Human herpesvirus 6A and axonal injury before the clinical onset of multiple sclerosis.

Recent research indicates that multiple sclerosis is preceded by a prodromal phase with elevated levels of serum neurofilament light chain (sNfL), a marker of axonal injury. The effect of environmental risk factors on the extent of axonal injury during this prodrome is unknown. Human herpesvirus 6A (HHV-6A) is associated with an increased risk of developing multiple sclerosis. The objective of this study was to determine if HHV-6A serostatus is associated with the level of sNfL in the multiple sclerosis prodrome, which would support a causative role of HHV-6A. A nested case-control study was performed by crosslinking multiple sclerosis registries with Swedish biobanks. Individuals with biobank samples collected before the clinical onset of multiple sclerosis were included as cases. Controls without multiple sclerosis were randomly selected, matched for biobank, sex, sampling date and age. Serostatus of HHV-6A and Epstein-Barr virus was analysed with a bead-based multiplex assay. The concentration of sNfL was analysed with single molecule array technology. The association between HHV-6A serology and sNfL was assessed by stratified t-tests and linear regressions, adjusted for Epstein-Barr virus serostatus and sampling age. Within-pair ratios of HHV-6A seroreactivity and sNfL were calculated for each case and its matched control. To assess the temporal relationship between HHV-6A antibodies and sNfL, these ratios were plotted against the time to the clinical onset of multiple sclerosis and compared using locally estimated scatterplot smoothing regressions with 95% confidence intervals (CI). Samples from 519 matched case-control pairs were included. In cases, seropositivity of HHV-6A was significantly associated with the level of sNfL (+11%, 95% CI 0.2-24%, P = 0.045) and most pronounced in the younger half of the cases (+24%, 95% CI 6-45%, P = 0.007). No such associations were observed among the controls. Increasing seroreactivity against HHV-6A was detectable before the rise of sNfL (significant within-pair ratios from 13.6 years versus 6.6 years before the clinical onset of multiple sclerosis). In this study, we describe the association between HHV-6A antibodies and the degree of axonal injury in the multiple sclerosis prodrome. The findings indicate that elevated HHV-6A antibodies both precede and are associated with a higher degree of axonal injury, supporting the hypothesis that HHV-6A infection may contribute to multiple sclerosis development in a proportion of cases.

Genetics of immune response to Epstein-Barr virus: prospects for multiple sclerosis pathogenesis.

Epstein-Barr virus (EBV) infection has been advocated as a prerequisite for developing multiple sclerosis (MS) and possibly the propagation of the disease. However, the precise mechanisms for such influences are still unclear. A large-scale study investigating the host genetics of EBV serology and related clinical manifestations, such as infectious mononucleosis (IM), may help us better understand the role of EBV in MS pathogenesis. This study evaluates the host genetic factors that influence serological response against EBV and history of IM and cross-evaluates them with MS risk and genetic susceptibility in the Swedish population. Plasma IgG antibody levels against EBV nuclear antigen-1 (EBNA-1, truncated=aa[325-641], peptide=aa[385-420]) and viral capsid antigen p18 (VCAp18) were measured using bead-based multiplex serology for 8744 MS cases and 7229 population-matched controls. The MS risk association for high/low EBV antibody levels and history of IM was compared to relevant clinical measures along with sex, age at sampling, and associated HLA allele variants. Genome-wide and HLA allele association analyses were also performed to identify genetic risk factors for EBV antibody response and IM history. Higher antibody levels against VCAp18 (OR=1.74, 95% CI=1.60-1.88) and EBNA-1, particularly the peptide (OR=3.13, 95% CI=2.93-3.35), were associated with an increased risk for MS. The risk increased with higher anti-EBNA-1 IgG levels up to twelve times the reference risk. We also identified several independent HLA haplotypes associated with EBV serology overlapping with known MS risk alleles (e.g., DRB1*15:01). Although there were several candidates, no variants outside the HLA region reached genome-wide significance. Cumulative HLA risk for anti-EBNA-1 IgG levels, particularly the peptide fragment, was strongly associated with MS. In contrast, the genetic risk for high anti-VCAp18 IgG levels was not as strongly associated with MS risk. IM history was not associated with class II HLA genes but negatively associated with A*02:01, which is protective against MS. Our findings emphasize that the risk association between anti-EBNA-1 IgG levels and MS may be partly due to overlapping HLA associations. Additionally, the increasing MS risk with increasing anti-EBNA-1 levels would be consistent with a pathogenic role of the EBNA-1 immune response, perhaps through molecular mimicry. Given that high anti-EBNA-1 antibodies may reflect a poorly controlled T-cell defense against the virus, our findings would be consistent with DRB1*15:01 being a poor class II antigen in the immune defense against EBV. Lastly, the difference in genetic control of IM supports the independent roles of EBNA-1 and IM in MS susceptibility.

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.

Seroreactivity against lytic, latent and possible cross-reactive EBV antigens appears on average 10 years before MS induced preclinical neuroaxonal damage.

BACKGROUND: Multiple sclerosis (MS) and presymptomatic axonal injury appear to develop only after an Epstein-Barr virus (EBV) infection. This association remains to be confirmed across a broad preclinical time range, for lytic and latent EBV seroreactivity, and for potential cross-reacting antigens. METHODS: We performed a case-control study with 669 individual serum samples obtained before clinical MS onset, identified through cross-linkage with the Swedish MS register. We assayed antibodies against EBV nuclear antigen 1 (EBNA1), viral capsid antigen p18, glycoprotein 350 (gp350), the potential cross-reacting protein anoctamin 2 (ANO2) and the level of sNfL, a marker of axonal injury. RESULTS: EBNA1 (latency) seroreactivity increased in the pre-MS group, at 15-20 years before clinical MS onset, followed by gp350 (lytic) seroreactivity (p=0.001-0.009), ANO2 seropositivity appeared shortly after EBNA1-seropositivity in 16.7% of pre-MS cases and 10.0% of controls (p=0.001).With an average lag of almost a decade after EBV, sNfL gradually increased, mainly in the increasing subgroup of seropositive pre-MS cases (p=8.10-5 compared with non-MS controls). Seropositive pre-MS cases reached higher sNfL levels than seronegative pre-MS (p=0.038). In the EBNA1-seropositive pre-MS group, ANO2 seropositive cases had 26% higher sNfL level (p=0.0026). CONCLUSIONS: Seroreactivity against latent and lytic EBV antigens, and in a subset ANO2, was detectable on average a decade before the appearance of a gradually increasing axonal injury occurring in the last decade before the onset of clinical MS. These findings strengthen the hypothesis of latent EBV involvement in the pathogenesis of MS.

Helicobacter hepaticus and Helicobacter bilis in liver and biliary cancers from ATBC and PLCO.

BACKGROUND: Helicobacter species (spp.) have been detected in human bile and hepatobiliary tissue Helicobacter spp. promote gallstone formation and hepatobiliary tumors in laboratory studies, though it remains unclear whether Helicobacter spp. contribute to these cancers in humans. We used a multiplex panel to assess whether seropositivity to Helicobacter (H.) hepaticus or H. bilis proteins was associated with the development of hepatobiliary cancers in the Finnish Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study, and US-based Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO). METHODS: We included 62 biliary and 121 liver cancers, and 190 age-matched controls from ATBC and 74 biliary and 105 liver cancers, and 364 age- and sex-matched controls from PLCO. Seropositivity to 14 H. hepaticus and H. bilis antigens was measured using a multiplex assay. Odds ratios (ORs) and 95% confidence intervals (CIs) were adjusted for major hepatobiliary cancer risk factors and Helicobacter pylori serostatus. RESULTS: Seropositivity to the H. bilis antigen, P167D, was associated with more than a twofold higher risk of liver cancer (OR: 2.38; 95% CI: 1.06, 5.36) and seropositivity to the H. hepaticus antigens HH0407 or HH1201, or H. bilis antigen, HRAG 01470 were associated with higher risk of biliary cancer (OR: 5.01; 95% CI: 1.53, 16.40; OR: 2.40; 95% CI: 1.00, 5.76; OR: 3.27; 95% CI: 1.14, 9.34, respectively) within PLCO. No associations for any of the H. hepaticus or H. bilis antigens were noted for liver or biliary cancers within ATBC. CONCLUSIONS: Further investigations in cohort studies should examine the role of Helicobacter spp. in the etiology of liver and biliary cancers.

COVID-19 and long-term impact on symptoms and Health-Related Quality of Life in Costa Rica: the RESPIRA cohort study.

BACKGROUND: Evidence continues to accumulate regarding the potential long-term health consequences of COVID-19 in the population. To distinguish between COVID-19-related symptoms and health limitations from those caused by other conditions, it is essential to compare cases with community controls using prospective data ensuring case-control status. The RESPIRA study addresses this need by investigating the lasting impact of COVID-19 on Health-related Quality of Life (HRQoL) and symptomatology in a population-based cohort in Costa Rica, thereby providing a robust framework for controlling HRQoL and symptoms. METHODS: The study comprised 641 PCR-confirmed, unvaccinated cases of COVID-19 and 947 matched population-based controls. Infection was confirmed using antibody tests on enrollment serum samples and symptoms were monitored monthly for 6 months post-enrolment. Administered at the 6-month visit (occurring between 6- and 2-months post-diagnosis for cases and 6 months after enrollment for controls), HRQoL and Self-Perceived Health Change were assessed using the SF-36, while brain fog, using three items from the Mental Health Inventory (MHI). Regression models were utilized to analyze SF-36, MHI scores, and Self-Perceived Health Change, adjusted for case/control status, severity (mild case, moderate case, hospitalized) and additional independent variables. Sensitivity analyses confirmed the robustness of the findings. RESULTS: Cases showed significantly higher prevalences of joint pain, chest tightness, and skin manifestations, that stabilized at higher frequencies from the fourth month post-diagnosis onwards (2.0%, 1.2%, and 0.8% respectively) compared to controls (0.9%, 0.4%, 0.2% respectively). Cases also exhibited significantly lower HRQoL than controls across all dimensions in the fully adjusted model, with a 12.4 percentage-point difference [95%CI: 9.4-14.6], in self-reported health compared to one year prior. Cases reported 8.0% [95%CI: 4.2, 11.5] more physical limitations, 7.3% [95%CI: 3.5, 10.5] increased lack of vitality, and 6.0% [95%CI: 2.4, 9.0] more brain fog compared to controls with similar characteristics. Undiagnosed cases detected with antibody tests among controls had HRQoL comparable to antibody negative controls. Differences were more pronounced in individuals with moderate or severe disease and among women. CONCLUSIONS: PCR-confirmed unvaccinated cases experienced prolonged HRQoL reductions 6 months to 2 years after diagnosis, this was particularly the case in severe cases and among women. Mildly symptomatic cases showed no significant long-term sequelae.

Common infections and neuroimaging markers of dementia in three UK cohort studies.

INTRODUCTION: We aimed to investigate associations between common infections and neuroimaging markers of dementia risk (brain volume, hippocampal volume, white matter lesions) across three population-based studies. METHODS: We tested associations between serology measures (pathogen serostatus, cumulative burden, continuous antibody responses) and outcomes using linear regression, including adjustments for total intracranial volume and scanner/clinic information (basic model), age, sex, ethnicity, education, socioeconomic position, alcohol, body mass index, and smoking (fully adjusted model). Interactions between serology measures and apolipoprotein E (APOE) genotype were tested. Findings were meta-analyzed across cohorts (Nmain  = 2632; NAPOE-interaction  = 1810). RESULTS: Seropositivity to John Cunningham virus associated with smaller brain volumes in basic models (ß = -3.89 mL [-5.81, -1.97], Padjusted  < 0.05); these were largely attenuated in fully adjusted models (ß = -1.59 mL [-3.55, 0.36], P = 0.11). No other relationships were robust to multiple testing corrections and sensitivity analyses, but several suggestive associations were observed. DISCUSSION: We did not find clear evidence for relationships between common infections and markers of dementia risk. Some suggestive findings warrant testing for replication.

Exposure to preference-matched alcohol advertisements from national sports broadcasts increases short-term alcohol consumption inclinations in risky drinkers.

BACKGROUND: In Australia, sports broadcasting is afforded special alcohol advertising rights during daytime hours, which raises public health concerns, including short-term increases in alcohol consumption among the broad viewership of national sporting codes. METHODS: We conducted a content analysis across a sample of nationally televised finals matches (N = 16) from the Australian Football League (AFL) and the National Rugby League (NRL) to determine the prevalence of alcohol advertising video clips during these broadcasts. We also conducted an online experiment exposing participants (N = 345) to a randomly selected alcohol advertisement and measured the immediate effects on self-reported alcohol craving and drinking intentions. RESULTS: The prevalence of alcohol advertising video clips during AFL broadcasts was 3.9% and 1.8% for NRL. While, overall, alcohol advertisement video clip exposure did not impact craving or drinking intentions, a modest increase in craving was found for a subsample of risky drinking participants (N = 107) who also reported a preference for the specific alcoholic beverage being advertised. CONCLUSIONS: Video alcohol advertisements occurred less than 1 in 20 advertisements on average and exposure to alcohol advertising elicited a low, yet measurable, short-term increase in alcohol inclinations, among vulnerable adult drinkers when a desirable alcoholic beverage advertisement is viewed. SO WHAT?: Given that alcohol advertisements are most likely to increase consumption among risky drinkers, health messaging during sports broadcasts needs to specifically target these individuals.

Association of Antibody Responses to Fusobacterium nucleatum and Streptococcus gallolyticus Proteins with Colorectal Adenoma and Colorectal Cancer.

BACKGROUND: Streptococcus gallolyticus subspecies gallolyticus (SGG) and Fusobacterium (F.) nucleatum have been implicated in colorectal carcinogenesis. Here, the association of immune responses to bacterial exposure with advancing stages of colorectal neoplasia was assessed by multiplex serology. METHODS: Immunoglobulin (Ig) A and G antibody responses to eleven proteins each of F. nucleatum and SGG were measured in plasma of controls (n = 100) and patients with colorectal cancer (CRC, n = 25), advanced adenoma (n = 82), or small polyps (n = 85). Multivariable logistic regression was used to evaluate the association of bacterial sero-positivity with colorectal neoplasia. In a cohort subset with matched data (n = 45), F. nucleatum sero-positivity was correlated with bacterial abundance in both neoplastic and matched normal tissue. RESULTS: IgG sero-positivity to Fn1426 of F. nucleatum was associated with an increased CRC risk (OR = 4.84; 95% CI 1.46-16.0), while IgA sero-positivity to any SGG protein or specifically Gallo0272 and Gallo1675 alone was associated with increased advanced adenoma occurrence (OR = 2.02, 95% CI 1.10-3.71; OR = 2.67, 95% CI 1.10-6.46; and OR = 6.17, 95% CI 1.61-23.5, respectively). Only F. nucleatum abundance in the normal mucosa positively correlated with the IgA response to the Fn1426 antigen (Correlation coefficient (r) = 0.38, p < 0.01). CONCLUSION: Antibody responses to SGG and F. nucleatum were associated with occurrence of colorectal adenomas and CRC, respectively. Further studies are needed to clarify the role these microbes or the immune response to their antigens may have in colorectal carcinogenesis stages.

Molecular mimicry between Anoctamin 2 and Epstein-Barr virus nuclear antigen 1 associates with multiple sclerosis risk.

Multiple sclerosis (MS) is a chronic inflammatory, likely autoimmune disease of the central nervous system with a combination of genetic and environmental risk factors, among which Epstein-Barr virus (EBV) infection is a strong suspect. We have previously identified increased autoantibody levels toward the chloride-channel protein Anoctamin 2 (ANO2) in MS. Here, IgG antibody reactivity toward ANO2 and EBV nuclear antigen 1 (EBNA1) was measured using bead-based multiplex serology in plasma samples from 8,746 MS cases and 7,228 controls. We detected increased anti-ANO2 antibody levels in MS (P = 3.5 × 10-36) with 14.6% of cases and 7.8% of controls being ANO2 seropositive (odds ratio [OR] = 1.6; 95% confidence intervals [95%CI]: 1.5 to 1.8). The MS risk increase in ANO2-seropositive individuals was dramatic when also exposed to 3 known risk factors for MS: HLA-DRB1*15:01 carriage, absence of HLA-A*02:01, and high anti-EBNA1 antibody levels (OR = 24.9; 95%CI: 17.9 to 34.8). Reciprocal blocking experiments with ANO2 and EBNA1 peptides demonstrated antibody cross-reactivity, mapping to ANO2 [aa 140 to 149] and EBNA1 [aa 431 to 440]. HLA gene region was associated with anti-ANO2 antibody levels and HLA-DRB1*04:01 haplotype was negatively associated with ANO2 seropositivity (OR = 0.6; 95%CI: 0.5 to 0.7). Anti-ANO2 antibody levels were not increased in patients from 3 other inflammatory disease cohorts. The HLA influence and the fact that specific IgG production usually needs T cell help provides indirect evidence for a T cell ANO2 autoreactivity in MS. We propose a hypothesis where immune reactivity toward EBNA1 through molecular mimicry with ANO2 contributes to the etiopathogenesis of MS.

DRB1-environment interactions in multiple sclerosis etiology: results from two Swedish case-control studies.

OBJECTIVE: We aimed to investigate the influence of environmental risk factors for multiple sclerosis (MS) in different genetic contexts, and study if interactions between environmental factors and human leucocyte antigen (HLA) genes differ in magnitude according to heterozygocity and homozygocity for HLA-DRB1*15:01. METHODS: Using population-based case-control studies (6985 cases, 6569 controls), subjects with different genotypes and smoking, EBNA-1 status and adolescent Body Mass status, were compared regarding MS risk, by calculating OR with 95% CI employing logistic regression. The interaction between different genotypes and each environmental factor was evaluated on the additive scale. RESULTS: The effect of each DRB1*15:01 allele on MS risk was additive on the log-odds scale for each additional allele. Interaction between DRB1*15:01 and each assessed environmental factor was of similar magnitude regardless of the number of DRB1*15:01 alleles, although ORs were affected. When any of the environmental factors were present in DRB1*15:01 carriers without the protective A*02:01 allele, a three-way interaction occurred and rendered high ORs, especially among DRB1*15:01 homozygotes (OR 20.0, 95% CI 13.1 to 30.5 among smokers, OR 21.9, 95% CI 15.0 to 31.8 among those with elevated EBNA-1 antibody levels, and OR 44.3, 95% CI 13.5 to 145 among those who reported adolescent overweight/obesity). CONCLUSIONS: The strikingly increased MS risk among DRB*15:01 homozygotes exposed to any of the environmental factors is a further argument in favour of these factors acting on immune-related mechanisms. The data further reinforce the importance of preventive measures, in particular for those with a genetic susceptibility to MS.

Low sun exposure acts synergistically with high Epstein-Barr nuclear antigen 1 (EBNA-1) antibody levels in multiple sclerosis etiology.

BACKGROUND: Among multiple sclerosis (MS) patients, an association has been observed between low levels of vitamin D and high Epstein-Barr nuclear antigen 1 (EBNA-1) antibody levels. However, whether sun exposure/vitamin D moderates the role of Epstein-Barr virus (EBV) infection in MS etiology is unclear. We aimed to investigate potential synergistic effects between low sun exposure and elevated EBNA-1 antibody levels regarding MS risk. METHODS: We used a population-based case-control study involving 2017 incident cases of MS and 2443 matched controls. We used logistic regression models to calculate the odds ratios of MS with 95% confidence intervals (CIs) in subjects with different sun exposure habits and EBNA-1 status. Potential interaction on the additive scale was evaluated by calculating the attributable proportion due to interaction (AP). RESULTS: Low sun exposure acted synergistically with high EBNA-1 antibody levels (AP 0.2, 95% CI 0.03-0.3) in its association to increased MS risk. The interaction was present regardless of HLA-DRB1*15:01 status. CONCLUSIONS: Low sun exposure may either directly, or indirectly by affecting vitamin D levels, synergistically reinforce pathogenic mechanisms, such as aspects of the adaptive immune response, related to MS risk conveyed by EBV infection.

Cytomegalovirus seropositivity is associated with reduced risk of multiple sclerosis-a presymptomatic case-control study.

BACKGROUND AND PURPOSE: Epstein-Barr virus (EBV) and human herpesvirus 6A (HHV-6A) are associated with increased risk of multiple sclerosis (MS). Conversely, infection with cytomegalovirus (CMV) has been suggested to reduce the risk of MS but supporting data from presymptomatic studies are lacking. Here, it was sought to increase the understanding of CMV in MS aetiology. METHODS: A nested case-control study was performed with presymptomatically collected blood samples identified through crosslinkage of MS registries and Swedish biobanks. Serological antibody response against CMV, EBV and HHV-6A was determined using a bead-based multiplex assay. Odds ratio (OR) with 95% confidence interval (CI) for CMV seropositivity as a risk factor for MS was calculated by conditional logistic regression and adjusted for EBV and HHV-6A seropositivity. Potential interactions on the additive scale were analysed by calculating the attributable proportion due to interaction (AP). RESULTS: Serum samples from 670 pairs of matched cases and controls were included. CMV seropositivity was associated with a reduced risk for MS (OR = 0.70, 95% CI 0.56-0.88, p = 0.003). Statistical interactions on the additive scale were observed between seronegativity for CMV and seropositivity against HHV-6A (AP 0.34, 95% CI 0.06-0.61) and EBV antigen EBNA-1 (amino acid 385-420) at age 20-39 years (AP 0.37, 95% CI 0.09-0.65). CONCLUSIONS: Cytomegalovirus seropositivity is associated with a decreased risk for MS. The protective role for CMV infection in MS aetiology is further supported by the interactions between CMV seronegativity and EBV and HHV-6A seropositivity.

Epstein-Barr virus and human papillomavirus serum antibodies define the viral status of nasopharyngeal carcinoma in a low endemic country.

Epstein-Barr virus (EBV) causes nasopharyngeal carcinoma (NPC) in endemic regions, where almost every tumor is EBV-positive. In Western populations, NPC is rare, and human papillomavirus infection (HPV) has been suggested as another viral cause. We validated multiplex serology with molecular tumor markers, to define EBV-positive, HPV-positive and EBV-/HPV-negative NPCs in the United Kingdom, and analyzed survival differences between those groups. Sera from NPC cases (n = 98) and age- and sex-matched controls (n = 142) from the Head and Neck 5000 clinical cohort study were analyzed. IgA and IgG serum antibodies against 13 EBV antigens were measured and compared with EBER in situ hybridization (EBER-ISH) data of 41 NPC tumors (29 EBER-ISH positive, 12 negative). IgG antibodies to EBV LF2 correctly diagnosed EBV-positive NPCs in 28 of 29 cases, while all EBER-ISH negative NPCs were seronegative to LF2 IgG (specificity = 100%, sensitivity = 97%). HPV early antigen serology was compared to HPV molecular markers (p16 expression, HPV DNA and RNA) available for 41 NPCs (13 positive, 28 negative). Serology matched molecular HPV markers in all but one case (specificity = 100%, sensitivity = 92%). EBV and HPV infections were mutually exclusive. Overall, 67% of the analyzed NPCs were defined as EBV-positive, 18% as HPV-positive and 14% as EBV/HPV-negative. There was no statistical evidence of a difference in survival between the three groups. These data provide evidence that both, EBV-positive and HPV-positive NPCs are present in a low incidence country, and that EBV and HPV serum antibodies correlate with the viral status of the tumor.

Seropositivity for Helicobacter pylori and hepatobiliary cancers in the PLCO study.

Helicobacter has been suggested to play a possible role in hepatitis, gallstones, and hepatobiliary tumours. We assessed whether seropositivity to 15 H. pylori proteins was associated with subsequent incidence of 74 biliary tract and 105 liver cancer cases vs. 357 matched controls in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO). Odds ratios and 95% confidence intervals were computed by conditional logistic regression after adjustment for known hepatobiliary cancer risk factors. H. pylori seropositivity was not associated with either biliary tract (1.76, 0.90-3.46) or liver cancer (0.87, 0.46-1.65). CagA seropositivity was associated with both endpoints, although the latter association was not statistically significant (biliary tract: 2.16, 1.03-4.50; liver cancer: 1.96, 0.98-3.93) and neither association was statistically significant after correcting for multiple comparisons. Together, these results suggest possible associations between H. pylori and hepatobiliary cancer and suggest the value of future studies investigating the association.Trial registration number: NCT00339495.

Serologic Response to Helicobacter pylori Proteins Associated With Risk of Colorectal Cancer Among Diverse Populations in the United States.

BACKGROUND & AIMS: Previous studies reported an association of the bacteria Helicobacter pylori, the primary cause of gastric cancer, and risk of colorectal cancer (CRC). However, these findings have been inconsistent, appear to vary with population characteristics, and may be specific for virulence factor VacA. To more thoroughly evaluate the potential association of H pylori antibodies with CRC risk, we assembled a large consortium of cohorts representing diverse populations in the United States. METHODS: We used H pylori multiplex serologic assays to analyze serum samples from 4063 incident cases of CRC, collected before diagnosis, and 4063 matched individuals without CRC (controls) from 10 prospective cohorts for antibody responses to 13 H pylori proteins, including virulence factors VacA and CagA. The association of seropositivity to H pylori proteins, as well as protein-specific antibody level, with odds of CRC was determined by conditional logistic regression. RESULTS: Overall, 40% of controls and 41% of cases were H pylori-seropositive (odds ratio [OR], 1.09; 95% CI, 0.99-1.20). H pylori VacA-specific seropositivity was associated with an 11% increased odds of CRC (OR, 1.11; 95% CI, 1.01-1.22), and this association was particularly strong among African Americans (OR, 1.45; 95% CI, 1.08-1.95). Additionally, odds of CRC increased with level of VacA antibody in the overall cohort (P = .008) and specifically among African Americans (P = .007). CONCLUSIONS: In an analysis of a large consortium of cohorts representing diverse populations, we found serologic responses to H pylori VacA to associate with increased risk of CRC risk, particularly for African Americans. Future studies should seek to understand whether this marker is related to virulent H pylori strains carried in these populations.

Validation of an Epstein-Barr Virus Antibody Risk Stratification Signature for Nasopharyngeal Carcinoma by Use of Multiplex Serology.

Serological testing for nasopharyngeal carcinoma (NPC) has recently been reinvigorated by the implementation of novel Epstein-Barr virus (EBV)-specific IgA and IgG antibodies from a proteome array. Although proteome arrays are well suited for comprehensive antigen selection, they are not applicable for large-scale studies. We adapted a 13-marker EBV antigen signature for NPC risk identified by proteome arrays to multiplex serology to establish an assay for large-scale studies. Taiwanese NPC cases (n = 175) and matched controls (n = 175) were used for assay validation. Spearman's correlation was calculated, and the diagnostic value of all multiplex markers was assessed independently using the area under the receiver operating characteristic curve (AUC). Two refined signatures were identified using stepwise logistic regression and internally validated with 10-fold cross validation. Array and multiplex serology showed strong correlation for each individual EBV marker, as well as for a 13-marker combined model on continuous data. Two refined signatures with either four (LF2 and BGLF2 IgG, LF2 and BMRF1 IgA) or two (LF2 and BGLF2 IgG) antibodies on dichotomous data were identified as the most parsimonious set of serological markers able to distinguish NPC cases from controls with AUCs of 0.992 (95% confidence interval [CI], 0.983 to 1.000) and 0.984 (95% CI, 0.971 to 0.997), respectively. Neither differed significantly from the 13-marker model (AUC, 0.992; 95% CI, 0.982 to 1.000). All models were internally validated. Multiplex serology successfully validated the original EBV proteome microarray data. Two refined signatures of four and two antibodies were capable of detecting NPC with 99.2% and 98.4% accuracy.

Differences in antibody levels to H. pylori virulence factors VacA and CagA among African Americans and whites in the Southeast USA.

PURPOSE: Helicobacter pylori (H. pylori) is the leading cause of gastric cancer. High antibody levels to H. pylori virulence factors Vacuolating cytotoxin A (VacA) and Cytotoxin-associated gene A (CagA) have been suggested as gastric cancer risk markers. In the USA, H. pylori sero-prevalence is twofold higher in African Americans compared to whites. We sought to assess whether African Americans also exhibit higher antibody levels to VacA and CagA. METHODS: Antibody responses to H. pylori proteins were measured by multiplex serology in 686 African Americans and whites of the Southern Community Cohort Study. Among VacA- and CagA-seropositives, we analyzed the association of race with antibody level using logistic regression models to produce odds ratios (OR) and 95% confidence intervals (CI). RESULTS: Sero-positive African Americans had significantly higher mean antibody levels to both VacA and CagA, which resulted in increased odds for the highest quartile of antibody levels compared to sero-positive whites (VacA, OR: 6.08; 95% CI 3.41, 10.86; CagA, OR: 3.77; 95% CI 1.61, 8.84). CONCLUSION: Our findings support future studies to assess the association of differential antibody responses by race with risk of gastric cancer in the USA, which could then aid in developing targeted H. pylori eradication strategies.

Performance of multiplex serology in discriminating active vs past Helicobacter pylori infection in a primarily African American population in the southeastern United States.

PURPOSE: To feasibly analyze associations of Helicobacter pylori (H. pylori) with disease in large cohort studies, assays are needed to assess H. pylori prevalence in existing biospecimens. However, serology has traditionally been unable to distinguish active from past infection. We sought to determine the sensitivity of seropositivity to H. pylori proteins to detect active infection. METHODS: We measured antibody responses to 13 H. pylori proteins using multiplex serology in serum samples of a training (n = 78) and validation set (n = 49) collected concurrently from patients undergoing urea breath test (UBT). To determine sensitivity of seropositivity to H. pylori proteins for active infection, a cutoff was applied to achieve 90% specificity. Antibody levels were retested in a subset of participants (n = 16) 6 months after baseline. RESULTS: With a specificity of 91%, seropositivity to H. pylori proteins VacA, GroEl, HcpC, and HP1564 ascertained active infection from 100% to 75% sensitivity. Positivity to a combination of these proteins (≥2 out of the 4) resulted in specificity of 90% and sensitivity of 100%. The validation set replicated results from the training set. Among those participants with successful H. pylori eradication after baseline, antibody levels decreased significantly for VacA, HcpC, and HP1564 when assessed 6 months later. CONCLUSION: Utilizing the cutoffs for seropositivity established through comparison with UBT, seropositivity to ≥2 of the H. pylori proteins VacA, GroEl, HcpC, and HP1564 determines active H. pylori infection at high specificity and sensitivity and may approximate the prevalence of active H. pylori infection in large cohorts.

Associations between Helicobacter pylori with nonalcoholic fatty liver disease and other metabolic conditions in Guatemala.

BACKGROUND: Previous studies have suggested an association between Helicobacter pylori (H pylori) and nonalcoholic fatty liver disease (NAFLD). The aim of the current study was to examine the association in Guatemala, a region with elevated prevalences of both H pylori and NAFLD. Associations between H pylori and other metabolic conditions were also examined, as were associations between H hepaticus and H bilis and the metabolic conditions. MATERIALS & METHODS: The analysis included 424 participants from a cross-sectional study in Guatemala. H pylori seropositivity was defined as positivity for ≥ 4 antigens. Seropositivities for H bilis and H hepaticus were defined as positivity for ≥ 2 antigens. NAFLD was estimated using the Fatty Liver Index and the Hepatic Steatosis Index. Other conditions examined were obesity, central obesity, hypercholesterolemia, low HDL, diabetes and metabolic syndrome (MetSyn). Prevalence odds ratios (POR) and 95% confidence intervals (CIs) were estimated. RESULTS: No overall associations between H pylori,H hepaticus, or H bilis and NAFLD or related metabolic conditions were found. Seropositivity for H pylori antigens CagA and VacA and H hepaticus antigen HH0713 was each significantly associated with NAFLD, however. In addition, associations were observed between the H pylori antigens HyuA, HP1564, and UreA and specified metabolic conditions. CONCLUSIONS: While no overall associations between H pylori or Helicobacter species with NAFLD or related conditions were observed, some selected Helicobacter spp. antigens were associated with NAFLD. Further research is warranted to examine whether H. species are associated with any metabolic condition.