Arie van der Ende

Quasispecies Development of Helicobacter pylori Observed in Paired Isolates Obtained Years Apart from the Same Host

Helicobacter pylori isolates show greater genetic diversity than other bacterial species studied, but the basis for this phenomenon is unknown. Whether detectable genomic mutation appears within an H. pylori population during persistent colonization was investigated. Paired H. pylori populations obtained across 7- to 10-year intervals from 13 patients were characterized by use of methods including polymerase chain reaction (PCR) genotyping for cagA, vacA, iceA, recA, and IS605; random arbitrarily primed DNA (RAPD)-PCR and amplified fragment length polymorphism (AFLP) analysis; and ELISA, to determine Lewis phenotypes. Genotyping, including recA sequence analysis, revealed that initial and follow-up populations represented the same population in 11 patients (85%). Nevertheless, distinct dissimilarities were shown within each of these 11 pairs by both RAPD-PCR and AFLP analyses. During follow-up, Lewis-y levels, but not Lewis-x levels, decreased significantly. The changes detected by RAPD-PCR and AFLP indicate that genetic drift occurs within H. pylori populations over the course of years of colonization of a single host.

Fit genotypes and escape variants of subgroup III Neisseria meningitidis during three pandemics of epidemic meningitis

The genetic variability at six polymorphic loci was examined within a global collection of 502 isolates of subgroup III, serogroup A Neisseria meningitidis. Nine “genoclouds” were identified, consisting of genotypes that were isolated repeatedly plus 48 descendent genotypes that were isolated rarely. These genoclouds have caused three pandemic waves of disease since the mid-1960s, the most recent of which was imported from East Asia to Europe and Africa in the mid-1990s. Many of the genotypes are escape variants, resulting from positive selection that we attribute to herd immunity. Despite positive selection, most escape variants are less fit than their parents and are lost because of competition and bottlenecks during spread from country to country. Competition between fit genotypes results in dramatic changes in population composition over short time periods.

Effects of Pneumococcal Conjugate Vaccine 2 Years after Its Introduction, the Netherlands

Vaccine-serotype disease decreased, but non–vaccine-serotype disease increased.

Invasive Pneumococcal Disease among Adults: Associations among Serotypes, Disease Characteristics, and Outcome

BACKGROUND The Streptococcus pneumoniae polysaccharide capsule may be related to invasive pneumococcal disease (IPD) course. METHODS We performed a retrospective cohort study with nationally representative surveillance data from 1075 hospitalized patients with IPD from the Netherlands from 1 June 2004 through 31 May 2006 in the prevaccination era. Serotypes were grouped according to invasive disease potential, rate of the most serious clinical syndromes of meningitis and bacteremia without focus, and case-fatality rates. Multivariable logistic regression analysis was performed to obtain odds ratios adjusted for baseline confounders for the association of serotypes and these outcomes, using the serotypes with the lowest rates as reference. RESULTS IPD caused by serogroups with low invasive disease potential concerned meningitis or bacteremia without focus in 22% of cases, and 74% of patients had an underlying comorbidity. For highly invasive serogroups these figures were 10% (P < .01) and 56% (P < .01). Individual serotypes varied in the relative rate by which they caused meningitis or bacteremia without focus. Compared with the reference group composed of serotypes 1, 5, 7F, 15B, 20, and 33F, the group of serotypes 3, 19F, 23A, 16F, 6B, 9N, and 18C was associated with increased case-fatality rates (group adjusted odds ratio, 2.6; 95% confidence interval, 1.5-4.7). CONCLUSIONS The serotype appeared to be independently associated with IPD severity in adults, which indicates that careful monitoring of IPD after implementation of conjugate vaccines is necessary.

The Stool Antigen Test for Detection of Helicobacter pylori after Eradication Therapy

Context Standard treatment regimens do not eradicate infection in approximately 10% to 20% of people with ulcers or gastritis caused by Helicobacter pylori. Symptoms do not reliably identify patients who have persistent infection despite treatment. Although positive results on a urea breath test done 4 weeks after treatment reliably identify persistent infection, a noninvasive test that detects successful eradication earlier would be useful. Contribution This multicenter study shows that a positive finding on a stool antigen test done as early as 1 week after treatment identifies about 95% (range, 70% to 100%) of cases of persistent infection. Generalization Cautions Findings are from patients with dyspepsia who were referred for endoscopy; 20% of patients were still infected at 1 month despite eradication therapy. The Editors Noninvasive tests for Helicobacter pylori are important in primary care, both for initial diagnosis of H. pylori infection and for confirmation of eradication. Current guidelines recommend noninvasive testing and treatment of young dyspeptic patients without alarm symptoms (such as dysphagia or weight loss that suggest underlying malignant disease) in a primary care setting by using low-cost noninvasive tests (1, 2). Randomized, controlled trials have shown that a test and eradicate strategy toward H. pylori is effective in patients with dyspepsia seen in primary care settings who have not undergone investigations such as endoscopy or radiographic studies (3). Post-therapy testing is also growing in importance. Resistant strains of H. pylori are now widely prevalent in the United States and Europe, and eradication therapy with current regimens fails in 10% to 20% of patients (4, 5). Furthermore, some patients with ulcer disease remain symptomatic despite successful eradication of H. pylori and healing of the ulcer (6). In patients with persistent symptoms, testing for persistent H. pylori infection is important to direct further therapy. Routine testing to confirm eradication in patients with complicated ulcer disease, such as bleeding peptic ulcer, is necessary because the risk for rebleeding is greatly increased in patients with persistent infection (7). The choice of tests in the post-therapy setting is limited. Serologic tests are unreliable in determining eradication (8). Endoscopic tests (rapid urease test, histologic examination, or culture) are reliable, but endoscopy is expensive and inconvenient. Until recently, the only noninvasive test that reliably demonstrated whether eradication was successful was the urea breath test (9). This test has high sensitivity and specificity in the post-therapy setting but cannot be used until 4 weeks after treatment. Moreover, the breath test is still not widely available in the United States. The fecal antigen test is a relatively new noninvasive test for detection of H. pylori (10). This test detects the presence of infection by measuring the fecal excretion of H. pylori antigens. It has been approved by the U.S. Food and Drug Administration for detection of H. pylori before and after therapy. We sought to determine whether a stool antigen test administered at various times after treatment correctly identifies persons in whom H. pylori infection persists despite eradication therapy. Methods We prospectively studied 84 patients infected with H. pylori at six clinical centers (31 in Bologna, Italy; 29 in Amsterdam, the Netherlands; 9 in Rome, Italy; 8 in Lisbon, Portugal; 4 in Madrid, Spain; and 3 in Milwaukee, Wisconsin). The sample consisted of consecutive patients with dyspepsia (defined as pain or discomfort centered in the upper abdomen) who were referred by primary care physicians for upper endoscopy (11). Consenting patients were enrolled if they tested positive for H. pylori on endoscopic tests. Patients enrolled in this study have not been enrolled in other studies. Patients were excluded if they had taken proton-pump inhibitors, H2-receptor antagonists, nonsteroidal anti-inflammatory agents, or antibiotics in the 4 weeks before the study. Failure to return for follow-up endoscopy was an a priori exclusion criterion. All patients gave written informed consent, and the study was approved by the human subjects review committee or equivalent at each participating institution. At baseline, patients underwent endoscopy with biopsy sampling for histologic examination (two samples from the antrum and two from the corpus), culture (two samples from the antrum and two from the corpus), and a rapid urease test (one sample from the antrum). All patients were infected with H. pylori at baseline, as demonstrated by positive results on both rapid urease testing and histologic examination or a positive culture for H. pylori. Within 24 hours of the endoscopy, all patients underwent a 13C or 14C urea breath test. The breath test was chosen according to local availability and experience, but in all cases a validated breath test analysis system was used. Cut-off values were determined according to the recommendations of the various manufacturers of these tests. Patients collected stool using a kit consisting of a plastic spoon that is used to scoop a small amount of stool from the toilet paper or toilet bowl into an airtight container. At all sites, the stool assay was performed by using the Premier Platinum HpSA test (Meridian Diagnostics, Inc., Cincinnati, Ohio). The assay is a microwell-based enzyme immunoassay that uses polyclonal antiH. pylori capture antibody adsorbed to microwells. Diluted patient samples and a peroxidase-conjugated polyclonal antibody were added to the wells and incubated at room temperature for 1 hour. A wash was performed to remove unbound material. Substrate was added and incubated for 10 minutes at room temperature. Color develops in the presence of bound enzyme. Stop solution was added, and the results were inspected spectrophotometrically at 450 nm within 15 minutes of adding the stop solution. Visual determination can also be used; this has been shown to have similar results (12). A positive control and a negative control are built into the test. The cut-off values were classified as negative (<0.140), indeterminate (0.140 to 0.159), or positive (>0.160). After completion of the baseline procedures, treatment was begun with ranitidine bismuth citrate (400 mg twice daily) or omeprazole (20 mg twice daily) in combination with amoxicillin (1 g twice daily) and clarithromycin (500 mg twice daily) for 7 to 10 days. Seven-day eradication therapy was used in Europe, where it is approved by the European Union and has been shown to be effective (5). Ten-day triple therapy with proton-pump inhibitors was used in the United States, where it is approved by the U.S. Food and Drug Administration. Patients collected stool for the stool antigen test on days 3, 7, 15, 21, 28, and 35 after completion of H. pylori eradication therapy. On day 35 after completion of eradication therapy, endoscopy was repeated and biopsy samples were again obtained for histologic examination, culture, and the rapid urease test, as performed at the baseline visit. The 13C or 14C urea breath test was repeated on day 35 by using the same method and cut-off values as at baseline. Patients were classified as being infected with H. pylori at baseline and having persistent infection on day 35 if culture of gastric biopsy specimens was positive for H. pylori or results of the rapid urease test and histologic examination were positive. All other patients were classified as negative. These criteria have been recommended by an expert panel for use in clinical trials of H. pylori eradication (13). At baseline, the sensitivity of the stool test and urea breath test were calculated by using the presence of infection (defined above) as the gold standard. At each time point after completion of therapy (days 3, 7, 15, 21, 28), predictive values were calculated by using continued infection on day 35 as the gold standard (positive result on culture or on rapid urease test and histologic examination). Trained investigators who were blinded to the results of the other diagnostic studies performed the stool assays. The first endoscopy procedure was performed before the stool and breath tests. Therapy was given on the basis of results on endoscopic testing. Endoscopists were blinded to the results of post-treatment stool studies and the breath test until all evaluations were completed. Long-Term Follow-up Patients in whom eradication of H. pylori was successful were eligible for entry into a long-term study evaluating the stool antigen test. For 6 months, stool antigen tests were done monthly and a urea breath test was obtained every 3 months. Statistical Analysis Statistical analysis was performed by using StatView for Windows, version 5.01 (SAS Institute, Inc., Cary, North Carolina). Results are presented as the mean (SD). Sensitivity, specificity, probabilities, and predictive values are presented with 95% exact binomial CIs. Equivocal stool tests are considered by inclusion in the denominator of sensitivity and specificity. Stool antigen concentrations at individual time points were compared by using theMannWhitney test with downward adjustment of the P values for repeated observations (14). Role of the Funding Source The manufacturer (Meridian Diagnostics, Inc.) provided the stool kits. The study had no other funding source. Collection, analysis, and interpretation of the data, including the decision to publish, were solely the decision of the authors; the manufacturer of the test had no role in this process. Results The mean age of the 84 study patients was 52 years (range, 18 to 81 years). Fifty-three patients were women, and 31 were men. Endoscopic findings were as follows: normal (7 patients), esophagitis (2 patients), erythema in the antrum (45 patients), erosions in the antrum (11 patients), erosive duodenitis (9 patients), duodenal ulcers (8 patients), gastric ulcer (2 p

Multi locus sequence typing of Chlamydiales: clonal groupings within the obligate intracellular bacteria Chlamydia trachomatis

BackgroundThe obligate intracellular growing bacterium Chlamydia trachomatis causes diseases like trachoma, urogenital infection and lymphogranuloma venereum with severe morbidity. Several serovars and genotypes have been identified, but these could not be linked to clinical disease or outcome. The related Chlamydophila pneumoniae, of which no subtypes are recognized, causes respiratory infections worldwide. We developed a multi locus sequence typing (MLST) scheme to understand the population genetic structure and diversity of these species and to evaluate the association between genotype and disease.ResultsA collection of 26 strains of C. trachomatis of different serovars and clinical presentation and 18 strains of C. pneumoniae were included in the study. For comparison, sequences of C. abortus, C. psittaci, C. caviae, C. felis, C. pecorum (Chlamydophila), C. muridarum (Chlamydia) and of Candidatus protochlamydia and Simkania negevensis were also included. Sequences of fragments (400 – 500 base pairs) from seven housekeeping genes (enoA, fumC, gatA, gidA, hemN, hlfX, oppA) were analysed. Analysis of allelic profiles by eBurst revealed three non-overlapping clonal complexes among the C. trachomatis strains, while the C. pneumoniae strains formed a single group. An UPGMA tree produced from the allelic profiles resulted in three groups of sequence types. The LGV strains grouped in a single cluster, while the urogenital strains were distributed over two separated groups, one consisted solely of strains with frequent occurring serovars (E, D and F). The distribution of the different serovars over the three groups was not consistent, suggesting exchange of serovar encoding ompA sequences. In one instance, exchange of fumC sequences between strains of different groups was observed. Cluster analyses of concatenated sequences of the Chlamydophila and Chlamydia species together with those of Candidatus Protochlamydia amoebophila and Simkania negevensis resulted in a tree identical to that obtained with 23S RNA gene sequences.ConclusionThese data show that C. trachomatis and C. pneumoniae are highly uniform. The difference in genetic diversity between C. trachomatis and C. pneumoniae is in concordance with a later assimilation to the human host of the latter. Our data supports the taxonomy of the order of Chlamydiales.

Effects of the 10-Valent Pneumococcal Nontypeable Haemophilus influenzae Protein D-Conjugate Vaccine on Nasopharyngeal Bacterial Colonization in Young Children: A Randomized Controlled Trial

This study evaluated effects of the 10-valent pneumococcal nontypeable Haemophilus influenzae protein D-conjugate vaccine (PHiDCV) compared with the 7-valent vaccine on nasopharyngeal bacterial colonization, specifically nontypeable Haemophilus influenzae (NTHi). PHiD-CV had no differential effect on nasopharyngeal NTHi colonization.

Community-acquired bacterial meningitis in adults in the Netherlands, 2006-14: a prospective cohort study.

BACKGROUND We studied causative pathogens, clinical characteristics, and outcome of adult community-acquired bacterial meningitis after the introduction of adjunctive dexamethasone treatment and nationwide implementation of paediatric conjugate vaccines. METHODS In this cohort study, we prospectively assessed adults (age >16 years) with community-acquired bacterial meningitis in the Netherlands, identified through the National Reference Laboratory for Bacterial Meningitis or individual physicians between Jan 1, 2006, and July 1, 2014. We identified independent predictors of an unfavourable outcome (Glasgow Outcome Scale score 1-4) by logistic regression. FINDINGS We assessed 1412 episodes of community-acquired bacterial meningitis. Incidence declined from 1·72 cases per 100,000 adults per year in 2007-08, to 0·94 per 100,000 per year in 2013-14. Streptococcus pneumoniae caused 1017 (72%) of 1412 episodes. Rates of adult bacterial meningitis decreased most sharply among pneumococcal serotypes included in paediatric conjugate vaccine, and in meningococcal meningitis. We found no evidence of serotype or serogroup replacement. The overall case fatality rate was 244 (17%) of 1412 episodes and unfavourable outcome occurred in 531 (38%) of 1412 episodes. Predictors of unfavourable outcome were advanced age, absence of otitis or sinusitis, alcoholism, tachycardia, lower score on the Glasgow Coma Scale, cranial nerve palsy, a cerebrospinal fluid white-cell count lower than 1000 cells per μL, a positive blood culture, and a high serum C-reactive protein concentration. Adjunctive dexamethasone was administered for 1234 (89%) of 1384 assessed episodes. The multivariable adjusted odds ratio of dexamethasone treatment for unfavourable outcome was 0·54 (95% CI 0·39-0·73). INTERPRETATION The incidence of adult bacterial meningitis has decreased substantially, which is partly explained by herd protection by paediatric conjugate vaccines. Adjunctive dexamethasone treatment was associated with substantially improved outcome. FUNDING European Research Council, National Institute of Public Health and the Environment, European Union, Academic Medical Center, and Netherlands Organization for Health Research and Development.

Clinical features, outcome, and meningococcal genotype in 258 adults with meningococcal meningitis: a prospective cohort study.

Abstract Meningococcal meningitis remains a life-threatening disease. Neisseria meningitidis is the leading cause of meningitis and septicemia in young adults and is a major cause of endemic bacterial meningitis worldwide. The Meningitis Cohort Study was a Dutch nationwide prospective observational cohort study of adults with community-acquired bacterial meningitis, confirmed by culture of cerebrospinal fluid, from October 1998 to April 2002. Patients underwent a neurologic examination at discharge, and outcome was graded with the Glasgow Outcome Scale. Serogrouping, multi-locus sequence typing, and susceptibility testing of meningococcal isolates were performed. The study identified 258 episodes of meningococcal meningitis in 258 patients. The prevalence of the classical triad of fever, neck stiffness, and change in mental status was low (70/258, 27%). When rash was added to the classical triad, 229 of 258 (89%) patients had at least 2 of 4 signs. Systolic hypotension was associated with rash (22/23 vs. 137/222, p = 0.002) and absence of neck stiffness (6/23 vs. 21/220, p = 0.05). Neuroimaging before lumbar puncture was an important cause of delay of therapy: antibiotics were not initiated before computed tomography (CT) scan in 85% of patients who underwent CT scan before lumbar puncture. Unfavorable outcome occurred in 30 of 258 (12%) patients, including a mortality rate of 7%. Neurologic sequelae occurred in 28 of 238 (12%) patients, particularly hearing loss (8%). Factors associated with sepsis and infection with meningococci of clonal complex 11 (cc11) are related with unfavorable outcome. Abbreviations: cc = clonal complex, CRP = C-reactive protein, CSF = cerebrospinal fluid, CT = computed tomography, ESR = erythrocyte sedimentation rate, GCS = Glasgow Coma Scale, IQR = interquartile range, MLST = multi-locus sequence typing, WBCC = white blood cell count.

Carriage of Streptococcus pneumoniae 3 years after start of vaccination program, the Netherlands.

To evaluate the effectiveness of the 7-valent pneumococcal conjugate vaccine (PCV7) program, we conducted a cross-sectional observational study on nasopharyngeal carriage of Streptococcus pneumoniae 3 years after implementation of the program in the Netherlands. We compared pneumococcal serotypes in 329 prebooster 11-month-old children, 330 fully vaccinated 24-month-old children, and 324 parents with age-matched pre-PCV7 (unvaccinated) controls (ages 12 and 24 months, n = 319 and n = 321, respectively) and 296 of their parents. PCV7 serotype prevalences before and after PCV7 implementation, respectively, were 38% and 8% among 11-month-old children, 36% and 4% among 24-month-old children, and 8% and 1% among parents. Non-PCV7 serotype prevalences were 29% and 39% among 11-month-old children, 30% and 45% among 24-month-old children, and 8% and 15% among parents, respectively; serotypes 11A and 19A were most frequently isolated. PCV7 serotypes were largely replaced by non-PCV7 serotypes. Disappearance of PCV7 serotypes in parents suggests strong transmission reduction through vaccination.