Advancing Toxoplasma gondii multiplex serology.

Toxoplasma gondii is a highly prevalent pathogen causing zoonotic infections with significant public health implications. Yet, our understanding of long-term consequences, associated risk factors, and the potential role of co-infections is still limited. Seroepidemiological studies are a valuable approach to address open questions and enhance our insights into T. gondii across human populations. Here, we present substantial advancements to our previously developed T. gondii multiplex serology assay, which is based on the immunodominant antigens SAG1 and P22. While our previous bead-based assay quantified antibody levels against multiple targets in a high-throughput fashion requiring only a small sample volume, impaired assay characteristics emerged in sample dilutions beyond 1:100 and when being transferred to magnetic beads. Both are now critical for inclusion in large-scale seroprevalence studies. Using the truncated versions, SAG1D1 and P22trunc, significantly enhanced signal-to-noise ratios were achieved with almost perfect concordance with the gold-standard Sabin-Feldman dye test. In sample dilutions of 1:100, the diagnostic accuracy of SAG1D1 and P22trunc reached sensitivities (true positive rates) of 98% and 94% and specificities (true negative rates) of 93% and 95%, respectively. Importantly, performance metrics were reproducible in a 1:1,000 sample dilution, using both magnetic and nonmagnetic beads. Thresholds for seropositivity were derived from finite mixture models and performed equally well as thresholds by receiver operating characteristic analysis. Our improved multiplex serology assay is therefore able to generate robust and reproducible performance metrics under various assay conditions. Inclusion of T. gondii antibody measurements with other pathogens, in multiplex serology panels will allow for large-scale seroepidemiological research. IMPORTANCE: Toxoplasma gondii is a pathogen of significant public health concern due to its widespread prevalence and zoonotic potential. However, our understanding of key aspects, such as risk factors for infection and disease, potential outcomes, and their trends, remains limited. Seroepidemiological studies in large cohorts are invaluable for addressing these questions but remain scarce. Our revised multiplex serology assay equips researchers with a powerful tool capable of delivering T. gondii serum antibody measurements with high sensitivity and specificity under diverse assay conditions. This advancement paves the way for the integration of T. gondii antibody measurements into multi-pathogen multiplex serology panels, promising valuable insights into public health and pathogen interactions.

Infectious pathogens and risk of esophageal, gastric and duodenal cancers and ulcers in China: A case-cohort study.

Infection by certain pathogens is associated with cancer development. We conducted a case-cohort study of ~2500 incident cases of esophageal, gastric and duodenal cancer, and gastric and duodenal ulcer and a randomly selected subcohort of ~2000 individuals within the China Kadoorie Biobank study of >0.5 million adults. We used a bead-based multiplex serology assay to measure antibodies against 19 pathogens (total 43 antigens) in baseline plasma samples. Associations between pathogens and antigen-specific antibodies with risks of site-specific cancers and ulcers were assessed using Cox regression fitted using the Prentice pseudo-partial likelihood. Seroprevalence varied for different pathogens, from 0.7% for Hepatitis C virus (HCV) to 99.8% for Epstein-Barr virus (EBV) in the subcohort. Compared to participants seronegative for the corresponding pathogen, Helicobacter pylori seropositivity was associated with a higher risk of non-cardia (adjusted hazard ratio [HR] 2.73 [95% CI: 2.09-3.58]) and cardia (1.67 [1.18-2.38]) gastric cancer and duodenal ulcer (2.71 [1.79-4.08]). HCV was associated with a higher risk of duodenal cancer (6.23 [1.52-25.62]) and Hepatitis B virus was associated with higher risk of duodenal ulcer (1.46 [1.04-2.05]). There were some associations of antibodies again some herpesviruses and human papillomaviruses with risks of gastrointestinal cancers and ulcers but these should be interpreted with caution. This first study of multiple pathogens with risk of gastrointestinal cancers and ulcers demonstrated that several pathogens are associated with risks of gastrointestinal cancers and ulcers. This will inform future investigations into the role of infection in the etiology of these diseases.

Helicobacter pylori multiplex serology and risk of non-cardia and cardia gastric cancer: a case-cohort study and meta-analysis.

BACKGROUND: Helicobacter pylori infection is a major cause of non-cardia gastric cancer (NCGC), but uncertainty remains about the associations between sero-positivity to different H. pylori antigens and risk of NCGC and cardia gastric cancer (CGC) in different populations. METHODS: A case-cohort study in China included ∼500 each of incident NCGC and CGC cases and ∼2000 subcohort participants. Sero-positivity to 12 H. pylori antigens was measured in baseline plasma samples using a multiplex assay. Hazard ratios (HRs) of NCGC and CGC for each marker were estimated using Cox regression. These were further meta-analysed with studies using same assay. RESULTS: In the subcohort, sero-positivity for 12 H. pylori antigens varied from 11.4% (HpaA) to 70.8% (CagA). Overall, 10 antigens showed significant associations with risk of NCGC (adjusted HRs: 1.33 to 4.15), and four antigens with CGC (HRs: 1.50 to 2.34). After simultaneous adjustment for other antigens, positive associations remained significant for NCGC (CagA, HP1564, HP0305) and CGC (CagA, HP1564, HyuA). Compared with CagA sero-positive only individuals, those who were positive for all three antigens had an adjusted HR of 5.59 (95% CI 4.68-6.66) for NCGC and 2.17 (95% CI 1.54-3.05) for CGC. In the meta-analysis of NCGC, the pooled relative risk for CagA was 2.96 (95% CI 2.58-3.41) [Europeans: 5.32 (95% CI 4.05-6.99); Asians: 2.41 (95% CI 2.05-2.83); Pheterogeneity<0.0001]. Similar pronounced population differences were also evident for GroEL, HP1564, HcpC and HP0305. In meta-analyses of CGC, two antigens (CagA, HP1564) were significantly associated with a higher risk in Asians but not Europeans. CONCLUSIONS: Sero-positivity to several H. pylori antigens was significantly associated with an increased risk of NCGC and CGC, with varying effects between Asian and European populations.

Performance of Dried Blood Spot Samples in SARS-CoV-2 Serolomics.

Numerous sero-epidemiological studies have been initiated to investigate the spread and dynamics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To address the concomitant need for serological high-throughput assays, a bead-based multiplex serology assay, specific for SARS-CoV-2, had been developed. SARS-CoV-2 serolomics allows for measuring antibody responses to almost the entire SARS-CoV-2 proteome in up to 2000 serum samples per day. To enlarge the pool of eligible sample collection methods, we here test the compatibility of serolomics with dried blood spot (DBS)-derived eluates. Antibody levels of nine SARS-CoV-2 antigens, including the nucleocapsid (N) and receptor-binding domain of the spike protein (S1-RBD), were measured in 142 paired DBS and serum samples. The numeric correlation between the two sample types was high, with a Pearson's r of 0.88 for both S1-RBD and N and intraclass correlation coefficients of 0.93 and 0.92, respectively. Systematically reduced antibody levels in DBS eluates were compensated by lowering the cutoffs for seropositivity accordingly. This enabled the concordant classification of SARS-CoV-2 seropositivity, without loss in sensitivity. Antibody levels against accessory SARS-CoV-2 antigens also showed a high concordance, demonstrating that DBS-derived eluates are eligible for SARS-CoV-2 serolomics. DBS cards facilitate the collection of blood samples, as they obviate the need for medically trained personnel and can be shipped at room temperature. In combination with SARS-CoV-2 serolomics, DBS cards enable powerful sero-epidemiological studies, thus allowing for the monitoring of patients and epidemiological analyses in resource-poor settings.

Sero-prevalence of 19 infectious pathogens and associated factors among middle-aged and elderly Chinese adults: a cross-sectional study.

OBJECTIVES: To systematically assess the sero-prevalence and associated factors of major infectious pathogens in China, where there are high incidence rates of certain infection-related cancers. DESIGN: Cross-sectional study. SETTING: 10 (5 urban, 5 rural) geographically diverse areas in China. PARTICIPANTS: A subcohort of 2000 participants from the China Kadoorie Biobank. PRIMARY MEASURES: Sero-prevalence of 19 pathogens using a custom-designed multiplex serology panel and associated factors. RESULTS: Of the 19 pathogens investigated, the mean number of sero-positive pathogens was 9.4 (SD 1.7), with 24.4% of participants being sero-positive for >10 pathogens. For individual pathogens, the sero-prevalence varied, being for example, 0.05% for HIV, 6.4% for human papillomavirus (HPV)-16, 53.5% for Helicobacter pylori (H. pylori) and 99.8% for Epstein-Barr virus . The sero-prevalence of human herpesviruses (HHV)-6, HHV-7 and HPV-16 was higher in women than men. Several pathogens showed a decreasing trend in sero-prevalence by birth cohort, including hepatitis B virus (HBV) (51.6% vs 38.7% in those born <1940 vs >1970), HPV-16 (11.4% vs 5.4%), HHV-2 (15.1% vs 8.1%), Chlamydia trachomatis (65.6% vs 28.8%) and Toxoplasma gondii (22.0% vs 9.0%). Across the 10 study areas, sero-prevalence varied twofold to fourfold for HBV (22.5% to 60.7%), HPV-16 (3.4% to 10.9%), H. pylori (16.2% to 71.1%) and C. trachomatis (32.5% to 66.5%). Participants with chronic liver diseases had >7-fold higher sero-positivity for HBV (OR=7.51; 95% CI 2.55 to 22.13). CONCLUSIONS: Among Chinese adults, previous and current infections with certain pathogens were common and varied by area, sex and birth cohort. These infections may contribute to the burden of certain cancers and other non-communicable chronic diseases.

Mycobacterial infection aggravates Helicobacter pylori-induced gastric preneoplastic pathology by redirection of de novo induced Treg cells.

The two human pathogens Helicobacter pylori and Mycobacterium tuberculosis (Mtb) co-exist in many geographical areas of the world. Here, using a co-infection model of H. pylori and the Mtb relative M. bovis bacillus Calmette-Guérin (BCG), we show that both bacteria affect the colonization and immune control of the respective other pathogen. Co-occurring M. bovis boosts gastric Th1 responses and H. pylori control and aggravates gastric immunopathology. H. pylori in the stomach compromises immune control of M. bovis in the liver and spleen. Prior antibiotic H. pylori eradication or M. bovis-specific immunization reverses the effects of H. pylori. Mechanistically, the mutual effects can be attributed to the redirection of regulatory T cells (Treg cells) to sites of M. bovis infection. Reversal of Treg cell redirection by CXCR3 blockade restores M. bovis control. In conclusion, the simultaneous presence of both pathogens exacerbates the problems associated with each individual infection alone and should possibly be factored into treatment decisions.

The relative and attributable risks of cardia and non-cardia gastric cancer associated with Helicobacter pylori infection in China: a case-cohort study.

BACKGROUND: Helicobacter pylori infection is a major cause of non-cardia gastric cancer (NCGC), but its causal role in cardia gastric cancer (CGC) is unclear. Moreover, the reported magnitude of association with NCGC varies considerably, leading to uncertainty about population-based H pylori screening and eradication strategies in high-risk settings, particularly in China, where approximately half of all global gastric cancer cases occur. Our aim was to assess the associations of H pylori infection, both overall and for individual infection biomarkers, with the risks of NCGC and CGC in Chinese adults. METHODS: A case-cohort study was done in adults from the prospective China Kadoorie Biobank study, aged 30-79 years from ten areas in China (Qingdao, Haikou, Harbin, Suzhou, Liuzhou, Henan, Sichuan, Hunan, Gansu, and Zhejiang), and included 500 incident NCGC cases, 437 incident CGC cases, and 500 subcohort participants who were cancer-free and alive within the first two years since enrolment in 2004-08. H pylori biomarkers were measured in stored baseline plasma samples using a sensitive immunoblot assay (HelicoBlot 2.1), with adapted criteria to define H pylori seropositivity. Cox regression was used to estimate adjusted hazard ratios (HRs) for NCGC and CGC associated with H pylori infection. These values were used to estimate the number of gastric cancer cases attributable to H pylori infection in China. FINDINGS: Of the 512 715 adults enrolled in the China Kadoorie Biobank between June, 2004, and July, 2008, 500 incident NCGC cases, 437 incident CGC cases, and 500 subcohort participants were selected for analysis. The seroprevalence of H pylori was 94·4% (95% CI 92·4-96·4) in NGCG, 92·2% (89·7-94·7) in CGC, and 75·6% (71·8-79·4) in subcohort participants. H pylori infection was associated with adjusted HRs of 5·94 (95% CI 3·25-10·86) for NCGC and 3·06 (1·54-6·10) for CGC. Among the seven individual infection biomarkers, cytotoxin-associated antigen had the highest HRs for both NCGC (HR 4·41, 95% CI 2·60-7·50) and CGC (2·94, 1·53-5·68). In this population, 78·5% of NCGC and 62·1% of CGC cases could be attributable to H pylori infection. H pylori infection accounted for an estimated 339 955 cases of gastric cancer in China in 2018. INTERPRETATION: Among Chinese adults, H pylori infection is common and is the cause of large numbers of gastric cancer cases. Population-based mass screening and the eradication of H pylori should be considered to reduce the burden of gastric cancer in high-risk settings. FUNDING: Cancer Research UK, Wellcome Trust, UK Medical Research Council, British Heart Foundation, Kadoorie Charitable Foundation, National Key Research and Development Program of China, and National Natural Science Foundation of China.

Development of High-Throughput Multiplex Serology to Detect Serum Antibodies against Coxiella burnetii.

The causative agent of Q fever, the bacterium Coxiella burnetii (C. burnetii), has gained increasing interest due to outbreak events and reports about it being a potential risk factor for the development of lymphomas. In order to conduct large-scale studies for population monitoring and to investigate possible associations more closely, accurate and cost-effective high-throughput assays are highly desired. To address this need, nine C. burnetii proteins were expressed as recombinant antigens for multiplex serology. This technique enables the quantitative high-throughput detection of antibodies to multiple antigens simultaneously in a single reaction. Based on a reference group of 76 seropositive and 91 seronegative sera, three antigens were able to detect C. burnetii infections. Com1, GroEL, and DnaK achieved specificities of 93%, 69%, and 77% and sensitivities of 64%, 72%, and 47%, respectively. Double positivity to Com1 and GroEL led to a combined specificity of 90% and a sensitivity of 71%. In a subgroup of seropositives with an increased risk for chronic Q fever, the double positivity to these markers reached a specificity of 90% and a sensitivity of 86%. Multiplex serology enables the detection of antibodies against C. burnetii and appears well-suited to investigate associations between C. burnetii infections and the clinical manifestations in large-scale studies.

Development of Helicobacter pylori Whole-Proteome Arrays and Identification of Serologic Biomarkers for Noncardia Gastric Cancer in the MCC-Spain Study.

BACKGROUND: Helicobacter pylori (H. pylori) is a bacterial carcinogen and the leading risk factor for noncardia gastric cancer (NCGC). Detecting antibodies against specific H. pylori proteins in peripheral blood can be applied to characterize infection and determine disease associations. Most studies analyzing the association between H. pylori infection and gastric cancer have focused on previously identified antigens, predominantly the virulence factor cytotoxin-associated gene A (CagA). Selecting antigens in an unbiased approach may, however, allow the identification of novel biomarkers. METHODS: Using a combination of multiple spotting technique and cell-free, on-chip protein expression, we displayed the H. pylori genome (strain 26695) on high-density microarrays. Immunogenic proteins were identified by serum pool incubations and henceforth analyzed in individual samples. To test its applicability, we used sera from a multicase-control (MCC)-Spain study. Serologic responses between NCGC cases and controls were assessed by conditional logistic regression estimating ORs and 95% confidence intervals. RESULTS: We successfully expressed 93% of the 1,440 H. pylori open reading frames in situ. Of these, 231 (17%) were found to be immunogenic. By comparing 58 NCGC cases with 58 matched controls, we confirmed a higher seroprevalence of CagA among cases (66%) than controls (31%). We further identified a potential novel marker, the Helicobacter outer membrane protein A (HopA). CONCLUSIONS: In this study, we provide evidence that our H. pylori whole-proteome microarray offers a platform for unbiased de novo identification of serologic biomarkers. IMPACT: Given its versatile workflow, antibody responses against other H. pylori strains and possible associations with diverse H. pylori-related outcomes can be systematically analyzed.

Helicobacter pylori seroprevalence in six different ethnic groups living in Amsterdam: The HELIUS study.

BACKGROUND: Helicobacter pylori prevalence varies greatly worldwide. We explored the prevalence of H. pylori and CagA seropositivity among adults aged 18-44 years living in the Netherlands by ethnicity and migration status (first vs second generation). MATERIALS AND METHODS: Participants from six different ethnic groups were selected from the population-based multi-ethnic HELIUS study in Amsterdam, the Netherlands. Serum samples were tested for H. pylori antigens using a validated Luminex-based multiplex serology assay. Prevalence ratios were estimated using Poisson regression analysis. RESULTS: A total of 4683 participants aged 18-44 years were randomly selected based on sex, ethnicity, and age. H. pylori seroprevalence was highest in the Ghanaian group (84%), followed by Moroccan (81%), Turkish (66%), African Surinamese (51%), South-Asian Surinamese (48%), and Dutch (17%) participants. All ethnic minority groups had a significantly higher risk of being H. pylori seropositive compared to the Dutch group. This association was strongest among participants born outside the Netherlands (first generation), but was still significant and apparent among second-generation participants. Among first-generation participants, all groups, except the Moroccans, had a significantly higher proportion of individuals with a cagA + H. pylori strain compared to the Dutch participants. CONCLUSION: Helicobacter pylori seroprevalence among first-generation migrants is high in the Netherlands and remains elevated among second-generation migrants (ie, those born in the Netherlands). High exposure to H. pylori, and especially to the more virulent cagA+ strain, highlights the need for tailored prevention of gastric diseases (notably peptic ulcers and cancers) among migrants.