Differences in Genome Content among Helicobacter pylori Isolates from Patients with Gastritis,Duodenal Ulcer,or Gastric Cancer Reveal Novel Disease-Associated Genes

Helicobacter pylori establishes a chronic infection in the human stomach, causing gastritis, peptic ulcer, or gastric cancer, and more severe diseases are associated with virulence genes such as the cag pathogenicity island (PAI). The aim of this work was to study gene content differences among H. pylori strains isolated from patients with different gastroduodenal diseases in a Mexican-Mestizo patient population. H. pylori isolates from 10 patients with nonatrophic gastritis, 10 patients with duodenal ulcer, and 9 patients with gastric cancer were studied. Multiple isolates from the same patient were analyzed by randomly amplified polymorphic DNA analysis, and strains with unique patterns were tested using whole-genome microarray-based comparative genomic hybridization (aCGH). We studied 42 isolates and found 1,319 genes present in all isolates, while 341 (20.5%) were variable genes. Among the variable genes, 127 (37%) were distributed within plasticity zones (PZs). The overall number of variable genes present in a given isolate was significantly lower for gastric cancer isolates. Thirty genes were significantly associated with nonatrophic gastritis, duodenal ulcer, or gastric cancer, 14 (46.6%) of which were within PZs and the cag PAI. Two genes (HP0674 and JHP0940) were absent in all gastric cancer isolates. Many of the disease-associated genes outside the PZs formed clusters, and some of these genes are regulated in response to acid or other environmental conditions. Validation of candidate genes identified by aCGH in a second patient cohort allowed the identification of novel H. pylori genes associated with gastric cancer or duodenal ulcer. These disease-associated genes may serve as biomarkers of the risk for severe gastroduodenal diseases.Infection with Helicobacter pylori is one of the most common bacterial infections in humans worldwide, and like the case for many other infections, rates are higher in developing countries (80 to 90%) than in developed countries (<50%) (20, 33, 39). The infection is associated with peptic ulcers, gastric carcinoma, and mucosa-associated lymphoma (32, 36, 39). Most infected people remain asymptomatic during their lifetime, and only about 15% develop gastroduodenal illness. Environmental, host, and bacterial factors all play a role in the outcome of the infection. A number of bacterial virulence factors associated with disease have been described for H. pylori, and the most consistently reported are the cag pathogenicity island (cag PAI) (4, 40, 52) and vacA (2, 43). Outer membrane proteins such as BabA2 (18), OipA (54), and SabB (11), as well as the iceA gene, have also been reported to be associated with disease. An outstanding characteristic of H. pylori is the high level of genetic diversity among isolates from different patients, even if they belong to the same ethnic population. Several molecular typing techniques have been used to genotype H. pylori strains from different populations, demonstrating genetic differences among populations and even among isolates from the same individual, suggesting the presence of mixed infection (3, 9, 19, 26, 35, 46). The ability of this bacterium to generate such genetic diversity is due to its natural competence, high recombination and mutation rates, or the occurrence of slipped-strand synthesis and phase variation (17, 24).H. pylori was the first bacterial species for which whole genome sequences of two independent strains were available (J99 and 26695). Their comparison showed that approximately 6 to 7% of the H. pylori genes present in one strain are absent in the other. These genes are called strain-specific genes, and almost half of them are located in hypervariable regions of the genome (1, 51). These regions contain a considerable number of restriction-modification genes and genes for transposases, topoisomerases, and outer membrane proteins. One of these regions is the cag PAI, whereas the others have been termed plasticity zones (PZs). Whole-genome DNA microarrays facilitated further analyses of the genomic contents of 15 H. pylori clinical isolates, revealing 362 genes (22% of all genes) that are not conserved among strains and represent variable or strain-specific genes (45). Similar microarray-based comparative genomic hybridization (aCGH) studies have been used to explore the genetic diversity in the H. pylori strain population colonizing the different regions of the stomach of a single host for both adults and children (46) and to correlate the genetic contents of H. pylori strains with pathogenesis in animal models (6, 25). These studies indicate that H. pylori strains gain or lose loci during chronic infection, suggesting a continuous genetic flux, mainly inside the PZs (14, 22, 26, 27, 28). The sequence of an H. pylori strain isolated from a patient with chronic atrophic gastritis was recently published, identifying additional strain-specific genes (38) and, by comparison with previous studies (22), suggesting that 121 genes are “chronic atrophic gastritis associated.”Other studies have reported that genes located in the PZs, such as jhp0947 and jhp0949, are associated with disease (12, 37, 47). The dupA gene was associated with an increased risk for duodenal ulcer (DU) and a low risk for gastric atrophy and cancer (31). The aim of the present study was to compare the genomic contents of H. pylori strains isolated from patients with nonatrophic gastritis (NAG), DU, or gastric cancer (GC) in a Mexican-Mestizo population in order to look for genes previously associated with severe gastroduodenal diseases and to identify novel disease biomarkers.     

Diverse Characteristics of the cagA Gene of Helicobacter pylori Strains Collected from Patients from Southern Vietnam with Gastric Cancer and Peptic Ulcer

The pathogenesis of gastroduodenal diseases is related to the diversity of Helicobacter pylori strains. CagA-positive strains are more likely to cause gastric cancer than CagA-negative strains. Based on EPIYA (Glu-Pro-Ile-Tyr-Ala) motifs at the carboxyl terminus corresponding to phosphorylation sites, H. pylori CagA is divided into East Asian CagA and Western CagA. The former type prevails in East Asia and is more closely associated with gastric cancer. The present study used full sequences of the cagA gene and CagA protein of 22 H. pylori strains in gastric cancer and peptic ulcer patients from Southern Vietnam to make a comparison of genetic homology among Vietnamese strains and between them and other strains in East Asia. A phylogenetic tree was constructed based on full amino acid sequences of 22 Vietnamese strains in accordance with 54 references from around the world. The cagA gene was found in all Vietnamese H. pylori strains. Twenty-one of 22 (95.5%) strains belonged to the East Asian type and had similar characteristics of amino acid sequence at the carboxyl terminus to other strains from the East Asian region. From evidence of East Asian CagA and epidemiologic cancerous lesions in Vietnam, H. pylori-infected Vietnamese can be classified into a high-risk group for gastric cancer, but further studies on the interaction among environmental and virulence factors should be done. Finally, phylogenetic data support that there is a Japanese subtype in the Western CagA type.Helicobacter pylori is a gram-negative bacterium that infects about 50% of the world''s population. Infections with H. pylori can result in chronic active gastritis and are a risk factor for peptic ulcers, gastric cancer and gastric mucosa-associated lymphoid tissue lymphoma (40, 52, 53, 56). The prevalence of H. pylori infections is not the same in different parts of the world. Recent studies reported that humans actually acquired H. pylori in the early days of their history, long before the migration of modern humans out of Africa, and the diverse distribution of H. pylori today is associated with waves of human migration in the past (19, 32, 36, 61, 62). The rate of H. pylori infections is high in Africa, East Asia, and South Asia; however, the incidence of gastric cancer is high in East Asia but not in South Asia or Africa; this may be explained partly by the diversity of H. pylori strains in these regions (62).In cases of gastroduodenal diseases, especially gastric cancer, the pathogenesis involves three major factors: H. pylori virulence factors, host factors, and environmental factors (1, 10, 14, 16, 21, 34, 57). Two H. pylori virulence factors that have been focused on in many studies all around the world are VacA and CagA. VacA is encoded by the vacA gene and found in all H. pylori strains. The vacA gene is classified into three major genotypes: s1/m1, s1/m2, and s2/m2. s1/m1 strains produce higher levels of cytotoxin than s1/m2 or s2/m2 strains (2, 3). CagA is encoded by cagA, located in the cagPAI (pathogenicity island) region of the H. pylori genome, and the presence of this protein is a marker of cagPAI. Unlike the vacA gene, the cagA gene has been found in only 50% to 70% of H. pylori strains infecting Western populations: however, 80% to 100% of H. pylori strains from East Asia have the cagA gene, with the cagPAI region, in their genome (17, 49, 55, 61, 64, 65). Patients infected with H. pylori strains possessing CagA were at greater risk of developing gastric adenocarcinoma than those uninfected or infected with CagA-negative strains (11, 41). CagA is injected directly from H. pylori into intragastric epithelial cells via the type IV secretion system. The injected CagA mimics eukaryotic adaptor proteins that recruit multiple host signaling factors into protein complexes that target cellular junctions, cell proliferation, and actin-cytoskeletal rearrangements (54). The translocated CagA undergoes tyrosine phosphorylation by Src and Abl family kinases and binds to SHP-2 in the human gastric mucosa (45, 47, 48). The repeated EPIYA (Glu-Pro-Ile-Tyr-Ala) motifs at the carboxyl-terminal end of CagA are the targets of tyrosine phosphorylation (8, 22, 24, 38, 63). CagA multimerization plays an important role in the pathophysiological activity of CagA in disturbing host cell functions via SHP-2 deregulation, and EPIYA polymorphisms of CagA greatly influence the magnitude and duration of phosphorylation-dependent CagA activity (37, 42). SHP-2, like its Drosophila melanogaster homolog Corkscrew, is known to play an important role in the mitogenic signal transduction that connects receptor tyrosine kinases and ras (20). It is possible that deregulation of SHP-2 by the translocation of CagA plays a role in the acquisition of a cellular-transformed phenotype at a relatively early stage in the carcinogenesis of gastric carcinoma. A recent study on generating CagA in transgenic mice has provided the first direct evidence of the role of CagA as a bacterium-derived oncoprotein that acts in mammals and further indicates the importance of tyrosine phosphorylation, which enables CagA to deregulate SHP-2, in the development of H. pylori-associated neoplasms (39). Based on characteristics of the EPIYA motif, the H. pylori CagA protein could be divided into a Western type and an East Asian type. The East Asian CagA protein exhibits stronger SHP-2-binding activity and so is more pathogenic than the Western CagA protein in H. pylori-infected patients (4, 7, 23, 37). Clinical data from East Asia, Japan, and South Korea indicated that the East Asian form of CagA was more closely related to persistent active inflammation, atrophic gastritis, and a higher risk of gastric cancer than the Western form (5, 6, 26, 44). It is quite clear that by studying the characteristics of H. pylori strains, especially the cagA gene and CagA protein, one can categorize H. pylori-infected patients into those at high risk of developing gastric cancer and those not.Vietnam is a developing country located in Southeast Asia. However, according to historical and migrational evidence, the Vietnamese are more closely related to people from East Asia than people from South Asia. Gastric cancer is one of the five most common cancers in Vietnam, including lung, stomach, liver, recto-colon and naso-pharynx cancer in males. In females, it ranks third behind breast and cervical-uterine cancer. The prevalence of gastric cancer in Northern Vietnam is as high as that in China or Korea. Its prevalence in Southern Vietnam is lower but still higher than that in Thailand and South Asia (46, 50). A few studies reported that cagA was found in nearly 100% of H. pylori-infected Vietnamese (60, 61), but no studies have examined the type of CagA protein or the full sequence of cagA in Vietnamese patients. The present study reports the diverse characteristics of cagA and classification of CagA in H. pylori-infected patients from Southern Vietnam based on the full genomic cagA sequence.     

High Prevalence of Clarithromycin-Resistant Helicobacter pylori Strains and Risk Factors Associated with Resistance in Madrid,Spain

Clarithromycin is one of the antibiotics used for the treatment of Helicobacter pylori infections, and clarithromycin resistance is the most important factor when it comes to predicting eradication failure. The present study analyzed H. pylori isolates for the presence of 23S rRNA gene mutations and determined the risk factors associated with resistance among H. pylori isolates collected in Madrid, Spain, in 2008. We studied 118 H. pylori strains isolated from the same number of patients. A total of 76.3% of the patients were born in Spain, 52.7% were children, 20.3% had previously been treated, and 66.1% were female. Clarithromycin resistance was determined by Etest. H. pylori strains were considered resistant if the MIC was ≥1 mg/liter. DNA extraction was carried out by use of the NucliSens easyMAG platform with NucliSens magnetic extraction reagents (bioMérieux). The DNA sequences of the 23S rRNA genes of clarithromycin-resistant and -sensitive strains were determined to identify specific point mutations. The vacA genotype and cagA status were determined by PCR. We found that 42 (35.6%) strains were resistant to clarithromycin by Etest. Etest results were confirmed by detection of the presence of point mutations in 34 (88.1%) of these strains. Eight H. pylori strains were resistant to clarithromycin by Etest but did not have a point mutation in the 23S rRNA gene. Mutation at A2143G was found in 85.3% of the strains, mutation at A2142G in 8.8%, and mutation at T2182C in 5.9%. Dual mutations were found in 8.8% of the strains. H. pylori clarithromycin-resistant strains were strongly associated with pediatric patients, with patients born in Spain, and with patients who had previously been treated (P ≤ 0.02). In addition, H. pylori strains resistant to clarithromycin more frequently presented the vacA s2/m2 genotype and were more likely to be cagA negative than susceptible strains (39.1% and 11.2%, respectively; P value < 0.001). We concluded that, in the present study, H. pylori clarithromycin-resistant strains are more frequently found in children, in patients mostly born in Spain, and in individuals who were previously treated for H. pylori infection and that these individuals are more likely colonized with a less virulent H. pylori strain.Helicobacter pylori is a microaerobic, Gram-negative spiral bacterium that colonizes the human stomach and is found in more than half of the world''s population (32). Infections with H. pylori are closely associated with chronic gastritis, peptic ulcer disease, and the development of gastric cancer (8, 32).All consensus guidelines recommend eradication of H. pylori for patients with symptoms (9, 28). Standard therapy combines a proton pump inhibitor (PPI) or ranitidine bismuth citrate and two antibiotics, chosen from among amoxicillin, clarithromycin, and metronidazole (24, 25). However, this therapy has been questioned because of the increased eradication failure rates. Many factors have been implicated as causes of treatment failure, including ineffective penetration of antibiotics into the gastric mucosa, antibiotic inactivation by the low stomach pH, a lack of patient compliance, and the emergence of acquired resistance to antibiotics by H. pylori (26, 27).In many cases, clarithromycin is the key component of these combination therapies. However, resistance to clarithromycin has become one of the major reasons for treatment failure (13). The prevalence of H. pylori resistance to clarithromycin varies among different countries, such as 10.6% to 25% in North America, 16% in Japan, and 1.7% to 23.4% in Europe (14, 19, 21). Overall, resistance to clarithromycin has been detected more in patients living in the south than in those living in the north of Europe (21). Fewer studies have focused on the prevalence of clarithromycin resistance among H. pylori strains from children compared with that among strains from the adult population. These studies will be useful for estimating the rate of clarithromycin-resistant H. pylori isolates among children and adults in Spain in the future (1, 23).Clarithromycin acts by binding to the peptidyltransferase region of 23S rRNA and inhibits protein synthesis (36). The resistance to clarithromycin in H. pylori has been shown to be due to point mutations in the peptidyltransferase region of domain V of the 23S rRNA. Two copies of the 23S rRNA gene are present in H. pylori, and the most common mutation is an A-to-G transition at position 2143 (A2143G) (13, 36), but several point mutations, at positions A2142G, A2144G, and T2182C, have been described. Recent reports have indicated that other mutations, such as A2115G, G2141A, C2147G, T2190C, C2195T, A2223G, and C2694A, might also be associated with clarithromycin resistance (20, 31). Other mechanisms of resistance, such as methylase production, the actions of macrolide-inactivating enzymes, and active efflux, have been described in several bacteria. Active efflux has also recently been described in H. pylori (22).Since the worldwide increase in the rate of clarithromycin resistance represents a problem of relevance, some studies have been performed in order to identify its relationship with bacterial genetic factors (12, 35, 38).Two genes (cytotoxin-associated gene A [cagA] and vacuolation-associated gene A [vacA]) have been identified to be the main virulence factors. cagA is located in the cag pathogenicity island (PAI), which encodes a type IV secretion system, and the presence of cagA is closely associated with more severe gastric diseases (2, 15, 34). The VacA toxin induces vacuole formation in the host cells. There is considerable variation in vacuolation activity among H. pylori strains, primarily due to differences in the vacA gene structure in the signal region (s1 and s2) and the middle region (m1 and m2). vacA s1/m1 and s1/m2 produce high and moderate levels of VacA toxin, respectively, whereas s2/m2 produces little or no toxin (11). A strong association between clarithromycin susceptibility and these virulence factors has been reported (12, 38).The focus of the present study was to evaluate the distribution of clarithromycin-resistant H. pylori strains and their association with genotypic markers, such as the cagA gene and allelic variants of the vac gene. We also examined the distribution of H. pylori clarithromycin resistance in relation to the patient''s age, place of birth, and history of treatment. Our main goal is to determine potential host and bacterial factors that may help in predicting resistance to clarithromycin among H. pylori isolates.     

Improved Performance of a Rapid Office-Based Stool Test for Detection of Helicobacter pylori in Children before and after Therapy

A modified version of a rapid office based one-step monoclonal immunoassay for detection of Helicobacter pylori antigen in stool samples from children was evaluated against biopsy specimen-based methods and compared to a monoclonal enzyme immunoassay using the same antigen. Blinded stool samples from 185 children (0.3 to 18.2 years) were investigated at the time of upper endoscopy prior to anti-H. pylori therapy; 62 children were H. pylori infected and 123 noninfected according to predefined reference standards. Samples obtained 6 to 8 weeks after anti-H. pylori therapy were available from 58 children (3.8 to 17.7 years) and were compared to results of the [¹³C]urea breath test (14/58 were positive). The rapid stool tests were performed by two independent readers. Of 243 rapid tests performed, 1 (0.4%) was invalid for technical reasons. Equivocal results (very weak line) were reported 16 times by reader 1 and 27 times by reader 2. When equivocal results were considered positive, the two observers agreed on 76 positive and 160 negative results and disagreed on 7 samples (2.9%). The sensitivity was 90.8% for reader 1 and 85.5% for reader 2, and the specificity was 91.0% and 93.4%, respectively. The monoclonal enzyme immunoassay revealed a sensitivity and specificity of 94.7% and 97.6%, respectively. The modified chromatographic immunoassay is a good alternative in settings or situations when the monoclonal enzyme immunoassay or the [¹³C]urea breath test are not available or feasible. In order to improve sensitivity, very weak lines should be considered positive test results.Several noninvasive methods are available for the diagnosis of H. pylori infection (5, 14). Serological tests are not appropriate, since they cannot distinguish between a present and previous infection and, in addition, they have a low sensitivity in children younger than 12 years of age (6, 13). The [¹³C]urea breath test (UBT) is the preferred noninvasive diagnostic tool and gives excellent performance for both adults and children, but specificity decreases in very young and mentally disabled children who are not able to cooperate with the test procedure (10, 11, 25). So far, tests for detection of H. pylori antigen in stool samples are the only noninvasive diagnostic tools which do not show an age dependence for the diagnostic accuracy (14, 15). This makes stool tests very attractive, particularly for young children and for epidemiological studies. Several tests have been developed, but validation studies showed differences in performance. An enzyme immunoassay (EIA) based on polyclonal antibodies that was developed by the Meridian Company has been validated in several studies, with controversial results (17, 20, 24). Lack of accuracy is obviously related to intertest variability (19). In contrast, EIA based on monoclonal antibodies showed consistently excellent results, with very high sensitivity and specificity in both children and adults (15, 21). A meta-analysis with head-to-head comparison has judged the monoclonal EIA superior to the polyclonal EIA (8).Recently, we reported on the performance of a one-step monoclonal chromatographic immunoassay for detection of H. pylori antigen in stool samples from symptomatic children compared to the results of a well-established monoclonal EIA using the same antigen, namely, the catalase of H. pylori (22). Evaluation against biopsy specimen-based diagnostic methods showed a moderate sensitivity but a good specificity. After publication of the data, the manufacturer modified the tests. The aim of this study was to evaluate this new version of the rapid office-based one-step stool test in symptomatic children against invasive diagnostic methods and to compare the results with those of the monoclonal EIA.     

Evaluation of a New Test,GenoType HelicoDR,for Molecular Detection of Antibiotic Resistance in Helicobacter pylori

The eradication rate of Helicobacter pylori by standard therapy is decreasing due to antibiotic resistance, mainly to clarithromycin. Our aim was to provide a new molecular test to guide the treatment of new and relapsed cases. We first studied 126 H. pylori strains for phenotypic (MIC) and genotypic resistance to clarithromycin (rrl mutation) and levofloxacin (gyrA mutation) and then developed a DNA strip genotyping test on the basis of the correlation results and literature data. Clinical strains (n = 92) and gastric biopsy specimens containing H. pylori (n = 105) were tested blindly with the new molecular test GenoType HelicoDR. The presence of mutations or the absence of hybridization with wild-type sequences was predictive, in rrl for clarithromycin resistance in 91 cases (mostly the A2147G mutation) and in gyrA for levofloxacin resistance in 58 cases (mutations at codon 87 or 91). Genotyping revealed a mix of genotypes in 33% of the cases, reflecting a coinfection or selection for resistant mutants. The sensitivity and specificity of detecting resistance were 94% and 99% for clarithromycin and 87% and 98.5% for levofloxacin, respectively. The concordance scores were 0.96 for clarithromycin and 0.94 for levofloxacin. With global resistance rates of 46% for clarithromycin and 25% for levofloxacin, which were observed for consecutive positive biopsy specimens from 2007 and 2008, the positive and negative predictive values for detecting resistance were 99% and 94% for clarithromycin and 96% and 96% for fluoroquinolone. GenoType HelicoDR is efficient at detecting mutations predictive of antibiotic resistance in H. pylori when applied to strains or directly to gastric biopsy specimens.Helicobacter pylori infection is a common chronic gastric infection worldwide with one-third prevalence (6). About 1 out of 10 humans infected with H. pylori suffers from various digestive diseases, such as duodenal and gastric ulcer and nonulcer dyspepsia; 1 out of 100 develops gastric adenocarcinoma; and ≤1 out of 1,000 may develop gastric mucosa-associated lymphoid tissue lymphoma. All consensus guidelines recommend eradication of H. pylori (6, 20) in symptomatic patients. Standard therapy combines a proton pump inhibitor, such as omeprazole, and two antibiotics, chosen from among amoxicillin, clarithromycin, and metronidazole (20). This therapy was assessed in studies in the early 1990s and demonstrated an eradication rate of H. pylori of over 80%. However, the eradication rate is decreasing, with as low as 60% success in some countries, and this is related to the increase in clarithromycin and metronidazole resistance reported worldwide (9, 10, 17). Fluoroquinolones, such as levofloxacin and moxifloxacin, are often used for rescue therapy in a third- or fourth-line treatment (20, 31).Antibiotics used for the treatment of H. pylori infection are usually not chosen on the basis of routine susceptibility testing, because H. pylori is a fastidious microorganism requiring 3 to 10 days in a microaerobic atmosphere, and susceptibility results are not reliable for all antibiotics (17, 22). Indeed, susceptibility breakpoints have been difficult to set due to the lack of standard methods for susceptibility testing and difficulties in assessing the correlation between susceptibility results and clinical outcomes. Phenotypic resistance is correlated with clinical and microbiological failure for clarithromycin, but not for metronidazole (21). The eradication rate drops from 88% in the case of a clarithromycin-susceptible strain to less than 20% in the case of clarithromycin resistance (7, 21). Fluoroquinolone resistance was also shown to be correlated with treatment failure (24). Because resistance rates vary according to the country and patient characteristics, the choice of antibiotics on the basis of susceptibility results might be an effective strategy to improve H. pylori eradication. Since susceptibility testing is cumbersome, molecular methods for detection of resistance may be cost-effective.The mutations leading to resistance are now well known for macrolides and fluoroquinolones, although they are still unclear for metronidazole and amoxicillin. Clarithromycin resistance in H. pylori is due to point mutations in the rrl gene encoding the 23S rRNA, with three major mutations described: A2146C, A2146G, and A2147G (the numeration is from genome sequencing of NC000921 and NC000915, positions 2146 and 2147, formerly described as 2142 and 2143 [reviewed in references 21 and 22]). The resistance of H. pylori to quinolones is due to point mutations in the so-called quinolone resistance-determining region of the gyrA gene coding for the A subunit of the DNA gyrase, mainly at codons 87 and 91 (corresponding to 83 and 87 in Escherichia coli numbering) (1, 4, 23, 30).Our objective was to develop and implement a molecular method to easily detect mutations predictive of clarithromycin and fluoroquinolone resistance in H. pylori. We based our test on the DNA strip methodology used with success for other pathogens, such as Mycobacterium tuberculosis (13). We first designed a prototype test using a panel test of 126 H. pylori strains for which the MICs of clarithromycin and fluoroquinolones and the rrl and gyrA genotypes had been determined. Then, the new test was applied blindly to clinical strains and gastric biopsy specimens containing H. pylori, and the results were compared to those of susceptibility testing done routinely. The specificity of the new test for H. pylori was evaluated by testing strains of Helicobacter species other than H. pylori, as well as negative biopsy specimens. The new test was concordant with reference tests for 94 to 98% of the samples, either performed on isolated strains or directly on gastric biopsy specimens containing H. pylori, and was easy to perform.     

Evaluation of Two Immunoblot Assays and a Western Blot Assay for the Detection of Antisyphilis Immunoglobulin G Antibodies

In the present study, two immunoglobulin G (IgG) immunoblot assays and one IgG Western blot assay were compared to the rapid plasma reagin test (RPR), the fluorescent treponemal antibody absorption test (FTA-ABS), and the Treponema pallidum particle agglutination assay (TP-PA). The agreement levels of the Viramed, Virotech, and MarDx assays were 97.0%, 96.4%, and 99.4%, and the agreements of samples inconclusive by FTA-ABS and resolved by TP-PA were 91.7%, 83.3%, and 69.4%, respectively.Syphilis, a disease caused by Treponema pallidum, is transmitted congenitally or through sexual intercourse (8-9). Non-treponema-based tests such as the rapid plasma reagin test (RPR) are used to detect syphilis infection (6, 9-10). These tests may produce false-positive results in pregnant women and patients with infections (3, 5-6, 9, 11). An algorithm has been developed for the serological diagnosis of syphilis which includes a non-treponema-based screening test and a treponema-based confirmatory assay (1-2, 7, 11). Traditional confirmatory assays include the fluorescent treponemal antibody absorption test (FTA-ABS) and the T. pallidum particle agglutination assay (TP-PA) (9).Western blot-based assays to detect immunoglobulin G (IgG) antibodies may prove useful, especially in cases where the FTA-ABS is inconclusive. In the present study, results of two immunoblot assays and one Western blot assay were compared to FTA-ABS/TP-PA and RPR results, as well as to each other.     

Variation in Susceptibility of Bloodstream Isolates of Candida glabrata to Fluconazole According to Patient Age and Geographic Location in the United States in 2001 to 2007

We examined the susceptibilities to fluconazole of 642 bloodstream infection (BSI) isolates of Candida glabrata and grouped the isolates by patient age and geographic location within the United States. Susceptibility of C. glabrata to fluconazole was lowest in the northeast region (46%) and was highest in the west (76%). The frequencies of isolation and of fluconazole resistance among C. glabrata BSI isolates were higher in the present study (years 2001 to 2007) than in a previous study conducted from 1992 to 2001. Whereas the frequency of C. glabrata increased with patient age, the rate of fluconazole resistance declined. The oldest age group (≥80 years) had the highest proportion of BSI isolates that were C. glabrata (32%) and the lowest rate of fluconazole resistance (5%).Candidemia is without question the most important of the invasive mycoses (6, 33, 35, 61, 65, 68, 78, 86, 88). Treatment of candidemia over the past 20 years has been enhanced considerably by the introduction of fluconazole in 1990 (7, 10, 15, 28, 29, 31, 40, 56-58, 61, 86, 90). Because of its widespread usage, concern about the development of fluconazole resistance among Candida spp. abounds (2, 6, 14, 32, 47, 53, 55, 56, 59, 60, 62, 80, 86). Despite these concerns, fluconazole resistance is relatively uncommon among most species of Candida causing bloodstream infections (BSI) (5, 6, 22, 24, 33, 42, 54, 56, 65, 68, 71, 86). The exception to this statement is Candida glabrata, of which more than 10% of BSI isolates may be highly resistant (MIC ≥ 64 μg/ml) to fluconazole (6, 9, 15, 23, 30, 32, 36, 63-65, 71, 87, 91). Suboptimal fluconazole dosing practices (low dose [<400 mg/day] and poor indications) may lead to an increased frequency of isolation of C. glabrata as an etiological agent of candidemia in hospitalized patients (6, 17, 29, 32, 35, 41, 47, 55, 60, 68, 85) and to increased fluconazole (and other azole) resistance secondary to induction of CDR efflux pumps (2, 11, 13, 16, 43, 47, 50, 55, 69, 77, 83, 84) and may adversely affect the survival of treated patients (7, 10, 29, 40, 59, 90). Among the various Candida species, C. glabrata alone has increased as a cause of BSI in U.S. intensive care units since 1993 (89). Within the United States, the proportion of fungemias due to C. glabrata has been shown to vary from 11% to 37% across the different regions (west, midwest, northeast, and south) of the country (63, 65) and from <10% to >30% within single institutions over the course of several years (9, 48). It has been shown that the prevalence of C. glabrata as a cause of BSI is potentially related to many disparate factors in addition to fluconazole exposure, including geographic characteristics (3, 6, 63-65, 71, 88), patient age (5, 6, 25, 35, 41, 42, 48, 63, 82, 92), and other characteristics of the patient population studied (1, 32, 35, 51). Because C. glabrata is relatively resistant to fluconazole, the frequency with which it causes BSI has important implications for therapy (21, 29, 32, 40, 41, 45, 56, 57, 59, 80, 81, 86, 90).Previously, we examined the susceptibilities to fluconazole of 559 BSI isolates of C. glabrata and grouped the isolates by patient age and geographic location within the United States over the time period from 1992 to 2001 (63). In the present study we build upon this experience and report the fluconazole susceptibilities of 642 BSI isolates of C. glabrata collected from sentinel surveillance sites throughout the United States for the time period from 2001 through 2007 and stratify the results by geographic region and patient age. The activities of voriconazole and the echinocandins against this contemporary collection of C. glabrata isolates are also reported.     

Evaluation of a New Selective Medium,BD BBL CHROMagar MRSA II,for Detection of Methicillin-Resistant Staphylococcus aureus in Different Specimens

The sensitivity of screening for methicillin-resistant Staphylococcus aureus (MRSA) can be improved by adding other specimen sites to nares. We describe an evaluation of a new selective medium, BBL CHROMagar MRSA II (CMRSAII), for its ability to detect MRSA from different specimen types. CMRSAII is a chromogenic medium which incorporates cefoxitin for the detection of MRSA. A study was performed at four clinical laboratories with the following specimens: 1,446 respiratory, 694 stool, 1,275 skin, and 948 wound specimens and 688 blood culture bottles containing Gram-positive cocci. The recovery of MRSA on traditional culture media was compared to results with CMRSAII. S. aureus was tested by cefoxitin disk diffusion. CMRSAII was interpreted as positive for MRSA at 24 h (range, 18 to 28 h) based solely on the visualization of mauve-colored colonies and at 48 h (range, 36 to 52 h) based on detection of mauve colonies with subsequent confirmation as S. aureus (by coagulase or latex agglutination testing). MRSA was recovered more frequently on CMRSAII (89.8% at 24 h and 95.6% at 48 h) than on traditional culture plates (83.1% at 24 h and 79.8% at 48 h) for all specimen types combined (P < 0.001). The percent sensitivities of CMRSAII at 24- and 48-h reads, respectively, were 85.5 and 92.4% for respiratory specimens, 87.9% and 98.3% for stool specimens, 88.4% and 96.1% for skin specimens, 92.1% and 94.6% for wound specimens, and 100% and 100% for positive blood cultures. The specificity was 99.8% for respiratory specimens and 100% for all others. In conclusion, CMRSAII is a reliable screening medium for multiple specimen types.Controlling the spread of multidrug-resistant microorganisms and especially methicillin-resistant Staphylococcus aureus (MRSA) has become a major infection control objective in the United States (4) and many European countries (3, 4, 21). A part of most programs to control the spread of MRSA is screening of patients (4, 8, 14), and screening has even become mandatory in some countries (11, 31).Traditionally, MRSA screening included mainly the culturing of naris swabs. However, it has been demonstrated that up to 35% of MRSA carriers may be colonized only from sites other than the nares, for example, the throat or the rectum (1, 2, 16).Usage of chromogenic media can improve the sensitivity and pace of MRSA detection (5, 6, 9, 10, 12, 13, 15, 17,19, 20, 22-24, 26-30); however, currently available media that have been marketed at this time are recommended only for nasal specimens.This study was designed to compare the performance of BBL CHROMagar MRSAII (CMRSAII), a chromogenic medium which incorporates cefoxitin, with traditional culture media in the recovery and identification of MRSA isolates from clinical specimens, including respiratory, lower gastrointestinal, and skin specimens as well as wound cultures and blood culture bottles with Gram-positive cocci. In addition, it was designed to determine whether CMRSAII results may be reported as presumptive or definitive with no (or one) confirmatory test at 24 and 48 h of incubation.(These data were presented in part at the 48th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 25 to 28 October 2008.)     

Helicobacter pylori Stimulates Dendritic Cells To Induce Interleukin-17 Expression from CD4+ T Lymphocytes

Helicobacter pylori is a human gastroduodenal pathogen that leads to active chronic inflammation characterized by T-cell responses biased toward a Th1 phenotype. It has been accepted that H. pylori infection induces a Th17 response. At mucosal sites, dendritic cells (DCs) have the capacity to induce effector T cells. Here, we evaluate the role of DCs in the H. pylori-induced interleukin-17 (IL-17) response. Immunohistochemistry and immunofluorescence were performed on human gastric mucosal biopsy samples and showed that myeloid DCs in H. pylori-infected patients colocalized with IL-23- and that IL-17-producing lymphocytes were present in H. pylori-infected antral biopsy samples. In parallel, human monocyte-derived DCs stimulated in vitro with live H. pylori cells produced significant levels of IL-23 in the absence of IL-12 release. The subsequent incubation of H. pylori-infected DCs with autologous CD4⁺ T cells led to gamma interferon (IFN-γ) and IL-17 expression. The inhibition of IL-1 and, to a lesser extent, IL-23 inhibited IL-17 production by T cells. Finally, isogenic H. pylori mutant strains not expressing major virulence factors were less effective in inducing IL-1 and IL-23 release by DCs and IL-17 release by T cells than parental strains. Altogether, we can conclude that DCs are potent inducers of IL-23/IL-17 expression following H. pylori stimulation. IL-1/IL-23 as well as H. pylori virulence factors seem to play an important role in mediating this response.Gram-negative Helicobacter pylori is a gastroduodenal pathogen identified as being the causative agent of a variety of disease including gastritis, peptic ulcer, gastric adenocarcinoma, and mucosa-associated lymphoma (23, 27, 41, 42). H. pylori infection of gastric mucosa leads to active chronic inflammation characterized by both a lymphocytic and neutrophil infiltrate with the induction of proinflammatory cytokines, mainly interleukin-1β (IL-1β), tumor necrosis factor alpha (TNF-α), IL-8, and IL-6 (13, 29).The H. pylori-specific gastric mucosal T-cell response is predominantly a CD4⁺ T-cell response polarized toward a T-helper 1 (Th1) phenotype with increased levels of gamma interferon (IFN-γ) (4, 38, 55). Although profound, this immune response does not clear the bacteria, and indeed, the cytokines secreted are more associated with pathogenesis (38, 45). Furthermore, neutrophil responses are associated with tissue damage and ulceration (7, 60). The release of the neutrophil chemoattractant IL-8 by gastric epithelial cells was previously shown to depend on the expression of an H. pylori virulence factor: the cytotoxin-associated gene (cag) pathogenicity island (PAI) (14, 62). The cag PAI encodes the immunodominant protein CagA and the type IV secretion system, which serves to transfer the bacterial CagA protein and other soluble factors, such as peptidoglycans, to the cytoplasm of the host cell (9, 52). Strains expressing the cag PAI have been associated with a more severe inflammatory response than that induced by cag PAI-negative strains (12). The cellular recognition of cag PAI-positive strains is mediated via signaling through the host-intracellular pathogen recognition molecule NOD1 (nucleotide-binding oligomerization domain 1), leading to NF-κB activation and the induction of proinflammatory responses (58).It was previously shown that H. pylori infection is also associated with a marked production of Th17 cytokines (2, 39, 44). By using real-time PCR and Western blotting, it was previously demonstrated that IL-17, a proinflammatory cytokine, is upregulated in H. pylori-infected stomach biopsy specimens in comparison to uninfected specimens (39). IL-17 is a cytokine that characterizes a distinct population of T cells, namely, Th17 (1, 28). IL-17 has been associated with chronic inflammatory conditions such as rheumatoid arthritis (10) and multiple sclerosis (37). In addition, IL-17 proinflammatory function leading to IL-8 stimulation raises the possibility that IL-17 may play a role during bacterial infections (39, 57). Major cytokines associated with the differentiation of human Th17 cells were identified to be IL-23, IL-1β, and IL-6 (11, 61). While IL-12 plays a key role in the differentiation of naïve T cells to Th1 cells, IL-23 promotes the expansion of Th17 cells. In contrast, IL-27, another IL-12 family member, has been shown to limit the development of Th17 cells (25). IL-12 and IL-23 are heterodimers with a shared subunit, p40. Both IL-23 and IL-12 are produced by activated antigen-presenting cells (APCs) such as DCs and macrophages (48, 53).DCs, which play a central role in the induction of adaptive immune responses, are widely distributed in tissues, including gastrointestinal mucosa (32, 33), and were previously shown to be capable of migrating through epithelial tight junctions to gain access to the gastrointestinal lumen (33, 49). Furthermore, we and others have shown that H. pylori interactions with DCs trigger maturation and activation events that lead to the production of cytokines, which are important for the induction and regulation of immune responses (5, 18, 34, 43, 46).Previous studies of DC activation by H. pylori have focused on the induction of the Th1-biased response. Much less is known about the mechanism of induction as well as the cells and cytokine stimuli responsible for the expression of IL-17 in Helicobacter infection. Here, we have reevaluated the role of DCs in the induction of immune responses to Helicobacter infection by addressing the interaction of H. pylori-infected DCs with CD4⁺ T lymphocytes in initiating a Th17 response.     

Concurrent Helicobacter bilis Infection in C57BL/6 Mice Attenuates Proinflammatory H. pylori-Induced Gastric Pathology

Because coinfections can alter helicobacter gastritis, we investigated whether enterohepatic Helicobacter bilis modulates Helicobacter pylori gastritis in C57BL/6 mice. Thirty mice per group were sham dosed, H. bilis or H. pylori infected, or H. bilis infected followed in 2 weeks by H. pylori and then evaluated at 6 and 11 months postinfection (mpi) for gastritis and premalignant lesions. Compared to H. pylori-infected mice, H. bilis/H. pylori-infected mice at 6 and 11 mpi had less severe gastritis, atrophy, mucous metaplasia and hyperplasia (P < 0.01) and, additionally, at 11 mpi, less severe intestinal metaplasia and dysplasia (P < 0.05). H. bilis/H. pylori-infected mice at 11 mpi exhibited less Ki67 labeling of proliferating epithelial cells, reduced numbers of FoxP3⁺ T-regulatory (T_REG) cells, and lower FoxP3⁺ mRNA levels than did H. pylori-infected mice (P < 0.05). Proinflammatory interleukin-1β (IL-1β), gamma interferon, and tumor necrosis factor alpha mRNA levels were attenuated in H. bilis/H. pylori-infected mice at 6 and 11 mpi (P < 0.01), although anti-inflammatory IL-10, IL-13, and transforming growth factor β1 mRNA levels were not consistently impacted by H. bilis coinfection. Decreased pathology in H. bilis/H. pylori-infected mice correlated with higher gastric H. pylori colonization at 6 mpi (P < 0.001) and lower Th1-associated immunoglobulin G2c responses to H. pylori at 6 and 10 mpi (P < 0.05). We hypothesized that reduced pathology in H. bilis/H. pylori-infected mice was due to H. bilis-primed T_REG cells in the lower bowel that migrated to the gastric compartment and inhibited Th1 responses to subsequent H. pylori infection. Thus, H. pylori-induced gastric lesions may vary in mouse models of unknown enteric helicobacter infection status and, importantly, variable sequelae to human H. pylori infection, particularly in developing countries, may occur where coinfection with lower bowel helicobacters and H. pylori may be common.Helicobacter pylori, first isolated by Warren and Marshall, induces a persistent infection and gastritis and is known to colonize the stomach of over 50% of the human population (2). In a subset of infected individuals, H. pylori is linked to the development of peptic ulcer disease, gastric adenocarcinoma, and gastric mucosa-associated lymphoid tissue lymphoma. It has been classified by the World Health Organization as a class I carcinogen (25). It is not clear why some individuals infected with H. pylori develop serious disease, while others do not. Host and environmental factors, as well as the virulence properties of H. pylori, appear to play an important role in determining disease outcome (17, 52, 60). Poor socioeconomic conditions promote early acquisition and infection with H. pylori and infection rates often approach >90% in these populations. Interestingly, some African countries with especially high prevalence rates of infection have lower-than-expected rates of gastric cancer. This paradox has been referred to as “the African Enigma” (24). The low incidence of gastric cancer has been linked to endemic parasites, diet, poor cancer registry data, and the low pathogenicity of some H. pylori strains (3a, 12, 31a).Like humans, mice respond immunologically to infectious agents with a repertoire of memory T cells that respond most efficiently after antigen priming (28) but also appear to modulate host responses to unrelated infections and likely are also effective in disease caused by organisms sharing common antigens. These cross-reactive T cells, when activated, not only modulate the immune response but also determine the eventual outcome of heterologous infections. This host immune response is often referred to as heterologous immunity (47). This phenomenon has been studied to a limited extent in mouse models of gastric helicobacter pathogenesis that have had varied pathological outcomes. In a C57BL/6 mouse model of H. felis gastritis, coinfection with an enteric helminth, Heligmosomoides polygyrus, stimulated a Th2 response that attenuated Th1-promoted gastric pathology (11). In contrast, in BALB/c mice which have a Th2-biased response to gastric helicobacter infection resulting in no discernible gastritis, coinfection with Toxoplasma gondii promoted a robust Th1 immune response, resulting in a progressive helicobacter-associated gastritis, gastric atrophy, and metaplasia (50). Recently, we have demonstrated that the colitis induced by Citrobacter rodentium resulted in a prolonged recovery of the disease in C57BL/6 mice when the animals were coinfected with H. hepaticus (34). It is also known that host immune responses resulting from infections with atypical mycobacteria can influence how mice or humans respond immunologically to BCG vaccination (9, 62). These examples of heterologous immunity suggest that disease outcomes can be impacted by modulation of Th1 and Th2 inflammatory responses (37).Subclinical lower bowel helicobacter infections are prevalent worldwide in mouse colonies; however, the persistent infection in certain inbred strains of mice often elicits demonstrable pathology (53). In susceptible mouse strains, enterohepatic helicobacters cause inflammatory bowel disease, colonic adenocarcinoma, hepatitis, cholecystitis, and hepatocellular carcinoma (8, 31, 33, 57). Non-H. pylori helicobacters are increasingly cited in association with human diarrheal disease, particularly in developing countries, as well as with hepatobiliary diseases in humans (10, 12, 13, 22, 38). These observations of enterohepatic helicobacter-associated disease in humans and the common occurrence of enteric helicobacter infections in mice suggest that helicobacter coinfections could impact murine studies involving H. pylori pathogenesis, vaccine strategies, and antimicrobial modalities. Thus, we initiated an experiment to ascertain whether coinfection with H. bilis, an enterohepatic helicobacter with a wide host range (10), could impact the progression of H. pylori-induced gastric disease and inflammatory responses in C57BL/6 mice (10, 12, 19, 46).     

Uptake of Helicobacter pylori Outer Membrane Vesicles by Gastric Epithelial Cells

Helicobacter pylori bacteria colonize the human stomach where they stimulate a persistent inflammatory response. H. pylori is considered noninvasive; however, lipopolysaccharide (LPS)-enriched outer membrane vesicles (OMV), continuously shed from the surface of this bacterium, are observed within gastric epithelial cells. The mechanism of vesicle uptake is poorly understood, and this study was undertaken to examine the roles of bacterial VacA cytotoxin and LPS in OMV binding and cholesterol and clathrin-mediated endocytosis in vesicle uptake by gastric epithelial cells. OMV association was examined using a fluorescent membrane dye to label OMV, and a comparison was made between the associations of vesicles from a VacA⁺ strain and OMV from a VacA⁻ isogenic mutant strain. Within 20 min, essentially all associated OMV were intracellular, and vesicle binding appeared to be facilitated by the presence of VacA cytotoxin. Uptake of vesicles from the VacA⁺ strain was inhibited by H. pylori LPS (58% inhibition with 50 μg/ml LPS), while uptake of OMV from the VacA⁻ mutant strain was less affected (25% inhibition with 50 μg/ml LPS). Vesicle uptake did not require cholesterol. However, uptake of OMV from the VacA⁻ mutant strain was inhibited by a reduction in clathrin-mediated endocytosis (42% with 15 μg/ml chlorpromazine), while uptake of OMV from the VacA⁺ strain was less affected (25% inhibition with 15 μg/ml chlorpromazine). We conclude that VacA toxin enhances the association of H. pylori OMV with cells and that the presence of the toxin may allow vesicles to exploit more than one pathway of internalization.Infection with the gastric pathogen Helicobacter pylori results in chronic gastritis (13) and is associated with increased risk for the development of peptic ulcer disease (35), gastric carcinoma (41, 57), and gastric lymphoma (5, 60). H. pylori persistence, in an environment where peristalsis and sloughing of cells are continually occurring, is mediated by a variety of adhesins present on the bacterial surface (14, 21, 36, 40). However, despite the ability to adhere to the gastric epithelium, the majority of organisms remain unattached to surface cells (32), leading to speculation that lipopolysaccharide (LPS)-enriched outer membrane vesicles (OMV) shed by these bacteria (15, 19, 26) contribute to H. pylori pathogenesis via the persistent delivery of bacterial virulence factors (including the vacuolating cytotoxin VacA) and antigens to the gastric mucosa (26, 27). Observations that H. pylori OMV modulate gastric epithelial cell proliferation (22), induce apoptosis (3), stimulate secretion of the proinflammatory cytokine interleukin-8 (22), increase micronucleus formation (8), and are at the luminal surface (15, 26) and within cells of the gastric epithelium (15) support this hypothesis.OMV shedding by Gram-negative bacteria is well described in the literature (reviewed by Kuehn and Kesty [33]), yet little is known of the mechanisms of vesicle adherence to and internalization within mammalian host cells. The adherence of enterotoxigenic Escherichia coli (ETEC) OMV to host cells is mediated via a heat-labile enterotoxin (LT) associated with these OMV (31), whereas leukotoxin, associated with OMV shed by Actinobacillus actinomycetemcomitans, is not involved in vesicle binding (12). The internalization of ETEC, Porphyromonas gingivalis, and Pseudomonas aeruginosa OMV has been shown to involve cholesterol-rich lipid rafts (6, 16, 31), and recently, Kaparakis and colleagues (25) reported that uptake of H. pylori OMV is also lipid raft dependent. This is in contrast to the uptake of Shigella flexneri OMV, which occurs via phagocytosis, with the proposed subsequent fusion of OMV with the phagosomal membrane and the release of vesicle contents into the cell cytoplasm (24).In this study, we sought to examine whether VacA cytotoxin associated with H. pylori OMV was involved in vesicle binding. We also examined the rate of OMV internalization and the involvement of LPS, cholesterol, and clathrin-mediated endocytosis in vesicle uptake by AGS gastric epithelial cells. We report that within 20 min essentially all VacA⁺ OMV associated with AGS cells were intracellular and that uptake was enhanced by the presence of vesicle-associated cytotoxin. Excess H. pylori LPS reduced vesicle uptake, having a more significant effect on VacA⁺ OMV than VacA⁻ vesicle uptake. Uptake of both VacA⁺ and VacA⁻ OMV did not require cholesterol. However, a reduction in clathrin-mediated endocytosis inhibited VacA⁻ OMV uptake and to a lesser extent VacA⁺ OMV internalization.     

Vancomycin MIC plus Heteroresistance and Outcome of Methicillin-Resistant Staphylococcus aureus Bacteremia: Trends over 11 Years

Vancomycin MICs (V-MIC) and the frequency of heteroresistant vancomycin-intermediate Staphylococcus aureus (hVISA) isolates are increasing among methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) isolates, but their relevance remains uncertain. We compared the V-MIC (Etest) and the frequency of hVISA (Etest macromethod) for all MRSA blood isolates saved over an 11-year span and correlated the results with the clinical outcome. We tested 489 isolates: 61, 55, 187, and 186 isolates recovered in 1996-1997, 2000, 2002-2003, and 2005-2006, respectively. The V-MICs were ≤1, 1.5, 2, and 3 μg/ml for 74 (15.1%), 355 (72.6%), 50 (10.2%), and 10 (2.1%) isolates, respectively. We detected hVISA in 0/74, 48/355 (13.5%), 15/50 (30.0%), and 8/10 (80.0%) isolates with V-MICs of ≤1, 1.5, 2, and 3 μg/ml, respectively (P < 0.001). The V-MIC distribution and the hVISA frequency were stable over the 11-year period. Most patients (89.0%) received vancomycin. The mortality rate (evaluated with 285 patients for whose isolates the trough V-MIC was ≥10 μg/ml) was comparable for patients whose isolates had V-MICs of ≤1 and 1.5 μg/ml (19.4% and 27.0%, respectively; P = 0.2) but higher for patients whose isolates had V-MICs of ≥2 μg/ml (47.6%; P = 0.03). However, the impact of V-MIC and hVISA status on mortality or persistent (≥7 days) bacteremia was not substantiated by multivariate analysis. Staphylococcal chromosome cassette mec (SCCmec) typing of 261 isolates (including all hVISA isolates) revealed that 93.0% of the hVISA isolates were SCCmec type II. These findings demonstrate that the V-MIC distribution and hVISA frequencies were stable over an 11-year span. A V-MIC of ≥2 μg/ml was associated with a higher rate of mortality by univariate analysis, but the relevance of the V-MIC and the presence of hVISA remain uncertain. A multicenter prospective randomized study by the use of standardized methods is needed to evaluate the relevance of hVISA and determine the optimal treatment of patients whose isolates have V-MICs of ≥2.0 μg/ml.The treatment of methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) bacteremia with vancomycin is often associated with a poor clinical outcome (6, 15, 28, 40). Treatment failure was reported among patients infected with isolates whose vancomycin MICs were ≥4 μg/ml (6, 9, 12, 25, 28, 42). This prompted the Clinical and Laboratory Standards Institute to lower the cutoffs for S. aureus susceptibility to ≤2 μg/ml for susceptible, 4 to 8 μg/ml for intermediate (vancomycin-intermediate S. aureus [VISA]), and 16 μg/ml for resistance (39). Within the susceptibility range, the MIC is reported to increase over time (14, 25, 35-40). This is often referred to as MIC creep (38). Additionally, isolates with heteroresistance (heteroresistant vancomycin-intermediate S. aureus [hVISA]) are emerging, and this has uncertain implications for laboratory detection and clinical management (2, 5, 15, 24, 40-42). The first isolate of hVISA to be identified was reported from Japan in 1997 (11). Since then, it has been reported worldwide at frequencies of 0 to 50% (2, 4, 6, 9, 12, 19, 20, 21, 24, 26, 27, 31, 40, 42, 44). This disparity in frequency is probably a result of its variable incidence and the different testing methodologies used. Likewise, the frequency of isolates with MICs of 1.5 to <4 μg/ml varies according to the testing method used (3, 32). The relevance of an MIC on the higher side of the susceptibility range and the presence of hVISA isolates remains uncertain (8, 19, 21). Therapeutic failure was reported in patients infected with isolates with vancomycin MICs of 2 μg/ml (6, 12, 28) and 1.5 or 1 μg/ml (25, 34, 37). Most clinical microbiology laboratories use automated testing methods that are known to underestimate the vancomycin MIC (13, 24). Additionally, most previous studies addressing the relevance of such isolates were observational and usually involved only a few patients and poorly selected controls (1, 4, 7, 9, 12, 14, 25, 35, 38, 42). At our institution, we found the frequency of hVISA isolates among isolates from patients with persistent MRSA bacteremia to be 14%; however, heteroresistance did not correlate with the mortality rate (19). In the current study, we tested all blood MRSA isolates collected over 11 years to determine whether the vancomycin MIC and the prevalence of hVISA have changed over time and to evaluate the effects of increasing vancomycin MICs and the hVISA frequency on patient outcomes.     

Pediatric Helicobacter pylori Isolates Display Distinct Gene Coding Capacities and Virulence Gene Marker Profiles

Helicobacter pylori strains display remarkable genetic diversity, and the presence of strains bearing the toxigenic vacA s1 allele, a complete cag pathogenicity island (PAI), cagA alleles containing multiple EPIYA phosphorylation sites, and expressing the BabA adhesin correlates with development of gastroduodenal disease in adults. To better understand the genetic variability present among pediatric strains and its relationship to disease, we characterized H. pylori strains infecting 47 pediatric North American patients. Prevalence of mixed infection was assessed by random amplified polymorphic DNA analysis of multiple H. pylori clones from each patient. Microarray-based comparative genomic hybridization was used to examine the genomic content of the pediatric strains. The cagA and vacA alleles were further characterized by allele-specific PCR. A range of EPIYA motif configurations were observed for the cagA gene, which was present in strains from 22 patients (47%), but only 19 (41%) patients contained a complete cag PAI. Thirty patients (64%) were infected with a strain having the vacA s1 allele, and 28 patients (60%) had the babA gene. The presence of a functional cag PAI was correlated with ulcer disease (P = 0.0095). In spite of declining rates of H. pylori infection in North America, at least 11% of patients had mixed infection. Pediatric strains differ in their spectrum of strain-variable genes and percentage of absent genes in comparison to adult strains. Most children were infected with H. pylori strains lacking the cag PAI, but the presence of a complete cag PAI, in contrast to other virulence markers, was associated with more severe gastroduodenal disease.It is estimated that >50% of the world''s population is colonized with Helicobacter pylori in the stomach, making it one of the most common bacterial pathogens of humans. H. pylori infection is generally acquired in childhood (24, 33) and can persist for life. Gastritis (inflammation of the gastric mucosa) results in all who are colonized with H. pylori, but some hosts remain asymptomatic, while others develop peptic ulcers, gastric adenocarcinomas, and mucosa-associated lymphoid tissue lymphoma. Gastric cancer is the second leading cause of cancer death worldwide, and 63% of gastric cancer cases in 2002 were attributable to H. pylori infection (38, 49). While severe disease most often presents in adulthood, children display H. pylori-associated gastritis and the incidence of ulcer disease among infected children was 6.8% in a European pediatric population (31). Many studies have examined bacterial, host, and environmental risk factors associated with development of H. pylori-associated diseases in adults, but similar studies in children have been limited.Genetic differences among H. pylori strains contribute to differences in disease outcome among infected individuals in adult populations. The gene encoding VacA, which induces vacuolation of host cells, is present in nearly all H. pylori strains, but a number of allele types have been defined. Strains having the type s1 vacA signal sequence and the m1 vacA middle region allele (vacA s1/m1) are associated with ulcer disease (9). The cag pathogenicity island (PAI) encodes a type IV secretion system (T4SS) (1, 15) that translocates the CagA protein effector, also encoded in the island, into host cells. Presence of the cag PAI is associated with increased inflammation, promoting host cell interleukin-8 (IL-8) production, and cagA-positive strains are associated with peptic ulcers (50) as well as gastric cancer (13). Inside the host cell, CagA protein becomes tyrosine phosphorylated at C-terminal EPIYA (Glu-Pro-Ile-Tyr-Ala) sites by src family kinases, deregulates SHP-2, and induces the hummingbird phenotype (26, 45). Strains having more C-type EPIYA motifs, the major phosphorylation site, induce stronger effects on host cells and are associated with gastric cancer (7, 12, 35). The presence of a functional allele of babA, a gene encoding an adhesin that mediates binding to Lewis B antigens expressed on gastric epithelial cells, is associated with duodenal ulcer and gastric adenocarcinoma (21).While these H. pylori genes and alleles have been associated with disease outcome in adults, studies in children have provided mixed results. A recent study identified two genes (jhp0562, coding for a putative glycosyltransferase, and jhp0870, coding for an outer membrane protein) associated with peptic ulcer disease in children, but not adults, suggesting a different spectrum of genetic risk factors in adults and children (37). Studies using a whole-genome microarray-based approach have been done to investigate the variability in genomic content of H. pylori strains, but these studies have included mostly strains from adult patients (25, 29, 41, 42). Studies of the genetic variability of pediatric H. pylori strains have largely been limited to genes previously associated with virulence in adult populations. To better understand the genetic variability present among pediatric strains, we used whole-genome microarray-based comparative genomic hybridization to examine the genomic content of H. pylori strains isolated from symptomatic North American children and compared the pediatric isolate genetic variability to that observed in adult strains. We then examined the frequency of known virulence genes and virulence alleles among the pediatric H. pylori strains and the associations of strain genotype with the clinical and histological characteristics of the patients.     

Results from the ARTEMIS DISK Global Antifungal Surveillance Study, 1997 to 2007: a 10.5-Year Analysis of Susceptibilities of Candida Species to Fluconazole and Voriconazole as Determined by CLSI Standardized Disk Diffusion

Fluconazole in vitro susceptibility test results for 256,882 isolates of Candida spp. were collected from 142 sites in 41 countries from June 1997 to December 2007. Data were collected for 197,619 isolates tested with voriconazole from 2001 to 2007. A total of 31 different species of Candida were isolated. Increased rates of isolation of the common non-albicans species C. glabrata (10.2% to 11.7%), C. tropicalis (5.4% to 8.0%), and C. parapsilosis (4.8% to 5.6%) were noted when the time periods 1997 to 2000 and 2005 to 2007 were compared. Investigators tested clinical isolates of Candida spp. by the CLSI M44-A disk diffusion method. Overall, 90.2% of Candida isolates tested were susceptible (S) to fluconazole; however, 13 of 31 species identified exhibited decreased susceptibility (<75% S), similar to that seen with the resistant (R) species C. glabrata and C. krusei. Among 197,619 isolates of Candida spp. tested against voriconazole, 95.0% were S and 3% were R. About 30% of fluconazole-R isolates of C. albicans, C. glabrata, C. tropicalis, C. rugosa, C. lipolytica, C. pelliculosa, C. apicola, C. haemulonii, C. humicola, C. lambica, and C. ciferrii remained S to voriconazole. An increase in fluconazole resistance over time was seen with C. parapsilosis, C. guilliermondii, C. lusitaniae, C. sake, and C. pelliculosa. Among the emerging fluconazole-R species were C. guilliermondii (11.4% R), C. inconspicua (53.2% R), C. rugosa (41.8% R), and C. norvegensis (40.7% R). The rates of isolation of C. rugosa, C. inconspicua, and C. norvegensis increased by 5- to 10-fold over the 10.5-year study period. C. guilliermondii and C. rugosa were most prominent in Latin America, whereas C. inconspicua and C. norvegensis were most common in Eastern European countries. This survey identifies several less-common species of Candida with decreased susceptibility to azoles. These organisms may pose a future threat to optimal antifungal therapy and underscore the importance of prompt and accurate species identification and antifungal susceptibility testing.Antifungal susceptibility testing is playing an increasing role as a means to track the development of antifungal resistance in epidemiological studies (2, 10, 12, 17, 27, 45-47, 55, 63). One of the important by-products of the standardization of antifungal susceptibility testing has been the ability to conduct surveillance for antifungal resistance using uniform methods (44). Meaningful large-scale surveys of antifungal susceptibility and resistance conducted over time would not be possible without a standardized broth microdilution (BMD) or disk diffusion (DD) method for performing the in vitro studies (12, 38, 60). Global surveillance programs such as the ARTEMIS antifungal surveillance program for DD testing (49, 57, 60) and MIC testing (12, 13), the European Confederation of Medical Mycology (ECMM) survey of candidemia (68), and the SENTRY Antifungal Surveillance Program (36-38) promote the use of standardized DD and BMD methods and provide useful and consistent antifungal susceptibility data from a broad international network of hospitals and laboratories.The ARTEMIS global antifungal surveillance program is among the most comprehensive and long-running fungal surveillance programs (12, 45, 57, 58, 60). The ARTEMIS program was designed to address many of the potential limitations of resistance surveillance studies (26): (i) it is both longitudinal (1997 to present) and global (142 participating sites in 41 countries) in scope, (ii) it employs standardized DD (7) and BMD (9) antifungal susceptibility test methods, (iii) both internal quality control (QC) performed in each participating laboratory and external quality assurance measures are used to validate test results (48, 50, 61), (iv) results are recorded electronically using the Biomic image analysis plate reader (Giles Scientific, Santa Barbara, CA) and are stored in a central database, and (v) both Candida and non-Candida (60) yeast isolates obtained from consecutive clinical samples from all body sites are tested locally, thus avoiding misleading results based on biased selective testing. Thus, the ARTEMIS program generates massive amounts of data that have been externally validated and that can be used to identify temporal and geographic trends in the species distribution of Candida and other opportunistic yeasts, as well as the resistance profiles of these organisms to fluconazole and voriconazole as determined by standardized Clinical and Laboratory Standards Institute (CLSI) DD methods.In the present study, we expand the ARTEMIS database to include the time period from June 1997 through December 2007 and a total of 256,882 isolates of Candida from 142 study sites in 41 countries. We provide comparative susceptibility data for fluconazole and voriconazole for more than 190,000 isolates collected from 2001 to 2007 and include an analysis of resistance rates by year, geographic location, hospital location, and specimen type for selected species.     

High-Throughput Identification of Bacteria and Yeast by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry in Conventional Medical Microbiology Laboratories

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is suitable for high-throughput and rapid diagnostics at low costs and can be considered an alternative for conventional biochemical and molecular identification systems in a conventional microbiological laboratory. First, we evaluated MALDI-TOF MS using 327 clinical isolates previously cultured from patient materials and identified by conventional techniques (Vitek-II, API, and biochemical tests). Discrepancies were analyzed by molecular analysis of the 16S genes. Of 327 isolates, 95.1% were identified correctly to genus level, and 85.6% were identified to species level by MALDI-TOF MS. Second, we performed a prospective validation study, including 980 clinical isolates of bacteria and yeasts. Overall performance of MALDI-TOF MS was significantly better than conventional biochemical systems for correct species identification (92.2% and 83.1%, respectively) and produced fewer incorrect genus identifications (0.1% and 1.6%, respectively). Correct species identification by MALDI-TOF MS was observed in 97.7% of Enterobacteriaceae, 92% of nonfermentative Gram-negative bacteria, 94.3% of staphylococci, 84.8% of streptococci, 84% of a miscellaneous group (mainly Haemophilus, Actinobacillus, Cardiobacterium, Eikenella, and Kingella [HACEK]), and 85.2% of yeasts. MALDI-TOF MS had significantly better performance than conventional methods for species identification of staphylococci and genus identification of bacteria belonging to HACEK group. Misidentifications by MALDI-TOF MS were clearly associated with an absence of sufficient spectra from suitable reference strains in the MALDI-TOF MS database. We conclude that MALDI-TOF MS can be implemented easily for routine identification of bacteria (except for pneumococci and viridans streptococci) and yeasts in a medical microbiological laboratory.Identification of bacteria and yeasts is generally based on conventional phenotypic methods, encompassing culture and growth patterns on specific media, Gram staining, and morphological and biochemical characteristics. Although results of Gram staining can be achieved within minutes, complete identification usually takes 1 or more days. In addition, tests may be difficult to interpret or inconclusive and require specialized staff. Recent molecular methods for microbial identification, such as real-time PCR, sequence analysis, or microarray analysis, have found some application in bacteriology. However, these methods do not provide the complete solution in routine bacterial identifications. To optimize care of patients with infectious diseases, there still is an urgent need for rapid and simple techniques for microbial identification.Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been used to analyze many different biological molecules. The application of microbial identification based on species-specific spectra of peptides and protein masses by mass spectrometry was first reported about 30 years ago (1). By further improvement of the technique, a rapid, accurate, easy-to-use, and inexpensive method has become available for identification of microorganisms (4, 14, 27). MALDI-TOF MS can be used for accurate and rapid identification of various microorganisms, such as Gram-positive bacteria (2, 3, 9, 10, 22, 26), Enterobacteriaceae (5), nonfermenting bacteria (6, 19-21), mycobacteria (12, 16, 24), anaerobes (10, 23), and yeasts (18, 25). Most studies have reported on MALDI-TOF MS identification of a single strain or family of microorganisms in a research setting. Only one study applied MALDI-TOF MS for identification of bacteria—but not yeasts—in conventional microbiology settings but did not evaluate the results for individual bacteria at the species level (27). In the present study, identification of bacteria by MALDI-TOF MS was extensively evaluated for both bacterial and yeast species identification in an academic medical microbiologic laboratory.     

Validation and Clinical Application of a Molecular Method for Identification of Histoplasma capsulatum in Human Specimens in Colombia,South America

The conventional means of diagnosis of histoplasmosis presents difficulties because of the delay to the time that the diagnosis is made, indicating the need for the implementation of molecular assays. We evaluated 146 clinical samples from 135 patients suspected of having histoplasmosis using a previously reported nested PCR assay for the Histoplasma capsulatum-specific 100-kDa protein (the Hc100 PCR). In order to determine the specificity of this molecular test, we also used samples from healthy individuals (n = 20), patients suspected of having respiratory disease with negative fungal cultures (n = 29), and patients with other proven infections (n = 60). Additionally, a sizable collection of DNA from cultures of H. capsulatum and other medically relevant pathogens was studied. A panfungal PCR assay that amplified the internal transcribed spacer 2 region was also used to identify all fungal DNAs. All PCR-amplified products were sequenced. Of the 146 clinical samples, 67 (45.9%) were positive by culture and PCR, while 9 samples negative by culture were positive by PCR. All the sequences corresponding to the 76 amplified products presented ≥98% identity with H. capsulatum. The Hc100 PCR exhibited a sensitivity of 100% and specificities of 92.4% and 95.2% when the results were compared to those for the negative controls and samples from other proven clinical entities, respectively; the positive predictive value was 83% and the negative predictive value was 100%; the positive and negative likelihood rates were 25 and 0, respectively. These results suggest that the Hc100 nested PCR assay for the detection of H. capsulatum DNA is a useful test in areas where mycosis caused by this organism is endemic.Histoplasmosis is the most important mycosis endemic in the Americas and occurs by inhalation of the infectious propagules (microconidia) produced by the dimorphic fungus Histoplasma capsulatum (19, 32). It is amply distributed in most countries, being more prevalent in specific regions of United States, such as the Mississippi and Ohio River Valleys (14, 19). A high prevalence of histoplasmosis has also been observed in Central America (Mexico, Panama, Honduras, Guatemala, and Nicaragua), the Caribbean (Jamaica, Puerto Rico, Cuba, and Martinique), and South America (Venezuela, French Guyana, Colombia, Peru, Brazil, and Argentina) (16, 25).The severity of histoplasmosis varies greatly depending on the intensity of the exposure to the fungus and on the immune status of the infected individual (18, 29). In patients with immunodeficiency disorders, and especially in those infected with HIV, histoplasmosis is considered an opportunistic infection (17, 20, 27); in addition, in a high proportion of cases, this fungal infection is manifested as a severe disseminated process which often leads to death if it is not treated promptly (17, 20, 27).The diagnosis of histoplasmosis is usually accomplished by culture and microscopic examination of respiratory tract, biopsy, and body fluid specimens; nevertheless, these techniques yield positive results in only approximately 50% of the cases (9, 16, 18, 32). In addition, culturing of the fungus usually takes from 2 to 6 weeks, thus delaying the times to diagnosis and the initiation of therapy. Immunological tests that detect antibodies and/or antigens are also of value and may give results faster than culture. However, they show variable values of sensitivity and specificity and may often be negative for immunodeficient patients (18). The detection of antigen in serum and urine samples appears to be a sensitive and specific diagnostic tool, especially in HIV-infected patients (81 to 95% sensitivity with urine) (8, 12, 13, 26), although antigen detection shows cross-reactivity with the causative agents of other mycoses (12, 13, 16, 18, 30, 31).In the last decade, several molecular approaches have been developed for the detection of H. capsulatum DNA in human clinical samples. Various studies have obtained high sensitivity and specificity values for PCR-based molecular tests, including a PCR (the Hc100 PCR) that detects a gene that codes for an H. capsulatum 100-kDa protein (Hc100), which is essential for the survival of H. capsulatum in human cells (3); a PCR that detects 18S rRNA (2); a PCR that detects the internal transcribed spacer (ITS) region of the rRNA gene complex (21); and a PCR that detects the M and H antigens (4, 15). Some of these PCR assays have been tested with paraffin-embedded biopsy samples (3), blood specimens (22), infected mouse tissues (2), and samples from in vitro cultures; however, the DNA-based diagnosis of this fungal infection has not yet been established as a regular diagnostic tool, nor is a PCR assay commercially available (19).In the present study, we evaluated over a 2-year period a cohort of patients with suspected or clinically diagnosed histoplasmosis, using a nested PCR targeting the gene coding for the 100-kDa protein previously described by Bialek et al. (3) and using fungal isolation in culture as the “gold standard” technique.(The results presented here are part of Cesar Muñoz′s master''s thesis for the Corporation of Biomedical Basic Sciences Master''s Program, Universidad de Antioquia, Medellín, Colombia.)     

Association of Single Nucleotide Polymorphisms in Cytotoxic T-Lymphocyte Antigen 4 and Susceptibility to Autoimmune Type 1 Diabetes in Tunisians

In addition to HLA and insulin genes, the costimulatory molecule CTLA-4 gene is a confirmed type 1 diabetes (T1D) susceptibility gene. Previous studies investigated the association of CTLA-4 genetic variants with the risk of T1D, but with inconclusive findings. Here, we tested the contributions of common CTLA-4 gene variants to T1D susceptibility in Tunisian patients and control subjects. The study subjects comprised 228 T1D patients (47.8% females) and 193 unrelated healthy controls (45.6% females). Genotyping for CTLA-4 CT60A/G (rs3087243), +49A/G (rs231775), and −318C/T (rs5742909) was performed by PCR-restriction fragment length polymorphism (RFLP) analysis. The minor-allele frequencies (MAF) for the three CTLA-4 variants were significantly higher in T1D patients, and significantly higher frequencies of homozygous +49G/G and homozygous CT60G/G genotypes were seen in patients, which was confirmed by univariate regression analysis (taking the homozygous wild type as a reference). Of the eight possible three-locus CTLA-4 haplotypes (+49A/G, −318C/T, and CT60A/G) identified, multivariate regression analysis confirmed the positive association of ACG (odds ratio [OR], 1.93; 95% confidence interval [CI], 1.26 to 2.94), GCG (OR, 2.40; 95% CI, 1.11 to 5.21), and GTA (OR, 4.67; 95% CI, 1.52 to 14.39) haplotypes with T1D, after confounding variables were adjusted for. Our results indicate that CTLA-4 gene variants are associated with increased T1D susceptibility in Tunisian patients, further supporting a central role for altered T-cell costimulation in T1D pathogenesis.Type 1 (insulin-dependent) diabetes (T1D) is the most prevalent form of diabetes in children and young adults and results from autoimmune CD4⁺ and CD8⁺ T-cell-directed destruction of insulin-producing pancreatic β islet cells in genetically susceptible individuals (3, 12), leading to irreversible hyperglycemia and related complications (13). There is a strong genetic component to T1D pathogenesis, evidenced by its clustering in families and by the contributions of a number of susceptibility gene variants to its pathogenesis (10, 12, 29). They include the human leukocyte antigen (HLA) locus, in particular the class II region (DR and DQ), which accounts for 40 to 50% of T1D familial clustering (1, 12, 18), and non-HLA susceptibility loci, several of which were mapped by genome-scanning (11, 29) and/or candidate gene (7, 18, 31) approaches. They include insulin promoter gene variants, which reportedly may modulate immunological tolerance by controlling the expansion of the autoreactive cell pool (26), and the T-cell costimulator cytotoxic T-lymphocyte antigen 4 (CTLA-4) transmembrane glycoprotein, which plays a key role in the fine tuning of T-cell immunity (9, 32, 33).CTLA-4 is a 40-kDa transmembrane glycoprotein expressed on resting and activated T cells and nonlymphoid cells (33), and along with the related CD28 costimulatory molecule, it regulates T-cell activation (and is itself primarily mediated by engagement of the T-cell receptor [TCR]) but does recognize major histocompatibility complex (MHC)-bound antigenic peptides (9, 33). CTLA-4 negatively regulates T-cell activation and effector function, in part by inhibiting Th1 (interleukin 2 [IL-2] and gamma interferon [IFN-γ]) cytokine production and IL-2 receptor α-chain (p55; Tac) expression by engaging antigen-presenting cell (APC)-bound B7.1 (CD80) and B7.2 (CD86) ligands (9, 33). Functionally, CTLA-4 attenuates T-cell signaling by interference with intracellular signal transduction events, including TCR signaling, and reduced CTLA-4 expression and/or activity results in uncontrolled T-cell-associated autoimmunity and lymphoproliferative disease (9, 21). In this regard, it was shown that CTLA-4 polymorphisms significantly influence the risk of autoimmune diseases, including Graves'' disease, systemic lupus erythematosus, autoimmune hypothyroidism, celiac disease, and type 1 diabetes (15, 21, 32).First observed in Italian subjects (25), and confirmed subsequently by case control and family studies, CTLA-4 polymorphic variants were linked with T1D pathogenesis (14, 20, 31, 32). While this association was detected in different ethnic groups (14, 23, 30), it appears more likely to be Caucasian selective (10, 29, 33) and absent from non-Caucasians (5, 6, 8, 19, 22). A recent report from the Type I Diabetes Genetics Consortium bearing on 2,300 affected sib pair families demonstrated that among the 24 single nucleotide polymorphisms (SNPs) genotyped in the CTLA-4 region, only the +49A/G and CT60 SNPs were replicated in the nine combined collections (27). In the present study, we investigated the association of three common CTLA-4 SNPs (−318C/T; +49A/G, and CT60A/G) and the corresponding haplotypes with T1D in Tunisian Arab patients.     

Clonal Analysis of Colonizing Group B Streptococcus,Serotype IV,an Emerging Pathogen in the United States

Colonizing group B Streptococcus (GBS) capsular polysaccharide (CPS) type IV isolates were recovered from vaginal and rectal samples obtained from 97 (8.4%) nonpregnant women of 1,160 women enrolled in a U.S. multicenter GBS vaccine study from 2004 to 2008. Since this rate was much higher than the rate of prevalence of 0.4 to 0.6% that we found in previous studies, the isolates were analyzed by using surface protein profile identification, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST) to characterize them and identify trends in DNA clonality and divergence. Of the 101 type IV isolates studied, 53 expressed α and group B protective surface (BPS) proteins, 27 expressed BPS only, 20 expressed α only, and 1 had no detectable surface proteins. The isolates spanned three PFGE macrorestriction profile groups, groups 37, 38, and 39, of which group 37 was predominant. The isolates in group 37 expressed the α and BPS proteins, while those in groups 38 and 39 expressed the α protein only, with two exceptions. MLST studies of selective isolates from the four protein profile groups showed that isolates expressing α,BPS or BPS only were of a new sequence type, sequence type 452, while those expressing α only or no proteins were mainly of a new sequence type, sequence type 459. Overall, our study revealed a limited diversity in surface proteins, MLST types, and DNA macrorestriction profiles for type IV GBS. There appeared to be an association between the MLST types and protein expression profiles. The increased prevalence of type IV GBS colonization suggested the possibility that this serotype may emerge as a GBS pathogen.Group B Streptococcus (GBS) (Streptococcus agalactiae) is a leading cause of neonatal infection in the United States, with maternal vaginal or rectal colonization often resulting in the transmission of GBS to the infant during the perinatal period (8, 23). GBS isolates are classified according to nine capsular polysaccharide (CPS) types: types Ia, Ib, and II to VIII and the recently proposed type IX (9, 15, 21, 23, 46, 52). Isolates that do not express any of the known CPS types are designated nontypeable (NT) (2, 6, 21, 40). In addition to CPS, GBS may express one or more surface-localized proteins, including the α and β components of the c protein (24); the alpha-like R proteins, specifically R1, R4(Rib), and R1,R4 (also known as Alp3) (14, 17, 19, 30, 40); and the group B protective surface (BPS) protein (12). Certain protein profiles are associated with each capsular polysaccharide CPS type (2), for example, the c(α only) protein with types Ia and II, c(α + β) with type Ib, and R4(Rib) with type III (2, 14). BPS, expressed by fewer than 3% of colonizing isolates, can be found alone or with another protein in type Ia, II, and V isolates (12, 14).In the United States, the predominant serotypes over the past 2 decades, constituting 70 to 75% of all GBS isolates, have been type Ia, type III, and the more recently emerged type V (14, 15, 20, 52). The remaining isolates consisted primarily of types Ib and II, with types IV, VI, VII, and VIII making up a small fraction of the isolates. We found type IV to represent between 0.4 and 0.6% of colonizing GBS isolates (14, 15), but only rare type IV isolates were found in invasive GBS disease during that same time period (14, 43, 52).In contrast to the previously low percentage of type IV isolates reported for the United States, recent studies in the United Arab Emirates, Turkey, and Zimbabwe showed large proportions of type IV isolates among their GBS isolates. In the United Arab Emirates, type IV was the predominant serotype among colonized pregnant women, representing 26.3% of the GBS isolates (1). In eastern Turkey, it was the second most common serotype, at 8.3%, among colonizing isolates (10), and in Zimbabwe, it was the fourth most common serotype, comprising 5.1% of GBS isolates from colonized pregnant women and 4.0% of all GBS isolates from various sites, including blood and cerebrospinal fluid (CSF), from hospitalized patients (36).Immunization studies of humans (3, 28) and protection studies with mice (37) have shown the potential of vaccines against the common GBS serotypes to prevent invasive neonatal GBS disease through the vaccination of pregnant women (3, 28). The GBS strains described here are from a phase II randomized, double-blinded clinical trial of a GBS serotype III-tetanus toxoid (CPS III-TT) vaccine to prevent the vaginal acquisition of GBS type III in nonpregnant women in three areas of the United States: Pittsburgh (PA), Georgia, and Texas (S. Hillier, unpublished data). Because we found type IV isolates for almost 10% of these patients, we examined the type IV isolates for surface proteins and clonality.Pulsed-field gel electrophoresis (PFGE) was used in this analysis because it is a widely used method that can further characterize GBS isolates within particular CPS type and/or protein profile groups (2, 4, 6, 48). Multilocus sequence typing (MLST) was performed in order to assess the general relatedness of strains within and across laboratories (25, 50). Together, the discriminatory power of PFGE and the objectivity of MLST gave insight into the GBS type IV population genetic structure and the identification of emerging clones (2, 5, 13, 18, 19).     

Diagnosis of Amebic Liver Abscess and Amebic Colitis by Detection of Entamoeba histolytica DNA in Blood,Urine, and Saliva by a Real-Time PCR Assay

The noninvasive diagnosis of amebic liver abscess is challenging, as most patients at the time of diagnosis do not have a concurrent intestinal infection with Entamoeba histolytica. Fecal testing for E. histolytica parasite antigen or DNA is negative in most patients. A real-time PCR assay was evaluated for detection of E. histolytica DNA in blood, urine, and saliva samples from amebic liver abscess as well as amebic colitis patients in Bangladesh. A total of 98 amebic liver abscess and 28 amebic colitis patients and 43 control subjects were examined. The real-time PCR assay detected E. histolytica DNA in 49%, 77%, and 69% of blood, urine, and saliva specimens from the amebic liver abscess patients. For amebic colitis the sensitivity of the real-time PCR assay for detection of E. histolytica DNA in blood, urine, and saliva was 36%, 61%, and 64%, respectively. All blood, urine, and saliva samples from control subjects were negative by the real-time PCR assay for E. histolytica DNA. When the real-time PCR assay results of the urine and saliva specimens were taken together (positive either in urine or saliva), the real-time PCR assay was 97% and 89% sensitive for detection of E. histolytica DNA in liver abscess and intestinal infection, respectively. We conclude that the detection of E. histolytica DNA in saliva and urine could be used as a diagnostic tool for amebic liver abscess.Entamoeba histolytica is a protozoan parasite that causes amebic diarrhea, colitis, and amebic liver abscess (ALA), mostly in developing countries (5, 7, 22, 25). Eighty percent of infected individuals remain asymptomatic carriers, while the other 20% develop clinically overt disease (7, 9, 22, 25). About 50 million symptomatic cases of amebiasis occur worldwide each year, resulting in 40,000 to 100,000 deaths annually (25). Mortality from amebiasis is mainly due to extra-amebic colitis, of which ALA is the most common.It is difficult to differentiate ALA from pyogenic liver abscess or other space-occupying lesions of the liver. Imaging techniques such as ultrasound, computed tomography, and magnetic resonance have excellent sensitivities for the detection of liver abscess arising from any cause, but there are no findings specific for ALA (13). Further complicating the diagnosis is the fact that most patients with an ALA do not have coexistent intestinal infection with E. histolytica (11). Therefore, detection of E. histolytica antigen or DNA in stool samples is not very helpful for the diagnosis of ALA (1, 6, 8, 12).The current means for diagnosis of ALA is the detection of antiamebic antibody by serological tests combined with aspiration of the abscess. The presence of serum antibodies against E. histolytica and the absence of bacteria in the abscess fluid are consistent with an ALA. A drawback of serologic tests is that the serum antibody levels in people from areas of endemicity remain positive for years after infection with E. histolytica (3, 16, 23). Therefore, antiamebic antibodies in the serum may be due to amebiasis in the past, limiting their specificity for the diagnosis of ALA. A further limitation to the current approach to ALA diagnosis is that collection of liver abscess pus is an invasive procedure that requires technical expertise and can be done only in specialized hospitals.Several groups have reported the detection of E. histolytica DNA in liver abscess pus, stool, and other clinical samples by PCR (14, 15, 18, 19, 21, 24, 26). A real-time PCR assay has also been used for detection of E. histolytica DNA in stool and liver abscess pus specimens (2, 10, 20). Real-time PCR has never been used for detection of E. histolytica DNA in urine, saliva, and blood specimens of ALA patients. In this study, we evaluated a real-time PCR assay to detect E. histolytica DNA in blood, urine, and saliva samples of amebic liver abscess and colitis patients in Bangladesh.