Natural Competence Promotes Helicobacter pylori Chronic Infection

Animal models are important tools for studies of human disease, but developing these models is a particular challenge with regard to organisms with restricted host ranges, such as the human stomach pathogen Helicobacter pylori. In most cases, H. pylori infects the stomach for many decades before symptoms appear, distinguishing it from many bacterial pathogens that cause acute infection. To model chronic infection in the mouse, a human clinical isolate was selected for its ability to survive for 2 months in the mouse stomach, and the resulting strain, MSD132, colonized the mouse stomach for at least 28 weeks. During selection, the cagY component of the Cag type IV secretion system was mutated, disrupting a key interaction with host cells. Increases in both bacterial persistence and bacterial burden occurred prior to this mutation, and a mixed population of cagY⁺ and cagY mutant cells was isolated from a single mouse, suggesting that mutations accumulate during selection and that factors in addition to the Cag apparatus are important for murine adaptation. Diversity in both alleles and genes is common in H. pylori strains, and natural competence mediates a high rate of interstrain genetic exchange. Mutations of the Com apparatus, a membrane DNA transporter, and DprA, a cytosolic competence factor, resulted in reduced persistence, although initial colonization was normal. Thus, exchange of DNA between genetically heterogeneous H. pylori strains may improve chronic colonization. The strains and methods described here will be important tools for defining both the spectrum of mutations that promote murine adaptation and the genetic program of chronic infection.     

Sublingual Immunization Protects against Helicobacter pylori Infection and Induces T and B Cell Responses in the Stomach

Sublingual (SL) immunization has been described as an effective novel way to induce mucosal immune responses in the respiratory and genital tracts. We examined the potential of SL immunization against Helicobacter pylori to stimulate immune responses in the gastrointestinal mucosa and protect against H. pylori infection. Mice received two SL immunizations with H. pylori lysate antigens and cholera toxin as an adjuvant, and after challenge with live H. pylori bacteria, their immune responses and protection were evaluated, as were immune responses prior to challenge. SL immunization induced enhanced proliferative responses to H. pylori antigens in cervicomandibular lymph nodes and provided at least the same level of immune responses and protection as corresponding intragastric immunization. Protection in SL-immunized mice was associated with strong H. pylori-specific serum IgG and IgA antibody responses in the stomach and intestine, with strong proliferation and gamma interferon (IFN-γ) and interleukin-17 (IL-17) production by spleen and mesenteric lymph node T cells stimulated with H. pylori antigens in vitro, and with increased IFN-γ and IL-17 gene expression in the stomach compared to levels in infected unimmunized mice. Immunohistochemical studies showed enhanced infiltration of CD4⁺ T cells and CD19⁺ B cells into the H. pylori-infected stomach mucosa of SL-immunized but not unimmunized H. pylori-infected mice, which coincided with increased expression of the mucosal addressin cell adhesion molecule (MAdCAM-1) and T and B cell-attracting chemokines CXCL10 and CCL28. We conclude that, in mice, SL immunization can effectively induce protection against H. pylori infection in association with strong T and B cell infiltration into the stomach.At least half of the world''s population is infected with Helicobacter pylori, one of the few microorganisms known to be able to colonize the human stomach. In 10 to 15% of infected individuals, chronic H. pylori infection causes duodenal ulcers, and infection with H. pylori has been shown to be a strong risk factor for the development of gastric adenocarcinoma and malignant mucosa-associated lymphomas (3, 17, 20). Although treatment with a combination of antibiotics and a proton pump inhibitor is usually effective in individual cases, limited treatment compliance, rapidly emerging antibiotic resistance, and frequent reinfection with H. pylori in countries where it is highly endemic make vaccination an increasingly attractive alternative or complement to standard therapy.Vaccination, given either preventively or therapeutically, is especially needed in countries with a high incidence of gastric cancer (20), reinfection (22), or antibiotic resistance. However, clinical trials of various oral or parenteral H. pylori vaccine candidates have not shown much promise to date, pointing to the need for identifying improved antigen-adjuvant formulations and/or alternative routes of immunization in the quest for an effective vaccine against H. pylori (33).The importance of cell-mediated mucosal immunity in protection against experimental H. pylori infection after vaccination is well established (1, 9, 10, 23, 35). In most studies, intragastric (IG) immunization has been used to achieve efficient stimulation of the gastrointestinal immune response. However, this route usually requires large amounts of antigen for efficient immunization, and the environment in the stomach and intestine may have adverse effects on the antigens and adjuvants used. Intranasal immunization against H. pylori has also been used in mice, but studies in humans have indicated that the nasal route of immunization is ineffective in stimulating immune responses in the intestine or stomach (12). In addition, intranasal immunization is associated with a risk of translocation of some types of antigens or adjuvants to the olfactory bulb of the brain, restricting its applicability in humans (31, 34).Sublingual (SL) immunization has recently emerged as an attractive novel approach for mucosal vaccination against pathogens (7, 8, 31). In a model of influenza virus infection, SL immunization with live or adjuvanted killed virus induced immune responses and protection against aerosol challenge with live virus. In contrast to intranasal immunization, SL immunization had no evidence of vaccine or adjuvant entering the brain (31). In another study, SL immunization was found to induce vaccine-specific antibody and T cell responses in the genital tract and, after SL immunization with human papillomavirus (HPV)-like particles, protection against genital HPV infection, indicating the potential of SL immunization to stimulate immune responses also in nonrespiratory mucosal tissues (7).In the present study, we examined whether SL immunization in mice can induce a mucosal immune response in the gastrointestinal tract. More specifically, we addressed the potential of SL immunization with H. pylori antigen and cholera toxin (CT) adjuvant to stimulate T and B cell responses in the stomach and protect against H. pylori infection. Our findings demonstrate that SL immunization induces strong systemic and stomach mucosal antibody and T cell responses and a high level of protection against H. pylori challenge. After SL immunization and H. pylori challenge, the stomach mucosa showed infiltration of both CD4⁺ T cells and CD19⁺ B cells and increased expression of gamma interferon (IFN-γ) and interleukin-17 (IL-17) compared to unimmunized infected mice. This was associated with increased expression of both the mucosal addressin cell adhesion molecule (MAdCAM-1) integrin and chemokines CXCL10 (10-kDa IFN-γ-induced protein) and CCL28 (mucosa-associated epithelial chemokine) in the immunized mice, which probably facilitated the migration of immunization-induced CD4⁺ T cells and CD19⁺ B cells into the stomach mucosa. Our results indicate that SL immunization against H. pylori effectively induces a strong immune response in the gastrointestinal tract mucosa and protects against infection, providing an attractive novel way of vaccinating against H. pylori infection.     

An Outbreak of Infection by Yersinia pseudotuberculosis in Nonhuman Primates

Between October 1970 and June 1971, at the National Center for Primate Biology, Yersinia pseudotuberculosis of serotypes I-B and III was isolated from 9 monkeys (one during life and 8 at necropsy) of the following species: Macaca cynomolgus, Macaca nemestrina, Macaca radiata and Cercocebus fulliginosus. All these animals had characteristic gastrointestinal lesions consisting of superficial erosions or ulcerations with masses of gram-negative coccobacilli and an acute inflammatory exudate. Involvement of mesenteric nodes, livers and spleens by similar lesions was common. A more granulomatous reaction was rarely seen. Similar lesions without bacteriologic confirmation were found at necropsy in 20 other animals. When guinea pigs were inoculated intraperitoneally with our isolates, they developed focal splenic and hepatic necrosis resembling the septicemic form of the disease which is seen rarely in man. When inoculated intraperitoneally, they developed mesenteric lymphadenitis resembling human nonspecific mesenteric lymphadenitis; no intestinal lesions could be detected in the animals inoculated orally. The granulomatous component of the inflammatory response was better developed in guinea pigs than in the monkeys. It is concluded that infection by Yersinia pseudotuberculosis in nonhuman primates and probably also in other species, including man, is primarily a gastrointestinal disease. The primary intestinal lesions may be conspicuous, as in the monkeys, or inconspicuous, as in the guinea pig and man. The acuteness of the inflammatory response in the monkeys, when compared to the more granulomatous reaction in guinea pigs, suggests that the great majority of the monkeys died from an overwhelming infection before they could develop hypersensitivity to the organism.     

Urease-Based Mucosal Immunization against Helicobacter heilmannii Infection Induces Corpus Atrophy in Mice

Mucosal immunization with Helicobacter heilmannii urease B or Helicobacter pylori urease, given nasally with cholera toxin, protects BALB/c mice against H. heilmannii infection and significantly reduces a preexisting infection. However, immunization aggravates gastric corpus atrophy. Our results underline the necessity of defining immunization regimens that do not enhance mucosal damage.     

Protection of Nonhuman Primates against Two Species of Ebola Virus Infection with a Single Complex Adenovirus Vector

Ebola viruses are highly pathogenic viruses that cause outbreaks of hemorrhagic fever in humans and other primates. To meet the need for a vaccine against the several types of Ebola viruses that cause human diseases, we developed a multivalent vaccine candidate (EBO7) that expresses the glycoproteins of Zaire ebolavirus (ZEBOV) and Sudan ebolavirus (SEBOV) in a single complex adenovirus-based vector (CAdVax). We evaluated our vaccine in nonhuman primates against the parenteral and aerosol routes of lethal challenge. EBO7 vaccine provided protection against both Ebola viruses by either route of infection. Significantly, protection against SEBOV given as an aerosol challenge, which has not previously been shown, could be achieved with a boosting vaccination. These results demonstrate the feasibility of creating a robust, multivalent Ebola virus vaccine that would be effective in the event of a natural virus outbreak or biological threat.The filoviruses, Ebola virus (EBOV) and Marburg virus (MARV), cause outbreaks of severe hemorrhagic fever disease in humans, with case-fatality rates that range up to 90%. Among the Ebolavirus genus, there are four distinct species: Zaire ebolavirus (ZEBOV), Sudan ebolavirus (SEBOV), Reston ebolavirus (REBOV), and Cote d''Ivoire ebolavirus (CIEBOV) (10), with a possible fifth species identified in a recent outbreak in the Bundibugyo region of Western Uganda (34). Of these, ZEBOV and SEBOV are known to cause lethal disease in humans. The persistence of these viruses in nature is not well understood. Sporadic outbreaks due to EBOV have been occurring in Central Africa since the 1970s, but since the mid-1990s, the incidence of outbreaks has increased more than 4-fold (6, 7, 8, 42-45), and EBOV has spread aggressively throughout the great ape sanctuaries of West and Central Africa, decimating wild populations of gorillas and chimpanzees (2). While the filoviruses infect both humans and great apes, due to the high mortality rates of the infection, neither is thought to serve as reservoirs for these viruses but only as accidental hosts (16). Recent findings suggest that African fruit bats may serve as a reservoir host for filoviruses (2, 22); however, little is known about the nature of transmission to humans and nonhuman primates from bats or the likelihood of other reservoir species. In outbreak situations, filoviruses are believed to transmit from person to person mainly through contact with bodily fluids from infected patients. However, recent studies of Ebola outbreaks in wild apes have suggested that there could be other modes of transmission, including aerosol (2, 36). Studies in nonhuman primates have shown that EBOV and MARV can be spread through aerosolized droplets under controlled laboratory conditions (18, 21). So, despite the low incidence of infections globally, the lethality and potential airborne transmission of filoviruses in heavily populated areas makes them a significant biological threat, resulting in their placement on the Centers for Disease Control and Prevention list of Category A Bioterrorism Agents and the Department of Health and Human Services (DHHS) list of select agents and toxins. Concern is further compounded by the potential for these agents to be obtained from the wild (2, 22). In a biological threat scenario, aerosol transmission will likely be the main mode of viral dissemination, and protection against aerosolized EBOV would be of utmost importance (3). However, most previous vaccine candidates have only been evaluated for efficacy against intramuscular or intraperitoneal challenge and not against an aerosol challenge in nonhuman primates.At present, there are no licensed vaccines or specific antiviral treatments available for EBOV or MARV infections. However, significant progress has been made over the past few years in developing vaccine candidates that can protect nonhuman primates (NHPs) from lethal EBOV and MARV challenges (11, 17, 20, 23, 32, 40, 41). Most of the candidates utilize recombinant vaccine approaches that direct the protective immune response toward the surface glycoprotein (GP) of a single species of EBOV. Importantly, each species of EBOV is antigenically distinct, based on the sequences of the viral GP (10), and therefore, vaccines targeted against the GP of one species of the virus will not provide cross-protection against infection by another (19). Unique among the vaccine candidates is the recombinant complex adenovirus vaccine (CAdVax) system, which provides multivalent protection of NHPs against multiple species of filoviruses (33). The CAdVax vaccine platform is based on a complex, replication-defective adenovirus 5 (Ad5) vector (28-30, 37, 38) that allows for the incorporation of multiple gene inserts into the vector''s genome. Using this design, a bivalent vaccine vector (EBO7) was developed that expresses modified GP gene sequences of SEBOV and ZEBOV. When included in a novel pan-filovirus vaccine formulation, this vaccine was 100% protective in NHPs against two species of EBOV (ZEBOV and SEBOV) and two different strains of MARV (Musoke and Ci67) (33).In the study presented here, we further tested the protective efficacy of the CAdVax-based EBO7 vaccine in macaques by comparing aerosol to parenteral challenge. Aerosol challenge is potentially even more lethal than parenteral infection, because it induces hemorrhagic pneumonia. This is particularly true of SEBOV aerosol challenge, against which protection has not previously been demonstrated. In our studies, we have found that for either route of infection, the vaccine-induced bivalent anti-EBOV responses were protective against lethal challenge with either SEBOV or ZEBOV. This is the first report of a vaccine that is capable of protecting against aerosol SEBOV challenge. In addition, we found that EBO7 was also capable of protecting macaques with preexisting immunity to adenovirus against ZEBOV challenge. These results provide further insight into the feasibility of developing a fully protective multivalent EBOV vaccine using the CAdVax vaccine platform.     

Cytokine Expression in Pediatric Helicobacter pylori Infection

Helicobacter pylori infection is one of the most common gastrointestinal infections worldwide and almost invariably causes chronic gastritis in the infected host. A predominant Th1 profile has been demonstrated in H. pylori-infected mucosa from adults, but no previous study has evaluated in situ cytokine expression in children. We therefore examined expression of proinflammatory, anti-inflammatory, and regulatory cytokines by immunohistochemistry in cryopreserved antral biopsy specimens from 10 H. pylori-infected and 10 uninfected children and correlated expression of cytokines with histology scores. Concomitant expression of interleukin-8 (IL-8), gamma interferon (IFN-γ), IL-4, transforming growth factor β, and tumor necrosis factor alpha was seen in 8/10 H. pylori-infected cases and in 5/10 noninfected cases; all H. pylori-infected subjects showed staining for at least two of the cytokines. The proportion of epithelial cytokine-specific staining did not differ significantly between the groups, either in surface or glandular epithelium. Furthermore, no significant differences were noticed between intraepithelial or lamina propria lymphocyte staining in the groups. There was, however, a tendency of higher numbers of IFN-γ- and IL-8-positive cells in the H. pylori-infected group. IFN-γ and IL-8 lamina propria lymphocyte expression correlated significantly with antrum chronic inflammation, but there was no correlation between histology scores and epithelial cytokine expression. When the same techniques were used, the cytokine response appeared to be smaller in H. pylori-infected children than in adults, and there was no clear Th1 dominance. These results therefore suggest a different mucosal immunopathology in children. It remains to be determined whether the gastric immune response is downregulated in children with H. pylori infection and whether this is relevant to the outcome of infection.     

Antibody-Mediated Protection against Infection with Helicobacter pylori in a Suckling Mouse Model of Passive Immunity

Studies of active immunization against Helicobacter pylori indicate that antibodies play a minor role in immunity. There is also evidence, however, that the translocation of antibodies in the stomach may be insufficient to achieve functional antibody levels in the gastric lumen. We have used a suckling mouse model of passive immunity to determine if perorally delivered antibodies can protect against infection with H. pylori. Female C57BL/6 mice were immunized parenterally with formalin-fixed cells of three clinical isolates of H. pylori (3HP) or the mouse-adapted H. pylori strain SS1 before mating. Their pups were challenged with the SS1 strain at 4 days of age and left to suckle before determination of bacterial loads 14 days later. Compared to age-matched controls, pups suckled by 3HP-vaccinated dams were significantly protected against infection (>95% reduction in median bacterial load; P < 0.0001). Pups suckled by SS1-vaccinated dams were also significantly protected in terms of both median bacterial load (>99.5% reduction; P < 0.0001) and the number of culture-negative pups (28% versus 2% for immune and nonimmune cohorts, respectively; P < 0.0001). Similar results were obtained with pups suckled by dams immunized with a urease-deficient mutant of SS1. Fostering experiments demonstrated that protection was entirely attributable to suckling from an immunized dam, and antibody isotype analysis suggested that protection was mediated by the immunoglobulin G fraction of immune milk. Analysis of the bacterial loads in pups sampled before and after weaning confirmed that infection had been prevented in culture-negative animals. These data indicate that antibodies can prevent colonization by H. pylori and suppress the bacterial loads in animals that are colonized.Helicobacter pylori colonizes the gastric mucosa of humans and commandeers host defenses to establish chronic active gastritis while increasing the host''s susceptibility to gastroduodenal ulceration or certain gastric malignancies (37). Although H. pylori induces profound systemic and mucosal immune responses, clearance of infection is infrequent, and there is no protection against reinfection following eradication by antimicrobial chemotherapy (15). Consequently, there are no obvious parameters of natural immunity on which to base effective vaccination strategies.Vaccination studies of animal models have suggested that antibody development is not necessary for protective immunity to H. pylori (19) and may even enhance colonization (5, 6). Conversely, cellular immunity, possibly in concert with innate immune factors, such as defensins (59), elicits protection or eradication by exaggerating the gastric inflammatory response induced by H. pylori, thus interrupting colonization without a need to interact with the bacteria directly (3). The importance of the inflammatory response for protection against H. pylori is supported by the association of postimmunization gastritis with vaccine efficacy (6, 23). Nevertheless, the failure of antibody to limit H. pylori colonization is yet to be fully explained. One reason for this failure may be the relatively low level of antibodies in the gastric lumen due to the apparent inability of the mucosal immune system to translocate sufficient quantities of antibody across the gastric mucosa.Although well characterized in the intestine, relatively little is known about antibody secretion into the stomach. Some studies of H. pylori infection have reported that levels of immunoglobulin A (IgA) in gastric juice are significantly lower than those found in the saliva or intestinal contents (33, 34). Evidence that these low levels of IgA are due to inadequate antibody secretion in the stomach includes the following: (i) H. pylori-specific antibodies in gastric juice of infected individuals are predominantly nonsecretory IgA (10); (ii) equivalent amounts of IgG and IgA in the stomach suggest that IgA may leak across the mucosa rather than being actively secreted (14, 18); and (iii) much of the secretory IgA (sIgA) in the stomach is derived from swallowed saliva (17, 54). In addition, compared to the small intestine, the normal mammalian stomach has barely detectable secretory component (SC), suggesting a limited capacity for translocation of polymeric IgA across the gastric mucosa (8, 33). Moreover, despite considerable upregulation of SC by gamma interferon following the development of gastritis, there is no corresponding increase in the concentration of sIgA in gastric juice (4). Consequently, the concentration of sIgA in the stomach is unlikely to be sufficient to prevent or eradicate colonization by H. pylori.On the other hand, there is evidence that passive immunization with antibodies delivered perorally may reduce the extent of gastric colonization by Helicobacter species. This therapeutic approach has shown some promise in adult mice given monoclonal IgA or hyperimmune bovine colostrum against Helicobacter felis (14, 41) or urease-specific, chicken-derived IgY against H. pylori (44). In addition, reports of delayed acquisition of H. pylori by Gambian infants that corresponded to their mothers’ levels of breast milk IgA specific for H. pylori (58) and the protection of infant mice against full colonization by H. felis while suckling from immunized dams (13) suggest that orally delivered antibodies may be beneficial in controlling gastric Helicobacter infections. Despite these favorable reports, there are no tightly controlled studies that conclusively show prevention of H. pylori infection by orally delivered immune antibodies in the absence of additional factors, such as famotidine (44). Moreover, no studies have investigated the refinement of vaccine preparations for use in the production of anti-H. pylori polyclonal antibody products.In this study, we used a suckling mouse model of infection to investigate whether H. pylori-specific antibodies delivered during lactation to the gastric lumen of infant mice can protect against H. pylori infection. The route and adjuvant used to immunize the dams were selected to evoke an immune response similar to that required for the production of commercial quantities of polyclonal monomeric antibodies, such as from hyperimmune bovine colostrum. The model allowed us for the first time to quantify the contribution of passively acquired H. pylori-specific antibodies to protection against infection and provided an opportunity to examine different vaccine preparations for their ability to elicit these antibodies.     

Oral immunization against Helicobacter pylori.

Helicobacter pylori, which has been associated with gastritis and duodenal ulcers, commonly chronically infects adults. Eradication of this microorganism, which is difficult to achieve, results in normalization of gastritis and marked reduction in the relapse rate of duodenal ulcers. Since eradication is difficult to achieve, prevention of initial colonization of the gastrointestinal tract may be a viable alternative for abrogation of H. pylori-associated gastroduodenal disease. To test the feasibility of this approach, mice and ferrets were orally immunized with killed H. pylori. Immunization induced immunoglobulin A and G anti-H. pylori antibodies in both gastrointestinal secretions and sera of mice. These responses were enhanced when cholera toxin was included in the immunization protocol as a mucosal adjuvant. In ferrets, addition of cholera toxin resulted in significant enhancement of anti-H. pylori antibody levels in sera and intestines. Thus, oral immunization with killed H. pylori may be feasible approach to protect hosts from this infection and the accompanying gastroduodenal disease.     

Lack of a Major Role of Staphylococcus aureus Panton-Valentine Leukocidin in Lower Respiratory Tract Infection in Nonhuman Primates

Panton-Valentine leukocidin (PVL) is a two-component cytolytic toxin epidemiologically linked to community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections, including serious invasive infections caused by the epidemic clone referred to as strain USA300. Although PVL has long been known to be a S. aureus virulence molecule in vitro, the relative contribution of this leukotoxin to invasive CA-MRSA infections such as pneumonia remains controversial. We developed a nonhuman primate model of CA-MRSA pneumonia and used it to test the hypothesis that PVL contributes to lower respiratory tract infections caused by S. aureus strain USA300. The lower respiratory tract disease observed in this monkey model mimicked the clinical and pathological features of early mild to moderate S. aureus pneumonia in humans, including fine-structure histopathology. In this experiment using a large sample of monkeys and multiple time points of examination, no involvement of PVL in virulence could be detected. Compared with the wild-type parental USA300 strain, the isogenic PVL deletion-mutant strain caused equivalent lower respiratory tract pathology. We conclude that PVL does not contribute to lower respiratory tract infection in this nonhuman primate model of human CA-MRSA pneumonia.Staphylococcus aureus is the most abundant cause of serious bacterial infections in the United States.^1–4 In addition, the organism acquires antibiotic resistance readily and methicillin-resistant S. aureus (MRSA) strains have been a major concern in health care facilities for decades.^3–7 The pathogen causes a wide spectrum of infections, ranging from mild skin problems to fatal invasive diseases.¹ The mortality rate associated with invasive MRSA infections is approximately 20%, the majority of which are health care– or hospital-associated.¹ Inasmuch as health care–associated S. aureus infections occur in individuals with predisposing risk factors, the health status of the patient plays a prominent role. In contrast, community-associated MRSA (CA-MRSA) strains generally cause infections in otherwise healthy individuals.CA-MRSA emerged unexpectedly in the 1990s and is now epidemic in the United States.^2,8,9 The prototype CA-MRSA strain in the United States, known as pulsed-field type USA400 (MW2), caused fatal pneumonia in children in the Midwest.^10,11 Whole-genome sequencing of a USA400 strain revealed the presence of a novel methicillin-resistance element SCCmecIV and lukS-PV and lukF-PV (lukS/F-PV) genes encoding a two-component cytolytic toxin known as Panton-Valentine leukocidin (PVL).¹² Although strains of USA400 remain a significant cause of CA-MRSA infections, especially in Canada,^13,14 they have been nearly replaced in the United States by the pulsed-field type USA300 epidemic clone (USA300).^9,15,16 Notably, SCCmecIV and PVL genes are also present in the genome of USA300,¹⁷ and thus, they are strongly linked by epidemiology to CA-MRSA infections in the United States. The great majority of USA300 infections involve skin and soft tissue, accounting for 75% to 95% of CA-MRSA infections.^8,16,18,19 However, this pathogen also has the ability to cause severe invasive disease, including cases of fatal pneumonia.^20,21Several lines of evidence suggest that PVL may be an important virulence factor in the pathogenesis of S. aureus pneumonia. Lina and colleagues first described an association of PVL with S. aureus strains that cause primary community-acquired pneumonia, some of which were fatal cases of necrotizing pneumonia.²² Subsequently, PVL-producing S. aureus, mostly methicillin-susceptible S. aureus strains, were reported to be associated with necrotizing pneumonia in young immunocompetent individuals.²³ Recent reports also document that PVL-positive strains of USA300 can cause necrotizing pneumonia, albeit these infections are infrequent and often associated with influenza or parainfluenza virus coinfection.²⁴Despite the epidemiological linkage, there is currently little direct experimental evidence that PVL is a primary virulence factor for CA-MRSA pneumonia. One study suggested that PVL promotes S. aureus pneumonia in a mouse infection model.²⁵ However, subsequent work by several investigators using wild-type USA300 and USA400 and isogenic PVL deletion mutant strains failed to support the idea that PVL contributes to experimental CA-MRSA pneumonia in mice or rats.^26–29Elucidating whether PVL has a role in pneumonia in humans is critical because the leukotoxin is widely considered to be a possible determining factor in CA-MRSA pneumonia, and therefore, it has become a target for antistaphylococcal therapeutics and vaccines.³⁰ We developed a nonhuman primate model of S. aureus lower respiratory tract infection and used it to test the hypothesis that USA300 wild-type and isogenic PVL mutant strains differ in their ability to cause CA-MRSA pneumonia.     

Helicobacter pylori Infection Introduces DNA Double-Strand Breaks in Host Cells

Gastric cancer is an inflammation-related malignancy related to long-standing acute and chronic inflammation caused by infection with the human bacterial pathogen Helicobacter pylori. Inflammation can result in genomic instability. However, there are considerable data that H. pylori itself can also produce genomic instability both directly and through epigenetic pathways. Overall, the mechanisms of H. pylori-induced host genomic instabilities remain poorly understood. We used microarray screening of H. pylori-infected human gastric biopsy specimens to identify candidate genes involved in H. pylori-induced host genomic instabilities. We found upregulation of ATM expression in vivo in gastric mucosal cells infected with H. pylori. Using gastric cancer cell lines, we confirmed that the H. pylori-related activation of ATM was due to the accumulation of DNA double-strand breaks (DSBs). DSBs were observed following infection with both cag pathogenicity island (PAI)-positive and -negative strains, but the effect was more robust with cag PAI-positive strains. These results are consistent with the fact that infections with both cag PAI-positive and -negative strains are associated with gastric carcinogenesis, but the risk is higher in individuals infected with cag PAI-positive strains.     

Utility of Dried Blood Spots in Detecting Helicobacter pylori Infection

Purpose: Identifying infectious pathogens by collecting intravenous blood (IVB) is a well-established procedure, however, the collection of IVB in field epidemiological study is challenging. The dried blood spot (DBS) as an alternative to IVB has been introduced, although, there is a limited study to demonstrate the utility of DBS stored at various storage conditions and transported at different periods. This is an observational study, which evaluates the effectiveness of DBS in field epidemiological studies to identify infectious pathogens. Materials and Methods: A total of 264 paired DBS samples prepared from IVB, stored at 4°C, −20°C after period 24, 48 and 72 h. Serologically, enzyme-linked immunosorbent assay [ELISA] IgG antibody detected against Helicobacter pylori infection from DBS and compared with IVB. Results: Quantitatively, IgG antibody reactivity showed >87% correlation between IVB and DBS samples stored at 4°C or −20°C within 48 h of transport duration. DBS stored at 4°C shows, equal sensitivity 87.5% and specificity 95% before 48 h of transport duration, while at −20°C storage similar sensitivity 87.5% observed but slightly less specificity 86.36% observed as compared to 24 h of transport duration. One-way analysis of variance showed, nonsignificant difference at both (−20°C and 4°C) the stored condition with P value (P > 0.851) and (P > 0.477). Kappa values showed good inter-rater reliability between DBS and IVB in a range (0.77–0.81). Conclusion: No significant difference was observed in detecting H. pylori when ELISA was conducted using IVB or DBS stored at 4°C and transported even after 48 h. This confirms that DBS collected even in compromised conditions in the field can be used for detecting infection.     

胃大部切除术后残胃幽门螺杆菌感染的临床研究

目的探讨胃大部切除术后残胃病变与幽门螺杆菌(Hp)感染的关系。方法采用快速尿素酶试验、组织切片W arth in-Starry银染及尿素呼气试验检测682例残胃病患者Hp感染情况,并进行流行病学分析。结果682例残胃病患者Hp感染检出率为27.42%,与年龄、性别以及术后时间无关;B illroth I式术后残胃Hp检出率(40.07%)明显高于B illroth II式术后残胃Hp检出率(17.37%),而伴有胆汁返流的残胃Hp感染阳性率(7.98%)明显低于不伴胆汁返流的残胃阳性率(33.53%)。同时,因溃疡、穿孔、出血行手术的残胃Hp检出率(47.88%)高于因胃癌手术的检出率(22.66%);不同病理学残胃中,残胃溃疡和残胃癌的Hp感染率明显高于慢性残胃炎及吻合口炎的Hp感染率。结论胃大部切除术后残胃病变与Hp感染密切相关,尤其是残胃癌,应及早进行Hp治疗,可减轻残胃病变,减少残胃癌的发生。     

Seroprevalence of Helicobacter pylori Infection in Urban and Rural Vietnam

Helicobacter pylori-associated diseases, such as peptic ulcer and gastric cancer, are common in Vietnam, but the prevalence of the infection is largely unknown. A validated enzyme-linked immunosorbent assay was used for seroepidemiology with 971 samples from the general population, ages 0 to 88 years, with 546 samples from an urban population (Hanoi), and with 425 samples from a poor, rural province (Hatay). The overall seroprevalence of the infection was 746 per 1,000, with a prevalence of 788 per 1,000 in Hanoi and 692 per 1,000 in Hatay (P = 0.0007). The risk for infection in the rural area of Hatay was 40% lower than in the urban population of Hanoi, with the odds ratio being 0.59 (95% confidence interval, 0.43 to 0.81). The study shows that the prevalence of H. pylori infection is high in Vietnam and especially high in a large urban area, such as the city of Hanoi.     

Molecular Identification of Entamoeba spp. in Captive Nonhuman Primates

This study describes the molecular identification of 520 Entamoeba-positive fecal samples from a large and diverse population of captive nonhuman primates (NHP). The results revealed the presence of Entamoeba histolytica (NHP variant only), E. dispar, E. moshkovskii, E. hartmanni, E. coli, and E. polecki-like organisms.Various Entamoeba species are frequently found in the stools of both captive (15, 22) and wild (5, 7) nonhuman primates (NHP). Although the majority of these Entamoeba spp. are considered to be harmless, care should be taken when E. histolytica, the causative agents of amoebiasis, is involved. Infection with this gastrointestinal parasite in NHP may cause hemorrhagic dysentery (6, 28) and extraintestinal pathologies (e.g., liver abscesses) and death (12, 16). Moreover, amoebiasis is of major concern in public health, resulting in similar pathologies in humans and causing up to 100,000 deaths worldwide each year (19). Currently, little is known about the occurrence of E. histolytica in NHP, and the role of these animals as a potential reservoir for zoonotic transmission remains unclear. Most of the previous studies were based on the detection of cysts or trophozoites in stools by using light microscopy. However, differentiation between E. histolytica and other Entamoeba spp. (such as E. coli, E. hartmanni, and E. polecki-like organisms) based on morphological features is difficult (8, 27) and when E. dispar or E. moshkovskii is involved, it can even be impossible (4). For this purpose, molecular methods are more appropriate. Furthermore, recent molecular analyses of E. histolytica indicate genetic differences between human and NHP isolates (21, 23, 24). Although these differences may contribute to the elucidation of zoonotic transmission pathways, little is known about the distribution of these E. histolytica variants in both humans and NHP. Therefore, the objective of the present study was to identify the Entamoeba spp. in a large and diverse population of captive NHP, including differentiation between the human and NHP variants of E. histolytica.A total of 520 stool samples containing Entamoeba cysts were selected for further molecular identification. These samples were obtained from previous epidemiological surveys (10; unpublished data) and were stored at −20°C. The animals were housed in nine zoological gardens and one sanctuary in Belgium and the Netherlands, representing 58 NHP groups belonging to 36 animal species (see Table S1 in the supplemental material). None of the animals showed clinical signs associated with gastrointestinal disorders. DNA was extracted using the QIAamp stool minikit according the instructions of the manufacturer (Qiagen) and the adaptations described previously (9). The identification of E. histolytica, E. dispar, E. moshkovskii, E. hartmanii, E. coli, and E. polecki-like organisms was based largely on a previously described PCR-reverse line hybridization blot (PCR-RLHB) protocol targeting the small-subunit rRNA gene (26). This assay was preferred since it allows the simultaneous detection of various Entamoeba species. The amplification reactions were performed in a volume of 25 μl containing 2.5 μl DNA, 0.5 μl of each primer (10 μM), 1 μl MgCl₂ (25 mM), 5 μl GoTaq Flexi buffer, 14.875 μl PCR-grade H₂O, and 0.125 μl GoTaq Flexi DNA polymerase. For E. histolytica, a novel probe (5′-YAT TRA ATR AAT TGG CCA TTT TGT A-3′) was designed based on the gene sequences of the human variant (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"X64142","term_id":"296694","term_text":"X64142"}}X64142) and the NHP variant (GenBank accession numbers {"type":"entrez-nucleotide","attrs":{"text":"AB197936","term_id":"134026420","term_text":"AB197936"}}AB197936 and {"type":"entrez-nucleotide","attrs":{"text":"AB282657","term_id":"145698413","term_text":"AB282657"}}AB282657) to ensure the detection of both variants. In each PCR-RLHB run, control DNA samples from E. histolytica (both variants), E. dispar, E. moshkovskii, E. hartmanni, E. coli, and E. polecki-like organisms were included. Samples showing hybridization with the E. histolytica probe were retained for additional differentiation between the human and the NHP variants by using novel variant-specific reverse primers (human variant primer, 5′-CAT TTC TAG AAA CTT TAC TTA CAT-3′; NHP variant primer, 5′-CAT TTC TAG AAA CTT TAC TTA TGC-3′) designed from sequences with the GenBank accession numbers mentioned above. The amplification conditions remained unchanged. In each PCR run, control DNA samples from both the human variant and the NHP variant of E. histolytica were included. PCR products were run on agarose gels, stained with ethidium bromide, and detected upon UV transillumination. Samples reacting only with the general Entamoeba probe and not with any of the species-specific probes were retained for further sequence analyses. To this end, the PCR preceeding the RLHB assay was repeated with unlabeled primers. The obtained PCR products were purified with QIAquick purification columns (Qiagen, Germany) and cloned into the pGEM-T Easy vector according to the instructions of the manufacturer (Promega, Madison, WI). Clones containing the expected amplicon of approximately 550 bp were sequenced using the BigDye Terminator kit (Applied Biosystems). Sequence reactions were analyzed with an ABI-3730xl sequencer (Applied Biosystems), and sequences were assembled using Seqman II (DNAstar, Madison, WI).The RLHB analysis revealed the presence of Entamoeba DNA in 372 (71.5%) of 520 samples. The distribution of the different Entamoeba spp. within these 372 samples is described in Table ​Table1.1. E. hartmanni (present in 51.9% of samples) was the most prevalent species, followed by E. polecki-like organisms (in 42.7% of samples), E. histolytica (in 36.0% of samples), and E. coli (in 21.5% of samples). E. dispar (present in 2.4% of samples) and E. moskovskii (present in 1.9% of samples) were found in only a small number of samples. Most samples (51.9%) carried mixed infections. A large proportion of the samples (18.8%) hybridized with the general Entamoeba probe but could not be assigned to any of the known Entamoeba spp. The E. histolytica variant-specific PCR revealed solely the NHP variant in 124 of the 132 E. histolytica-positive samples. For the remaining 8 samples, no amplification was found in either PCR protocol.

TABLE 1.

Numbers of mono- and mixed infections with Entamoeba spp. in 372 samples based on a PCR-RLHB protocol targeting the small-subunit rRNA gene

4214名健康体检者幽门螺杆菌检验结果分析

目的 探讨大连地区体检人群幽门螺杆菌尿素酶抗体阳性率及分布特点.方法 用胶体金法对2013年9月至2015年9月期间在大连市第三人民医院做体检的4214名健康体检者的幽门螺杆菌尿素酶抗体进行检测,以性别和年龄进行分组,对其检验结果进行统计学分析.结果 4214名健康体检者幽门螺杆菌总阳性率为21.76％ (917/4214);其中男性和女性健康体检者阳性率分别为23.98％ (480/2002)、19.76％ (437/2212),男性和女性健康体检者幽门螺杆菌阳性率比较,差异有统计学意义(P＜0.05);再将这些体检者分为＜45岁、45～ 59岁、60 ～ 74岁和75～ 89岁4个组,幽门螺杆菌阳性率在4个年龄组分别为19.13％ (447/2337)、24.77％(357/1441)、26.09％(102/391)和24.44％(11/45),组间幽门螺杆菌尿素酶抗体阳性率差异具有统计学意义(P＜0.05).结论 大连地区体检人群幽门螺杆菌尿素酶抗体阳性率分布与性别和年龄有关.     

Natural acquisition of Helicobacter pylori infection in newborn rhesus macaques

Helicobacter pylori infection is usually acquired in childhood, but precise estimates of the age of acquisition are difficult to obtain in young children. Since serial endoscopic biopsies are not feasible in human infants, we examined acquisition of H. pylori infection that is known to occur in socially housed nonhuman primates. By 12 weeks of age, 8 of 20 newborns (40%) were culture positive for H. pylori, and prevalence reached 90% by 1 year of age. Newborns from infected dams were more commonly infected than those from uninfected dams, particularly during the peripartum period, suggesting that close contact during this time may facilitate transmission. Transient infection was uncommon and occurred only after the first positive culture. These results suggest that in a high-prevalence environment, persistent H. pylori infection may be acquired at an earlier age than was previously thought. Since clean, potable water was readily available, contamination of water supply is not essential for widespread infection at an early age in areas where hygiene is otherwise poor. Furthermore, breastfeeding seems to offer little protection, since newborn macaques breastfeed during the first year of life and typically are fully weaned only when another newborn arrives the following spring.