Highly contiguous genomes of human clinical isolates of Giardia duodenalis reveal assemblage- and sub-assemblage-specific presence-absence variation in protein-coding genes.

Giardia duodenalis (syn. G. intestinalis, G. lamblia) is a widespread gastrointestinal protozoan parasite with debated taxonomic status. Currently, eight distinct genetic sub-groups, termed assemblages A-H, are defined based on a few genetic markers. Assemblages A and B may represent distinct species and are both of human public health relevance. Genomic studies are scarce and the few reference genomes available, in particular for assemblage B, are insufficient for adequate comparative genomics. Here, by combining long- and short-read sequences generated by PacBio and Illumina sequencing technologies, we provide nine annotated genome sequences for reference from new clinical isolates (four assemblage A and five assemblage B parasite isolates). Isolates chosen represent the currently accepted classification of sub-assemblages AI, AII, BIII and BIV. Synteny over the whole genome was generally high, but we report chromosome-level translocations as a feature that distinguishes assemblage A from B parasites. Orthologue gene group analysis was used to define gene content differences between assemblage A and B and to contribute a gene-set-based operational definition of respective taxonomic units. Giardia is tetraploid, and high allelic sequence heterogeneity (ASH) for assemblage B vs. assemblage A has been observed so far. Noteworthy, here we report an extremely low ASH (0.002%) for one of the assemblage B isolates (a value even lower than the reference assemblage A isolate WB-C6). This challenges the view of low ASH being a notable feature that distinguishes assemblage A from B parasites, and low ASH allowed assembly of the most contiguous assemblage B genome currently available for reference. In conclusion, the description of nine highly contiguous genome assemblies of new isolates of G. duodenalis assemblage A and B adds to our understanding of the genomics and species population structure of this widespread zoonotic parasite.

Extensive testing of a multi-locus sequence typing scheme for Giardia duodenalis assemblage A confirms its good discriminatory power.

BACKGROUND: The flagellated parasite Giardia duodenalis is a major and global cause of diarrhoeal disease. Eight genetically very distinct groups, known as assemblages A to H, have been recognized in the G. duodenalis species complex, two of which (assemblages A and B) infect humans and other mammalian hosts. Informative typing schemes are essential to understand transmission pathways, characterize outbreaks and trace zoonotic transmission. In this study, we evaluated a published multi-locus sequence typing (MLST) scheme for G. duodenalis assemblage A, which is based on six polymorphic markers. METHODS: We genotyped 60 human-derived and 11 animal-derived G. duodenalis isolates collected in Europe and on other continents based on the published protocol. After retrieving previously published genotyping data and excluding isolates whose sequences showed allelic sequence heterozygosity, we analysed a dataset comprising 146 isolates. RESULTS: We identified novel variants at five of the six markers and identified 78 distinct MLST types in the overall dataset. Phylogenetic interpretation of typing data confirmed that sub-assemblage AII only comprises human-derived isolates, whereas sub-assemblage AI comprises all animal-derived isolates and a few human-derived isolates, suggesting limited zoonotic transmission. Within sub-assemblage AII, isolates from two outbreaks, which occurred in Sweden and Italy, respectively, had unique and distinct MLST types. Population genetic analysis showed a lack of clustering by geographical origin of the isolates. CONCLUSION: The MLST scheme evaluated provides sufficient discriminatory power for epidemiological studies of G. duodenalis assemblage A.

Establishment of a specimen panel for the decentralised technical evaluation of the sensitivity of 31 rapid diagnostic tests for SARS-CoV-2 antigen, Germany, September 2020 to April 2021.

IntroductionThe detection of SARS-CoV-2 with rapid diagnostic tests (RDT) has become an important tool to identify infected people and break infection chains. These RDT are usually based on antigen detection in a lateral flow approach.AimWe aimed to establish a comprehensive specimen panel for the decentralised technical evaluation of SARS-CoV-2 antigen rapid diagnostic tests.MethodsWhile for PCR diagnostics the validation of a PCR assay is well established, there is no common validation strategy for antigen tests, including RDT. In this proof-of-principle study we present the establishment of a panel of 50 pooled clinical specimens that cover a SARS-CoV-2 concentration range from 1.1 × 109 to 420 genome copies per mL of specimen. The panel was used to evaluate 31 RDT in up to six laboratories.ResultsOur results show that there is considerable variation in the detection limits and the clinical sensitivity of different RDT. We show that the best RDT can be applied to reliably identify infectious individuals who present with SARS-CoV-2 loads down to 106 genome copies per mL of specimen. For the identification of infected individuals with SARS-CoV-2 loads corresponding to less than 106 genome copies per mL, only three RDT showed a clinical sensitivity of more than 60%.ConclusionsSensitive RDT can be applied to identify infectious individuals with high viral loads but not to identify all infected individuals.

Implementation of Automated Blood Culture With Quality Assurance in a Resource-Limited Setting.

Background: Blood cultures (BC) have a high clinical relevance and are a priority specimen for surveillance of antimicrobial resistance. Manual BC are still most frequently used in resource-limited settings. Data on automated BC performance in Africa are scarce. We implemented automated BC at a surveillance site of the African Network for improved Diagnostics, Epidemiology and Management of Common Infectious Agents (ANDEMIA). Methods: Between June 2017 and January 2018, pairs of automated BC (BacT/ALERT®FA Plus) and manual BC (brain-heart infusion broth) were compared at a University hospital in Bouaké, Côte d'Ivoire. BC were inoculated each with a target blood volume of 10 ml from the same venipuncture. Automated BC were incubated for up to 5 days, manual BC for up to 10 days. Terminal subcultures were performed for manual BC only. The two systems were compared regarding yield, contamination, and turnaround time. For quality assurance, isolates were retested in a German routine microbiological laboratory. Results: BC sampling was increased from on average 24 BC to 63 BC per month. A total of 337 matched pairs of BC were included. Automated BC was positive in 36.5%, manual BC in 24.0% (p-value < 0.01), proportion of contamination was 47.9 and 43.8%, respectively (p-value = 1.0). Turnaround time of positive BC was shortened by 2.5 days with automated compared to manual BC (p < 0.01). Most common detected pathogens in both systems were Klebsiella spp. (26.0%) and Staphylococcus aureus (18.2%). Most contaminants were members of the skin flora. Retesting of 162 isolates was concordant in 79.6% on family level. Conclusions: Implementing automated BC in a resource-limited setting is possible and improves microbiological diagnostic performance. Automated BC increased yield and shortened turnaround times. Regular training and mentorship of clinicians has to be intensified to increase number and quality of BC. Pre-analytical training to improve diagnostic stewardship is essential when implementing a new microbiological method. Retesting highlighted that manual identification and antimicrobial susceptibility testing can be of good quality and sustainable. The implementation of automated tools should be decided individually according to economic considerations, number of samples, stable supply chain of consumables, and technical sustainability.

Suitability of current typing procedures to identify epidemiologically linked human Giardia duodenalis isolates.

BACKGROUND: Giardia duodenalis is a leading cause of gastroenteritis worldwide. Humans are mainly infected by two different subtypes, i.e., assemblage A and B. Genotyping is hampered by allelic sequence heterozygosity (ASH) mainly in assemblage B, and by occurrence of mixed infections. Here we assessed the suitability of current genotyping protocols of G. duodenalis for epidemiological applications such as molecular tracing of transmission chains. METHODOLOGY/PRINCIPAL FINDINGS: Two G. duodenalis isolate collections, from an outpatient tropical medicine clinic and from several primary care laboratories, were characterized by assemblage-specific qPCR (TIF, CATH gene loci) and a common multi locus sequence typing (MLST; TPI, BG, GDH gene loci). Assemblage A isolates were further typed at additional loci (HCMP22547, CID1, RHP26, HCMP6372, DIS3, NEK15411). Of 175/202 (86.6%) patients the G. duodenalis assemblage could be identified: Assemblages A 25/175 (14.3%), B 115/175 (65.7%) and A+B mixed 35/175 (20.0%). By incorporating allelic sequence heterozygosity in the analysis, the three marker MLST correctly identified 6/9 (66,7%) and 4/5 (80.0%) consecutive samples from chronic assemblage B infections in the two collections, respectively, and identified a cluster of five independent patients carrying assemblage B parasites of identical MLST type. Extended MLST for assemblage A altogether identified 5/6 (83,3%) consecutive samples from chronic assemblage A infections and 15 novel genotypes. Based on the observed A+B mixed infections it is estimated that only 75% and 50% of assemblage A or B only cases represent single strain infections, respectively. We demonstrate that typing results are consistent with this prediction. CONCLUSIONS/SIGNIFICANCE: Typing of assemblage A and B isolates with resolution for epidemiological applications is possible but requires separate genotyping protocols. The high frequency of multiple infections and their impact on typing results are findings with immediate consequences for result interpretation in this field.

Prevalence and antimicrobial susceptibility of Arcobacter species in human stool samples derived from out- and inpatients: the prospective German Arcobacter prevalence study Arcopath.

BACKGROUND: Arcobacter species, particularly A. butzleri, but also A. cryaerophilus constitute emerging pathogens causing gastroenteritis in humans. However, isolation of Arcobacter may often fail during routine diagnostic procedures due to the lack of standard protocols. Furthermore, defined breakpoints for the interpretation of antimicrobial susceptibilities of Arcobacter are missing. Hence, reliable epidemiological data of human Arcobacter infections are scarce and lacking for Germany. We therefore performed a 13-month prospective Arcobacter prevalence study in German patients. RESULTS: A total of 4636 human stool samples was included and Arcobacter spp. were identified from 0.85% of specimens in 3884 outpatients and from 0.40% of specimens in 752 hospitalized patients. Overall, A. butzleri was the most prevalent species (n = 24; 67%), followed by A. cryaerophilus (n = 10; 28%) and A. lanthieri (n = 2; 6%). Whereas A. butzleri, A. cryaerophilus and A. lanthieri were identified in outpatients, only A. butzleri could be isolated from samples of hospitalized patients. Antimicrobial susceptibility testing of Arcobacter isolates revealed high susceptibilities to ciprofloxacin, whereas bimodal distributions of MICs were observed for azithromycin and ampicillin. CONCLUSIONS: In summary, Arcobacter including A. butzleri, A. cryaerophilus and A. lanthieri could be isolated in 0.85% of German outpatients and ciprofloxacin rather than other antibiotics might be appropriate for antibiotic treatment of infections. Further epidemiological studies are needed, however, to provide a sufficient risk assessment of Arcobacter infections in humans.

Characterization of Arcobacter strains isolated from human stool samples: results from the prospective German prevalence study Arcopath.

BACKGROUND: Arcobacter constitute emerging food- and waterborne pathogens causing gastroenteritis in humans, but the underlying mechanisms are only incompletely understood. We therefore characterized Arcobacter isolates derived from human stool samples that had been collected during a prospective prevalence study in Germany in vitro. Thirty-six bacterial isolates belonging to the species A. butzleri (n = 24), A. cryaerophilus (n = 10) and A. lanthieri (n = 2) were genotyped by ERIC-PCR, the presence of 10 putative virulence genes was assessed and cytotoxic effects on the human intestinal cell line HT-29/B6 were analyzed applying the WST-assay. RESULTS: Genotyping revealed high genetic diversity within the species A. butzleri, A. cryaerophilus and A. lanthieri. Both, A. butzleri and A. lanthieri encoded for a large number of putative virulence genes, while fewer genes were detectable in A. cryaerophilus isolates. Notably, the three cytolethal distending toxin (CDT) genes cdtA, cdtB and cdtC were abundant in both A. lanthieri isolates. Furthermore, all A. butzleri and A. lanthieri, but only one of the A. cryaerophilus isolates exerted cytotoxic effects. CONCLUSIONS: Our study provides evidence for the abundance of putative virulence genes in Arcobacter isolates and prominent cytotoxic effects of A. butzleri and A. lanthieri in vitro. The presence of cdtA, cdtB, cdtC in A. lanthieri points towards CDT secretion as potential mechanism underlying cytotoxicity as opposed to A. butzleri. However, the association of the Arcobacter virulence factors detected and human morbidity should be addressed in future studies.

Clostridioides difficile in Outpatients: Application of a Diagnostic Algorithm Recommended by the European Society of Clinical Microbiology and Infectious Diseases.

This study aimed at evaluating in outpatients an algorithm for the laboratory diagnosis of Clostridioides (Clostridium) difficile infection (CDI), i.e., enzyme immunoassays (EIAs) detecting bacterial glutamate dehydrogenase (GDH) and toxin A/B, followed by polymerase chain reaction (PCR) analyses of samples with discordant EIA results. In total, 9802 examinations of stool samples by GDH and toxin EIAs performed in 7263 outpatients and 488 inpatients were analyzed retrospectively. Samples with discordant EIA results had been tested by a commercially available PCR assay detecting genes of the C. difficile-specific triose phosphate isomerase (tpi) and toxin B (tcdB). Concordant EIA results (686 C. difficile-positive, 8121 negative) were observed for 8807 (89.8%; 95% CI, 89.2-90.4%) samples. Of 958 samples with discordant EIA results, 895 were analyzed using PCR and 580 of 854 GDH-positive/borderline, toxin-negative samples (67.9%; 95% CI, 64.7-71.0%) were positive for tpi and tcdB, while 274 samples (32.1%; 95% CI, 29.0-35.3%) were tcdB-negative. In contrast, 35 of 41 GDH-negative, toxin-positive/borderline samples (85.4%; 95% CI, 71.2-93.5%) were tcdB-negative. Still, 6 samples (14.6%; 95% CI, 6.5-28.8%) yielded positive PCR results for both genes. In conclusion, around 90% of the samples were analyzed appropriately by only applying EIAs. Approximately one third of the PCR-analyzed samples were tcdB-negative; thus, patients most likely did not require CDI treatment.

Accurate Detection of Helicobacter pylori Antigen in Human Stool Specimens by Two Novel Immunoassays.

Stool antigen tests are recommended for the diagnosis of Helicobacter pylori infection. Here, we compared two novel assays, i.e., one enzyme immunoassay (EIA) and one immunochromatography assay (ICA), with a chemiluminescence immunoassay (CLIA) that had previously been compared with rapid urease test, histology, and urea breath test. Two hundred sixty-six frozen stool samples with defined CLIA results (42 positives, 219 negatives, and 5 samples with borderline results) collected between January and May 2018 were thawed and immediately tested by EIA, ICA, and CLIA. In 248 samples with repeatedly positive/negative CLIA results, EIA and ICA were positive for 40 and 37 of 41 CLIA-positive samples and yielded negative results for 206 and 201 of 207 CLIA-negative samples, respectively. There was a high positive percent agreement (EIA, 97.6%; 95% confidence interval (95% CI), 86.3-100%; ICA, 90.2%; 95% CI, 76.9-96.7%), as well as a negative percent agreement between the assays (EIA, 99.5%; 95% CI, 97.0-100%; ICA, 97.1%; 95% CI, 93.7-98.8%). This was further supported by kappa values indicating very good agreement (CLIA vs. EIA, 0.971; CLIA vs. ICA, 0.857). In conclusion, both EIA and ICA comprise valuable assays for the detection of H. pylori antigen in stool samples.

No evidence of carbapenemase-producing Enterobacteriaceae in stool samples of 1,544 asylum seekers arriving in Rhineland-Palatinate, Germany, April 2016 to March, 2017.

INTRODUCTION: Since 2015, increased migration from Asia and Africa to Europe has raised public health concerns about potential importation of extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBL-PE), specifically those producing carbapenemases (C-PE), into European hospitals. AIMS: To inform infection control practices about ESBL-PE prevalence in asylum seekers and to investigate whether C-PE prevalence exceeds that in the German population. METHODS: Cross-sectional study from April 2016-March 2017. Routinely collected stool samples from asylum seekers were tested for antibiotic resistant Enterobacteriaceae. Country/region of origin and demographic characteristics were explored as risk factors for faecal colonisation. RESULTS: Of 1,544 individuals, 294 tested positive for ESBL-PE colonisation (19.0%; 95% confidence intervals (CI): 17.0-21.0). Asylum seekers originating from Afghanistan/Pakistan/Iran had a prevalence of 29.3% (95% CI: 25.6-33.2), from Syria 20.4% (95% CI: 16.1-25.2) and from Eritrea/Somalia 11.9% (95% CI: 8.7-15.7). CTX-M-15 (79%) and CTX-M-27 (10%) were the most common ESBL determinants. Highest ESBL-PE prevalences were observed in boys under 10 years and women aged 20-39 years (interaction: p = 0.03). No individuals tested positive for C-PE. Faecal C-PE colonisation prevalence in asylum seekers was not statistically significantly different from prevalence reported in German communities. CONCLUSION: In absence of other risk factors, being a newly arrived asylum seeker from a region with increased faecal ESBL-PE colonisation prevalence is not an indicator for C-PE colonisation and thus not a reason for pre-emptive screening and isolation upon hospital admission.

Detection of Enterobius vermicularis in greater Berlin, 2007-2017: seasonality and increased frequency of detection.

The pinworm (Enterobius vermicularis) causes mostly mild infections characterised by nocturnal anal pruritus, mainly in children. Still, the infection is stigmatising and sleep disturbances may lead to lack of concentration. For Germany, no epidemiological data are available. Laboratory data of all patients for whom detection of E. vermicularis by cellulose tape test had been requested between 2007 and November 2017 were analysed retrospectively. E. vermicularis was detected in 971/5578 (17.4%) samples collected from 3991 patients. The detection rate increased significantly within the period of investigation. It was higher in male than in female patients (20.0 vs. 15.4%). Children 4 to 10 years old and, if also examined, their relatives were most frequently affected. Control investigations at an interval of at least 1 month, which could indicate insufficient therapy or re-infection, were performed in 90/714 patients (12.6%). While parasite detection in children < 6 years was evenly distributed throughout the year, in older patients, it peaked between October and December. In conclusion, in the area of investigation, the frequency of E. vermicularis is higher in males than in females and is subject to a hitherto undescribed seasonality. The causes of the increased frequency of parasite detection warrant further investigations.

Giardia duodenalis in small animals and their owners in Germany: A pilot study.

Giardia duodenalis is a relevant gastrointestinal protozoan pathogen of humans and animals. This species complex consists of eight genetically different assemblages. Assemblages A and B are pathogenic to humans and pets, thus confer zoonotic potential. The risk of zoonotic transmission has been controversially discussed. The aim of this monocentric cross-sectional pilot study was to investigate G. duodenalis assemblages in humans and pets living in common households in Berlin/Brandenburg (Germany). Samples from dogs, cats and humans sharing the same households were screened for Giardia infection by antigen-detecting assays. All human samples were additionally analysed by a Giardia-specific qPCR. Cyst quantification and sequences of different gene loci (triosephosphate isomerase (tpi), glutamate dehydrogenase (gdh), ß-giardin (bg) and for dogs SSUrDNA) were analysed. A total of 38 households (31 households with dogs and seven with cats) with 69 human individuals participated in the study. Initial antigen-detecting assays revealed Giardia-positive results for 13 (39%) canine, one (14%) feline and one human sample. Reanalysis of the human samples by qPCR revealed two more positive specimens (4%). Two of these three samples were identified as assemblage B at all tested loci. Success rate of assemblage typing for pet samples was generally low and comprised mainly the SSUrDNA locus only. Overall, six of 13 Giardia-positive canine samples were typable (2× A, 1× co-infection: A and B, 1× C; 2× D). One pair of samples (dog and human) from the same household had a similar but not identical assemblage B sequence at tpi locus. Assemblage A was also detected in the dog specimen, which hampered sequence analysis. In conclusion, although exhibiting limitations due to the sample size, our study highlights the need for better and standardized typing tools to distinguish G. duodenalis strains with higher resolution in order to perform proper case-control studies for a realistic estimation of zoonotic risk.

Epidemiology and morbidity of hookworm-related cutaneous larva migrans (HrCLM): Results of a cohort study over a period of six months in a resource-poor community in Manaus, Brazil.

BACKGROUND: Hookworm-related cutaneous larva migrans (HrCLM) is a neglected parasitic skin disease, widespread in resource-poor communities in tropical and subtropical countries. Incidence and risk factors have never been investigated in a cohort study. METHODOLOGY/PRINCIPAL FINDINGS: To understand the seasonal epidemiology of HrCLM, an open cohort of 476 children in a resource-poor community in Manaus, Brazil was examined for HrCLM monthly over a period of 6 months. Monthly prevalence and intensity of infection were correlated with the amount of monthly precipitation. Multivariable Cox regression analysis indicated male sex (hazard ratio [HR] 3.29; 95% confidence interval [CI] 1.95-5.56), walking barefoot on sandy ground (HR 2.30; 95% CI 1.03-5.16), poverty (HR 2.13; 95% CI 1.09-4.17) and age between 10 and 14 years (HR 1.87; 95% CI 1.01-3.46) as predictors of HrCLM. Monthly incidence rates ranged between 0.21 and 1.05 cases per person-year with an overall incidence of 0.52 per person-year. CONCLUSIONS/SIGNIFICANCE: HrCLM is a frequent parasitic skin disease in this resource-poor community. Every second child theoretically becomes infected during one year. Boys, 10 to 14 years old, belonging to the poorest households of the community, are the most vulnerable population group. Even in the tropical monsoonal climate of Amazonia there is a considerable seasonal variation with monthly incidence and number of lesions peaking in the rainy season.

Highly sensitive and specific detection of Giardia duodenalis, Entamoeba histolytica, and Cryptosporidium spp. in human stool samples by the BD MAX™ Enteric Parasite Panel.

Detection of intestinal protozoan parasites by light microscopy is cumbersome, needs experienced personnel, and may lack sensitivity and/or specificity as compared with molecular-based stool assays. Here, we evaluated the BD MAX™ Enteric Parasite Panel, i.e., a multiplex real-time PCR assay for simultaneous detection of Giardia duodenalis, Entamoeba histolytica, and cryptosporidia (Cryptosporidium parvum and C. hominis), by examining 200 positive human stool samples (138 × G. duodenalis, 27 × E. histolytica, 35 × Cryptosporidium spp.) and 119 controls including 18 samples with E. dispar. The majority of the samples, i.e., 153/200 (76.5%) positive samples and 66/119 (55.5%) controls, were confirmed by multiplex in-house PCR detecting the same parasites as the BD MAX™ Enteric Parasite Panel. The BD MAX™ assay did not yield false-positive results. Sensitivity and specificity were 97.8% (95% CI, 93.3-99.4%) and 100% (95% CI, 97.4-100%) for G. duodenalis, 100% (95% CI, 84.5-100%) and 100% (95% CI, 98.4-100%) for E. histolytica, and 100% (95% CI, 87.7-100%) and 100% (95% CI, 98.3-100%) for cryptosporidia, and similar data were obtained when only the 219 PCR-confirmed samples were analyzed. Thus, the BD MAX™ Enteric Parasite Panel provides a highly sensitive and specific tool for the laboratory diagnosis of three predominant protozoan parasites causing enteritis.

Increasing metronidazole and rifampicin resistance of Helicobacter pylori isolates obtained from children and adolescents between 2002 and 2015 in southwest Germany.

BACKGROUND: Increasing antibiotic resistance has been reported for Helicobacter pylori, but data on the prevalence of antibiotic resistance of H. pylori in pediatric patients and the development of resistance over time are sparse. METHODS: Data for 610 H. pylori isolates obtained between 2002 and 2015 from gastric biopsies of 582 (mainly treatment-naïve) pediatric patients from southwest Germany were analyzed retrospectively regarding the antibiotic susceptibility determined by Etest and patients' characteristics. RESULTS: Overall resistance to metronidazole, clarithromycin, and rifampicin was 28.7%, 23.2%, and 13.3%, respectively, while resistance to amoxicillin was rare (0.8%). Simultaneous resistance to metronidazole and clarithromycin was observed for 7.7% of the isolates, and 2.3% were resistant to metronidazole, clarithromycin, and rifampicin. Differences between primary vs secondary resistance existed for metronidazole (24.7% vs 38.8%, P=.01) and clarithromycin (17.2% vs 54.1%, P=.0001). From 2002-2008 to 2009-2015, resistance to metronidazole increased from 20.8% to 34.4% (P=.003) and to rifampicin from 3.9% to 18.8% (P=.0001); this was not associated with increased numbers of patients previously treated for H. pylori infection in the second study period. In contrast, resistance to clarithromycin did not change significantly over time. Resistance was not associated with age, sex, or family origin in Europe. CONCLUSIONS: The considerable antibiotic resistance of H. pylori isolates argues for standard antibiotic susceptibility testing of H. pylori in pediatric patients prior to the initiation of antibiotic therapy.

Usage of Murine T-cell Hybridoma Cells as Responder Cells Reveals Interference of Helicobacter Pylori with Human Dendritic Cell-mediated Antigen Presentation.

Direct effects of Helicobacter pylori (H. pylori) on human CD4+ T-cells hamper disentangling a possible bacterial-mediated interference with major histocompatibility complex class II (MHC-II)-dependent antigen presentation to these cells. To overcome this limitation, we employed a previously described assay, which enables assessing human antigen-processing cell function by using murine T-cell hybridoma cells restricted by human leukocyte antigen (HLA) alleles. HLA-DR1+ monocyte-derived dendritic cells were exposed to H. pylori and pulsed with the antigen 85B from Mycobacterium tuberculosis (M. tuberculosis). Interleukin-2 (IL-2) secretion by AG85Baa97-112-specific hybridoma cells was then evaluated as an integral reporter of cognate antigen presentation. This methodology enabled revealing of interference of H. pylori with the antigen-presenting capacity of human dendritic cells.