boucher et al., 1994; Shahamat et al., 2004). The changes in the morphology of H. pylori, accompanied by physiological changes, make it very difficult to recover this bacterium by routine culturing methods (Shahamat et al., 2004). However, under special laboratory procedures, it is possible to resuscitate the coccoid cells (Shahamat et al., 1993). The morphological changes may represent a survival strategy of H. pylori and a number of other non-spore forming bacteria in the environment, something similar to the spores of spore forming bacteria. This may therefore be the method of transfer of H. pylori to the new hosts and may also be the reason that H. pylori survives in the environment (Hulter et al., 1996). Roe et al. (1999) studied the changes in the antigenic profiles and morphology of H. pylori during coccoid conversion and concluded that the pattern of antigenic fractions of H. pylori changes during coccoid conversion and protein bands at 120 kDa (CagA) and 35 kDa was not detected in coccoid forms. Figueroa et al. (2002) studied IgG response to the antigens of coccoid forms of H. pylori. They reported identical serum IgG response to the antigens from both coccoid and spiral forms. They also observed higher OD in ELISA on coccoid antigen preparation. These authors concluded that the higher OD obtained on the antigens from coccoid forms was related to the overexpression of one or more major epitopes in the coccoid forms. In this study, we analyzed IgM responses to the antigens prepared from coccoid and spiral forms of H. pylori cells. We induced conversion from spiral to coccoid form in three selective strains of H. pylori (Table. 2) and compared IgM responses to antigens preared from spiral and coccoid forms of H. pylori. The resulting data showed a higher number of false negatives on antigens prepared from coccoid cells. These findings clearly showed that during the conversion certain antigenic fractions of H. pylori disappear. Since the number of false negatives on antigens prepared from coccoid forms of 43629 strain (A CagA positive strain, Figs. 1 and 2) was higher (3/12), we concluded that the 120 kDa fraction is lost during coccoid conversion. This finding confirms the Roe et al. (1999) study. It also explains the reason for the higher OD obtained in ELISA on antigens prepared from coccoid forms in the Figueroa et al. (2002) study. The major antigen of H. pylori is urease and in the absence of CagA this protein has a higher binding chance to microwells in ELISAs. Since a significant number of sera obtained
from individuals infected by H. pylori do not have antibody to CagA, the higher OD in ELISA is related to the higher binding of antibodies to urease fractions. H. pylori, like other Gram-negative bacteria, may lose certain periplasmic proteins if subjected to osmotic shock. Our years of experience, working with H. pylori, has clearly shown that harvesting and subsequent suspension of H. pylori should be performed using buffer solutions which have the right pH and osmolarity to prevent leakage of certain antigenic fractions. Apel et al. (1988) were the first to analyze the 120 kDa (CagA) protein in H. pylori and found it to be a surface located protein. Odenbreit et al. (1999) suggested that CagA is a protein located in the periplasmic space. The result of our study suggests that, during the conversion to coccoid form, some strains of H. pylori either completely or partially lose CagA, which is the most important protein and has the greatest clinical and pathological impact on H. pylori infections. This phenomenon was also observed by Carbone et al. (2005). They were able to detect CagA from seven of thirty six environmental samples (19.4%). Since the coccoid form of H. pylori retains its urease activity, it is able to induce immune response, as previously suggested in many studies. This study confirms our previous works and suggests using antigens containing CagA for detection of antibodies to H. pylori in clinical specimens. It also shows that antigen prepared from coccoid form of H. pylori may not be a useful tool for detection of anti-H. pylori antibodies in patient sera. Furthermore, Weaver et al. (1999) reported the presence of coccoid form of H. pylori cells in stool. Hulter et al. (1996) suggested that H. pylori may be in the form of coccoid in stool and is exerted and survives in the environment in this form. As previously stated, in the coccoid form, H. pylori is viable but nonculturable. This may be the main reason associated with the difficulty of culturing the organism from stool. Presence of coccoid forms in stool supports fecal–oral transmission of H. pylori and the high prevalence of H. pylori infection in the developing world. It seems likely that many strains of H. pylori lose CagA protein in the coccoid stage and regain it after a period of time and conversion to spiral forms. As stated above, CagA positive strains of H. pylori are associated with the development of gastric cancer (Argen et al., 2005). It is shown that in animals coccoid forms of H. pylori can develop gastric inflammation with the same severity as infection with the spiral form (Nilsson et al., 2002). This ability of the coccoid cells indicates that H. pylori can persist in the environment in a dormant state for a long period of time, and when ingested, this bacterium may regain infectivity and produce the gastric disease. With fecally contaminated waters, the potential exists for fecal– oral transmission of H. pylori via water (Lu et al., 2002). Acknowledgment This research was partially supported by Micro Detect Inc. grant COMB05106. References Alem, M., Foley, T.J., Dooley, C.P., 1993a. Antibody response to H. pylori antigen fractions. “93rd General Meeting of ASM” (Abstract V-13). Atlanta, GA.
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