External quality assessment schemes in bacteriology support public health in Germany-results from 2006 to 2023.

External Quality Assessment schemes (EQAS) are mandatory to ensure quality standards in diagnostic methods and achieve laboratory accreditation. As host institution for two German culture-based bacteriology EQAS (RV-A and RV-B), we investigated the obtained data of 590 up to 720 surveys per year in RV-A and 2,151 up to 2,929 in RV-B from 2006 to 2023. As educational instruments, they function to review applied methodology and are valuable to check for systemic- or method-dependent failures in microbiology diagnostics or guidelines. Especially, containment of multi-resistant bacteria in times of rising antibiotic resistance is one major point to assure public health. The correct identification and reporting of these strains is therefore of high importance to achieve this goal. Moreover, correct antimicrobial susceptibility testing (AST) per se is important for selecting appropriate therapy, to restrict broad-spectrum antibiotics and minimize resistance development. The reports of participating laboratories displayed a high level of correct identification results in both schemes with mostly consistent failure rates around 2.2% (RV-A) and 3.9% (RV-B) on average. In contrast, results in AST revealed increasing failure rates upon modification of AST requirements concerning adherence to standards and subsequent bacterial species-specific evaluation. Stratification on these periods revealed in RV-A a moderate increase from 1.3% to 4.5%, while in RV-B failure rates reached 14% coming from 4.3% on average. Although not mandatory, subsequent AST evaluation and consistent reporting are areas of improvement to benefit public health.

Single-base resolution quantitative genome methylation analysis in the model bacterium Helicobacter pylori by enzymatic methyl sequencing (EM-Seq) reveals influence of strain, growth phase, and methyl homeostasis.

BACKGROUND: Bacterial epigenetics is a rapidly expanding research field. DNA methylation by diverse bacterial methyltransferases (MTases) contributes to genomic integrity and replication, and many recent studies extended MTase function also to global transcript regulation and phenotypic variation. Helicobacter pylori is currently one of those bacterial species which possess the highest number and the most variably expressed set of DNA MTases. Next-generation sequencing technologies can directly detect DNA base methylation. However, they still have limitations in their quantitative and qualitative performance, in particular for cytosine methylation. RESULTS: As a complementing approach, we used enzymatic methyl sequencing (EM-Seq), a technology recently established that has not yet been fully evaluated for bacteria. Thereby, we assessed quantitatively, at single-base resolution, whole genome cytosine methylation for all methylated cytosine motifs in two different H. pylori strains and isogenic MTase mutants. EM-Seq reliably detected both m5C and m4C methylation. We demonstrated that three different active cytosine MTases in H. pylori provide considerably different levels of average genome-wide single-base methylation, in contrast to isogenic mutants which completely lost specific motif methylation. We found that strain identity and changed environmental conditions, such as growth phase and interference with methyl donor homeostasis, significantly influenced quantitative global and local genome-wide methylation in H. pylori at specific motifs. We also identified significantly hyper- or hypo-methylated cytosines, partially linked to overlapping MTase target motifs. Notably, we revealed differentially methylated cytosines in genome-wide coding regions under conditions of methionine depletion, which can be linked to transcript regulation. CONCLUSIONS: This study offers new knowledge on H. pylori global and local genome-wide methylation and establishes EM-Seq for quantitative single-site resolution analyses of bacterial cytosine methylation.

A multiserological line assay to potentially discriminate current from past Helicobacter pylori infection.

OBJECTIVES: Early diagnosis is important in controlling Helicobacter pylori-induced gastritis and progression to gastric malignancy. Serological testing is an efficient non-invasive diagnostic method, but currently does not allow differentiation between active and past infections. To fill this diagnostic gap we investigated the diagnostic value of a panel of ten H. pylori-specific antibodies in individuals with different H. pylori infection status within a German population. METHODS: We used the recomLine Helicobacter IgG 2.0 immunoblotting assay to analyse ten H. pylori-specific antibodies in serum samples collected from 1108 volunteers. From these, 788 samples were used to build exposure and infection status models and 320 samples for model validation. H. pylori infection status was verified by histological examination. We applied logistic regression to select antibodies correlated to infection status and developed, with independent validation, discriminating models and risk scores. Receiving operating characteristic analysis was performed to assess the accuracy of the discriminating models. RESULTS: Antibody reactivity against cytotoxin-associated gene A (CagA), H. pylori chaperone (GroEL), and hook-associated protein 2 homologue (FliD) was independently associated with the risk of H. pylori exposure with ORs and 95% CIs of 99.24 (46.50-211.80), 46.17 (17.45-122.17), and 22.16 (8.46-55.04), respectively. A risk score comprising these three selected antibodies differentiated currently H. pylori infected or eradicated participants from negatives with an area under the curve of 0.976 (95% CI: 0.965-0.987) (Model 1). Seropositivity for vacuolating cytotoxin A (VacA), GroEL, FliD, H. pylori adhesin A (HpaA), and γ-glutamyl transpeptidase (gGT) was associated with a current infection with an area under the curve of 0.870 (95% CI: 0.837-0.903), which may help discriminate currently infected patients from eradicated ones (Model 2). DISCUSSION: The recomLine assay is sensitive and specific in determining H. pylori infection and eradication status and thus represents a valuable tool in the management of H. pylori infection.

Methylome evolution suggests lineage-dependent selection in the gastric pathogen Helicobacter pylori.

The bacterial pathogen Helicobacter pylori, the leading cause of gastric cancer, is genetically highly diverse and harbours a large and variable portfolio of restriction-modification systems. Our understanding of the evolution and function of DNA methylation in bacteria is limited. Here, we performed a comprehensive analysis of the methylome diversity in H. pylori, using a dataset of 541 genomes that included all known phylogeographic populations. The frequency of 96 methyltransferases and the abundance of their cognate recognition sequences were strongly influenced by phylogeographic structure and were inter-correlated, positively or negatively, for 20% of type II methyltransferases. Low density motifs were more likely to be affected by natural selection, as reflected by higher genomic instability and compositional bias. Importantly, direct correlation implied that methylation patterns can be actively enriched by positive selection and suggests that specific sites have important functions in methylation-dependent phenotypes. Finally, we identified lineage-specific selective pressures modulating the contraction and expansion of the motif ACGT, revealing that the genetic load of methylation could be dependent on local ecological factors. Taken together, natural selection may shape both the abundance and distribution of methyltransferases and their specific recognition sequences, likely permitting a fine-tuning of genome-encoded functions not achievable by genetic variation alone.

Vaccination with Helicobacter pylori attachment proteins protects against gastric cancer.

Most cases of gastric cancer are caused by chronic Helicobacter pylori infection, but the lack of early onco-diagnostics and a high risk for antibiotic resistance hampers early intervention through eradication of H. pylori infection by antibiotics. We reported on a protective mechanism where H. pylori gastric mucosal attachment can be reduced by natural antibodies that block the binding of its attachment protein BabA. Here we show that challenge infection with H. pylori induced response of such blocking antibodies in both human volunteers and in rhesus macaques, that mucosal vaccination with BabA protein antigen induced blocking antibodies in rhesus macaques, and that vaccination in a mouse model induced blocking antibodies that reduced gastric mucosal inflammation, preserved the gastric juice acidity, and fully protected the mice from gastric cancer caused by H. pylori.

A retrospective evaluation of the Euroarray STI-11 multiplex system for the detection of eight STI causing agents.

With an incidence of more than > 1,000,000/day, sexually transmitted diseases remain a major challenge for health care systems worldwide. To reduce disease burden, complications, and spread, rapid diagnosis permitting early therapy is pivotal. The range of pathogens is wide and co-infections are common. This complicates pre-analytics, which are based on different laboratory techniques with potentially long turnaround times, e.g., cultivation and multistep serologies. Multiplex PCR provides the opportunity to overcome these limitations. In this study, we evaluated a novel assay, the Euroarray STI-11 microarray (EA; Euroimmun Medizinische Labordiagnostika), for the detection of eight obligate or facultative pathogens. Three-hundred-thirteen clinical specimens, which had been tested and pre-characterized for STI causing agents as part of routine diagnostics, were used as cases and controls in this retrospective study. The EA detected 34/44 Chlamydia trachomatis, 48/50 HSV-1, 50/50 HSV-2, 48/48 Mycoplasma hominis, 45/47 Neisseria gonorrhoeae, 9/11 Treponema pallidum, 46/46 Ureaplasma parvum, and 49/49 Ureaplasma urealyticum infections, respectively. 293 samples were EA positive, with polymicrobial infections (positive for two to six microbial or viral agents) detected in 130/293 cases. Specificities were 100% in the respective control groups (n = 18-48 depending on targeted pathogen) except for N. gonorrhoeae (25/26) and U. urealyticum (44/45). The broad spectrum of obligate and facultative pathogens targeted by the EA makes it a valuable tool in the setting of STI diagnostics and surveillance. The test has the potential to diagnose diseases neglected or overlooked in routine clinical practice. Besides a low sensitivity for C. trachomatis, the EA demonstrated high performance for all analyzed parameters. Further studies are warranted in order to capture a larger variety of the tested pathogens.

Helicobacter pylori infection.

Helicobacter pylori infection causes chronic gastritis, which can progress to severe gastroduodenal pathologies, including peptic ulcer, gastric cancer and gastric mucosa-associated lymphoid tissue lymphoma. H. pylori is usually transmitted in childhood and persists for life if untreated. The infection affects around half of the population in the world but prevalence varies according to location and sanitation standards. H. pylori has unique properties to colonize gastric epithelium in an acidic environment. The pathophysiology of H. pylori infection is dependent on complex bacterial virulence mechanisms and their interaction with the host immune system and environmental factors, resulting in distinct gastritis phenotypes that determine possible progression to different gastroduodenal pathologies. The causative role of H. pylori infection in gastric cancer development presents the opportunity for preventive screen-and-treat strategies. Invasive, endoscopy-based and non-invasive methods, including breath, stool and serological tests, are used in the diagnosis of H. pylori infection. Their use depends on the specific individual patient history and local availability. H. pylori treatment consists of a strong acid suppressant in various combinations with antibiotics and/or bismuth. The dramatic increase in resistance to key antibiotics used in H. pylori eradication demands antibiotic susceptibility testing, surveillance of resistance and antibiotic stewardship.

Genome and population dynamics during chronic infection with Helicobacter pylori.

Helicobacter pylori is responsible for one of the most prevalent bacterial infections worldwide. Chronic infection typically leads to chronic active gastritis. Clinical sequelae, including peptic ulcers, mucosa-associated lymphoid tissue lymphoma or, most importantly, gastric adenocarcinoma develop in 10-15% of cases. H. pylori is characterized by extensive inter-strain diversity which is the result of a high mutation rate, recombination, and a large repertoire of restriction-modification systems. This diversity is thought to be a major contributor to H. pylori's persistence and exceptional aptitude to adapt to the gastric environment and evade the immune system. This review covers efforts in the last decade to characterize and understand the multiple layers of H. pylori's diversity in different biological contexts.

Intraprocedural gastric juice analysis as compared to rapid urease test for real-time detection of Helicobacter pylori.

BACKGROUND: Endofaster is an innovative technology that can be combined with upper gastrointestinal endoscopy (UGE) to perform gastric juice analysis and real-time detection of Helicobacter pylori (H. pylori). AIM: To assess the diagnostic performance of this technology and its impact on the management of H. pylori in the real-life clinical setting. METHODS: Patients undergoing routine UGE were prospectively recruited. Biopsies were taken to assess gastric histology according to the updated Sydney system and for rapid urease test (RUT). Gastric juice sampling and analysis was performed using the Endofaster, and the diagnosis of H. pylori was based on real-time ammonium measurements. Histological detection of H. pylori served as the diagnostic gold standard for comparing Endofaster-based H. pylori diagnosis with RUT-based H. pylori detection. RESULTS: A total of 198 patients were prospectively enrolled in an H. pylori diagnostic study by Endofaster-based gastric juice analysis (EGJA) during the UGE. Biopsies for RUT and histological assessment were performed on 161 patients (82 men and 79 women, mean age 54.8 ± 19.2 years). H. pylori infection was detected by histology in 47 (29.2%) patients. Overall, the sensitivity, specificity, accuracy, positive predictive value, and negative predictive value (NPV) for H. pylori diagnosis by EGJA were 91.5%, 93.0%, 92.6%, 84.3%, and 96.4%, respectively. In patients on treatment with proton pump inhibitors, diagnostic sensitivity was reduced by 27.3%, while specificity and NPV were unaffected. EGJA and RUT were comparable in diagnostic performance and highly concordant in H. pylori detection (κ-value = 0.85). CONCLUSION: Endofaster allows for rapid and highly accurate detection of H. pylori during gastroscopy. This may guide taking additional biopsies for antibiotic susceptibility testing during the same procedure and then selecting an individually tailored eradication regimen.

Performance of galactomannan testing from endotracheal aspirate to guide bronchoalveolar lavage in the diagnosis of invasive aspergillosis.

PURPOSE: Invasive aspergillosis is a major threat to immunocompromised individuals. Galactomannan (GM) is used as a biomarker for invasive aspergillosis. Investigations recommended in current guidelines include GM testing of bronchoalveolar lavage (BAL) fluids. GM testing of endotracheal aspirate, the sampling of which is less invasive, less resource-intensive and less aerosol-generating, is not validated. We compared the performance of endotracheal aspirate GM as a screening tool to predict BAL fluid GM-positivity in patients with suspected invasive aspergillosis. METHODS: Of each patient, a pair of corresponding endotracheal aspirate and BAL fluid samples was tested and compared for GM results. Two sample sets were included. The first consisted of 140 consecutive BAL fluid/endotracheal aspirate pairs obtained from 133 patients. The pairs of the second sample set (n = 38) were selected based on the criterion that the BAL tested positive for GM. All specimens were obtained in a German 2,000 bed tertiary care center. RESULTS: Among BAL fluid GM-positive samples, endotracheal aspirate GM demonstrated poor specificity (72%) but high sensitivity (92% in predicting BAL fluid GM of ≥ 0.50 and 91% for BAL fluid GM of ≥ 1.00) and an excellent negative predictive value (98%). The use of a marginally elevated cutoff of 0.63 resulted in an improved specificity (72-81%), without loss of sensitivity. CONCLUSIONS: For screening purposes, one might consider testing endotracheal aspirate for GM, which could help avoid unnecessary BAL.

Repeated out-of-Africa expansions of Helicobacter pylori driven by replacement of deleterious mutations.

Helicobacter pylori lives in the human stomach and has a population structure resembling that of its host. However, H. pylori from Europe and the Middle East trace substantially more ancestry from modern African populations than the humans that carry them. Here, we use a collection of Afro-Eurasian H. pylori genomes to show that this African ancestry is due to at least three distinct admixture events. H. pylori from East Asia, which have undergone little admixture, have accumulated many more non-synonymous mutations than African strains. European and Middle Eastern bacteria have elevated African ancestry at the sites of these mutations, implying selection to remove them during admixture. Simulations show that population fitness can be restored after bottlenecks by migration and subsequent admixture of small numbers of bacteria from non-bottlenecked populations. We conclude that recent spread of African DNA has been driven by deleterious mutations accumulated during the original out-of-Africa bottleneck.

Performance of Multiplex PCR and ß-1,3-D-Glucan Testing for the Diagnosis of Candidemia.

Bloodstream infections caused by Candida yeasts (candidemia) are associated with high morbidity and mortality. Diagnosis remains challenging, with the current gold standard­isolation from blood culture (BC)­being limited by low sensitivity and long turnaround time. This study evaluated the performance of two nonculture methods: PCR and ß-1,3-D-glucan (BDG) testing. The sera of 103 patients with BC-proven candidemia and of 46 controls were analyzed with the Fungiplex Candida Real-Time PCR and the Wako ß-Glucan Test. The BDG assay demonstrated higher sensitivity than the multiplex PCR (58% vs. 33%). This was particularly evident in ICU patients (60% vs. 28%) and in C. albicans candidemia (57% vs. 37%). The earlier prior to BC sampling the sera were obtained, the more the PCR sensitivity decreased (46% to 18% in the periods of 0−2 and 3−5 days before BC, respectively), while BDG testing was independent of the sampling date. No positive PCR results were obtained in sera sampled more than five days before BC. Specificities were 89% for BDG and 93% for PCR testing. In conclusion, BDG testing demonstrated several advantages over PCR testing for the diagnosis of candidemia, including higher sensitivity and earlier diagnosis. However, BC remains essential, as BDG does not allow for species differentiation.

Management of Helicobacter pylori infection: the Maastricht VI/Florence consensus report.

Helicobacter pyloriInfection is formally recognised as an infectious disease, an entity that is now included in the International Classification of Diseases 11th Revision. This in principle leads to the recommendation that all infected patients should receive treatment. In the context of the wide clinical spectrum associated with Helicobacter pylori gastritis, specific issues persist and require regular updates for optimised management.The identification of distinct clinical scenarios, proper testing and adoption of effective strategies for prevention of gastric cancer and other complications are addressed. H. pylori treatment is challenged by the continuously rising antibiotic resistance and demands for susceptibility testing with consideration of novel molecular technologies and careful selection of first line and rescue therapies. The role of H. pylori and antibiotic therapies and their impact on the gut microbiota are also considered.Progress made in the management of H. pylori infection is covered in the present sixth edition of the Maastricht/Florence 2021 Consensus Report, key aspects related to the clinical role of H. pylori infection were re-evaluated and updated. Forty-one experts from 29 countries representing a global community, examined the new data related to H. pylori infection in five working groups: (1) indications/associations, (2) diagnosis, (3) treatment, (4) prevention/gastric cancer and (5) H. pylori and the gut microbiota. The results of the individual working groups were presented for a final consensus voting that included all participants. Recommendations are provided on the basis of the best available evidence and relevance to the management of H. pylori infection in various clinical fields.

The Helicobacter pylori UvrC Nuclease Is Essential for Chromosomal Microimports after Natural Transformation.

Helicobacter pylori is a Gram-negative bacterial carcinogenic pathogen that infects the stomachs of half of the human population. It is a natural mutator due to a deficient DNA mismatch repair pathway and is naturally competent for transformation. As a result, it is one of the most genetically diverse human bacterial pathogens. The length of chromosomal imports in H. pylori follows an unusual bimodal distribution consisting of macroimports with a mean length of 1,645 bp and microimports with a mean length of 28 bp. The mechanisms responsible for this import pattern were unknown. Here, we used a high-throughput whole-genome transformation assay to elucidate the role of nucleotide excision repair pathway (NER) components on import length distribution. The data show that the integration of microimports depended on the activity of the UvrC endonuclease, while none of the other components of the NER pathway was required. Using H. pylori site-directed mutants, we showed that the widely conserved UvrC nuclease active sites, while essential for protection from UV light, one of the canonical NER functions, are not required for generation of microimports. A quantitative analysis of recombination patterns based on over 1,000 imports from over 200 sequenced recombinant genomes showed that microimports occur frequently within clusters of multiple imports, strongly suggesting they derive from a single strand invasion event. We propose a hypothetical model of homologous recombination in H. pylori, involving a novel function of UvrC, that reconciles the available experimental data about recombination patterns in H. pylori. IMPORTANCE Helicobacter pylori is one of the most common and genetically diverse human bacterial pathogens. It is responsible for chronic gastritis and represents the main risk factor for gastric cancer. In H. pylori, DNA fragments can be imported by recombination during natural transformation. The length of those fragments determines how many potentially beneficial or deleterious alleles are acquired and thus influences adaptation to the gastric niche. Here, we used a transformation assay to examine imported fragments across the chromosome. We show that UvrC, an endonuclease involved in DNA repair, is responsible for the specific integration of short DNA fragments. This suggests that short and long fragments are imported through distinct recombination pathways. We also show that short fragments are frequently clustered with longer fragments, suggesting that both pathways may be mechanistically linked. These findings provide a novel basis to explain how H. pylori can fine-tune the genetic diversity acquired by transformation.

Serologic biomarkers in Candida and Aspergillus infections of the central nervous system: A comparison of galactomannan, mannan and ß-1,3-D-gucan testing from serum and cerebrospinal fluid.

BACKGROUND: The incidence of Aspergillus and Candida CNS infection, which are characterised by high mortality rates, is underestimated. This underdiagnosis presumably results from the limitations of available diagnostic tools and the need for invasive sampling. Little is known about the role of serologic biomarkers in the setting of CNS aspergillosis and candidiasis. PATIENTS, MATERIALS AND METHODS: Serum and cerebrospinal fluid (CSF; 10) samples of 19 patients, whose CNS specimens yielded growth of Aspergillus or Candida, were analysed for different biomarkers for fungal infection, that is galactomannan (GM), galactomannoprotein (GP), mannan, anti-mannan-antibodies and ß-1,3-D-glucan (BDG). Serum and CSF specimens of time-matched patients (two each for every case of fungal CNS infection) were included as controls. RESULTS: Galactomannan, GP and BDG seropositivity was found in one, two and three of five cases of CNS aspergillosis. BDG and mannan/anti-mannan-antibody sensitivity in proven CNS candidiasis was 40% and 20%, respectively. Applying the serum cut-off, sensitivity in CSF testing was 100% for GM and BDG and 50% for mannans. While serum specificity for all assays ranged from 89 to 97%, specificity for CSF BDG was only 70%. No false-positive GM results from CSF were obtained. CONCLUSION: Sensitivity for diagnosing CNS aspergillosis and CNS candidiasis from serum is mediocre for all serological biomarkers. GM testing in CSF proved excellent performance. With a sensitivity of 100% but a specificity of only 70%, CSF BDG might be most useful when used in patients with a high pre-test probability.

[Helicobacter pylori-New aspects of forthcoming guidelines]. / Helicobacter pylori ­ neue Aspekte kommender Leitlinien.

The diagnostics and treatment of Helicobacter pylori infections are subject to continuous changes and adaptations. Due to the increase of resistance rates to frequently used antibiotics, especially clarithromycin and the lack of new antibacterial substances as well as new developments in the diagnostics, particularly new procedures for resistance testing, the guidelines have to be updated regularly. In this article new directions and trends of the forthcoming European and German guidelines are summarized, categorized and discussed by the authors involved in the compilation of future guidelines.