IgG ELISA Research Articles

Killed whole-cell Staphylococcus aureus formulation in Montanide ISA266 and Alum adjuvants: different vaccine formulations varied in the vaccine's potency and efficacy.

Immunotherapy can be a sensible alternative because invasive Staphylococcus aureus infection mortality, morbidity, and cost are still alarmingly high despite the development of multiple new medications to treat methicillin-resistant S. aureus infections. Herein, killed whole-cell Staphylococcus aureus was formulated in Montanide ISA266 and Alum adjuvants, and the potency and efficacy of the vaccine were studied. After the preparation of two kinds of whole-cell vaccine (bacterin and lysate), 20 µg of each vaccine candidate was formulated in Montanide ISA266 and Alum adjuvants, then subcutaneously injected in distinct groups. Blood samples were taken two weeks after each booster injection, and two booster shots were given at 2-week intervals. Sera were examined by ELISA for total IgG, isotypes (IgG1 and IgG2a), and cytokine production (IFN-γ and IL-4), respectively, to ascertain the kind of induced immune response. Experimental mice were challenged intraperitoneally with 5 × 108CFU of bacteria 2 weeks after their last immunization, and the mortality rate and bacterial load were measured. Both immunogens elicited strong humoral immune responses, producing antibodies that improved opsonic capability, IFN-γ, and IL-4 production and protectivity in response to the experimental challenge. Compared to other immunized groups, the lysate formulation with Montanide ISA266 produced a greater antibody titer and IgG1 isotype and showed the highest vaccine potency. Additionally, combining the whole-cell vaccine (bacterin and lysate) with the adjuvant Montanide ISA266 increased IFN-γ and IL-4 cytokines response and protection in the experimental challenge. These findings show that avoiding S. aureus infection using active vaccination with inactivated whole-cell vaccines (bacterin and lysate) may be a successful strategy. The type of adjuvant in the vaccine formulation is important and influences vaccine potency and efficacy.

P-185. Seroprevalence and Clinical Spectrum of Toxocara Infections: A Retrospective Study from a Tertiary Care Center in Lebanon

Abstract Background Toxocara is a prevalent zoonotic disease worldwide. It exhibits a broad spectrum of clinical manifestations, ranging from asymptomatic cases to severe conditions like myelitis and, in rare instances, fatal outcomes such as aortic thrombosis. Despite its prevalence, studies focusing on the seroprevalence, clinical manifestations, and laboratory findings of Toxocara in the MENA region are limited, highlighting a significant gap in research. Methods This is a retrospective, single-center cohort study conducted at the American University of Beirut Medical Center. The charts of 227 patients who were tested for Toxocara via ELISA IgG, presenting either as inpatients or outpatients from January 2002 to September 2022, were examined. Results The majority of the patients were middle-aged, generally exhibiting few co-morbidities as evidenced by 75% (75/100) of the study population being classified as “mild” on the Charlson co-morbidity index and only 16% (16/100) being immunosuppressed. 95.3% (61/64) of the patients were residents of Lebanon, most of whom were living in either Beirut or Mount Lebanon (22/59 (37.3%) and 20/59 (33.9%), respectively). 78.5% (62/79) of the patients presented with symptoms, and 67.7% (42/62) primarily complained of neurologic symptoms, mostly tingling or weakness in the extremities (37/43 (86%)). Asymptomatic patients were typically investigated for incidental eosinophilia (15/17 (88.2%)). Except for a lower overall age (44 vs 55, p=0.005), no statistically significant variable was detected between symptomatic and asymptomatic patients in terms of demographic characteristics. Among patients with Toxocara myelitis, most of the MRIs examined revealed enhancing (7/8 (87.5%)) longitudinal lesions (6/10 (60%)), mainly involving the thoracic levels of the spine (4/10 (40%)). Conclusion According to our center's experience, most patient who presented were symptomatic, mainly complaining of neurologic symptoms, with those diagnosed with Toxocara myelitis mainly showing longitudinal enhancing lesions involving the thoracic levels. Further studies are crucial to ascertain the actual prevalence and clinical presentations of Toxocara in the region. Disclosures All Authors: No reported disclosures

P-1312. MPox Seroprevalence and Seroincidence among People who Inject Drugs in the Mexico-U.S. Border Region

Abstract Background We assessed seroprevalence and seroincidence against orthopoxvirus (OPXV) among people who inject drugs (PWID) in the U.S./Mexico border region during the COVID-19 pandemic. Methods Between 09/21-11/23, PWID who lived in either San Diego (SD; N=129) or Tijuana (TJ; N=343) underwent a baseline and follow-up visit involving interviewer-administered surveys and venipuncture. Sera were batch tested at the U.S. Center for Disease Control and Prevention using an OPXV generic IgG ELISA using JYNNEOS® vaccine as the viral antigen. Optical densities (OD) ≥0.1 were conservatively considered seropositive. Mpox seroprevalence was assessed after excluding known sources of anti-OPXV antibodies Results Of 472 participants enrolled, median age was 45 (IQR:38-53), 72.9% were male (3.2% of whom reported sex with men), 11.3% had HIV and 25.8% were unhoused. Although no symptoms consistent with Mpox were reported during the study, 50 (10.6%) had seropositive results at baseline, follow-up or both. Baseline OPXV seroprevalence was 19.4% in SD and 3.2% in TJ, and Mpox seroincidence was 8.97 (95%CI:4.09-13.85) per 100 person-years in TJ vs. 1.81 (95%CI: 0.00-5.35) in SD, with a median of 5.8 months of follow-up (IQR: 4.53 – 6.31 months). Excluding participants who reported Mpox vaccination (N=3), smallpox vaccination (N=73), or those born prior to 1971 who could have received smallpox vaccination (N=140), likely Mpox seroprevalence was 7.8% (23/296), with 13/23 seronegative at baseline but seropositive at follow-up. Of these 13 seroconveters, 5 were female of whom 2 were HIV+. Conclusion These preliminary data suggest a undetected Mpox circulation in the Mexico-U.S. border region between 2022 and 2023 and that asymptomatic or mildly symptomatic Mpox infection may be more common than previously recognized. Although the clinical implications of these findings remain unclear, expansion of Mpox vaccination to PWID may be warranted. Disclosures David Smith, MD, MAS, Bayer: Advisor/Consultant|Gilead: Advisor/Consultant|Model Medicines: Advisor/Consultant|Red Queen Therapuetics: Advisor/Consultant

P-2326. The Under-recognized Burden of Lassa Virus Infection in Liberia

Abstract Background Lassa virus (LASV) is a persistent and lethal threat in endemic regions of West Africa. While severe disease due to Lassa fever (LF) is responsible for 5000-10,000 deaths annually, there is evidence of a wide spectrum of clinical manifestation of LASV infection. Methods The Coalition for Epidemic Initiative (CEPI) sponsored ENABLE study is a prospective, observational, cohort study of the prevalence and incidence of LASV infection and LF in five West African countries: Nigeria, Liberia, Sierra Leone, Guinea, and Benin. LASV seroprevalence was determined by LASV nucleoprotein (NP) IgG at entry (ReLASV NP IgG ELISA). Participants were assessed every 2 weeks in-person for fever with and without LF symptoms. Febrile events triggered LASV RT-PCR (RealStar Lassa Virus RT-PCR Altona 2.0). To assess for subclinical LASV infection, serum was collected every 6 months for 2 years from a subset (Infection Cohort) for LASV NP IgG. Results In Liberia, 5,005 were enrolled between April 2021 and December 2023; 25 withdrew before data collection. Of the remaining 4,980 participants, 1,018 were in the Infection Cohort and 591 of these provided samples at all 5 time points. At entry, 2,259 (45%) were LASV NP IgG positive. Entry LASV NP IgG seropositivity increased with age with the lowest prevalence among those 2-5 years (101 of 510, 20%, CI: 17-24%) rising to a peak among participants 30-39 years (322 of 564, 57%, CI: 53-61%). Of the 591 with serological results at all 5 timepoints, 314 (53%) were LASV NP IgG negative at study entry and 68 (22%) had at least a single change in serostatus from seronegative to seropositive. Over 24-months, 1,176 febrile events in 818 participants were identified and of these 10 (1%) had LASV RNA detected. Conclusion In this 24-month longitudinal study of individuals in a LASV hyperendemic area of West Africa, close to half were LASV seropositive at enrollment, and across two years of follow-up, 1 in 5 participants who were LASV seronegative at entry had observed seroconversion. However, only a small number of LF cases (n=14) were identified through symptom-driven testing dependent on fever. These findings indicate that in this region of Liberia LASV infection is common, increases during the first decades of life, but is rarely clinically detected. Disclosures Katie R. Mollan, MS, Gilead Sciences: Grant/Research Support|Ridgeback Biotherapeutics: Grant/Research Support

P-2152. Strongyloides stercoralis Seroprevalence among Solid Organ Transplant Candidates and Recipients in a Multicenter Hospital System in Arizona

Abstract Background Solid organ transplant (SOT) recipients have a higher risk of Strongyloidiasis associated complications. Universal screening for Strongyloides among SOT candidates and recipients is not well defined in the Southwest United States. We assessed our center’s seropositivity rate for pre- and post-SOT using Strongyloides Ab (strAb) test. Methods We conducted a multicenter, IRB-approved retrospective study. Data were collected from January 2019 to March 2024 for patients ≥18 years old before and after SOT. Most of the testing at our center was performed utilizing the Strongyloides ELISA IgG test kit, Gold Standard Diagnostics, Davis, CA, USA. Results There were 387 patients with a median age of 61 (range 24-85), the majority being white non-Hispanics, 221 (57%) and 241 (62%) males. There were 152 pre-SOT tests performed: kidney (61), liver (54), lung (17), and other (20), in contrast to the 292 post-SOT: kidney (153), liver (45), lung (41), heart (31), and others (22). The majority (248/387) of patients reported historical travel, with 154 (62%) to Strongyloides endemic regions. There were 269 pre-SOT serology, mostly (85%) positive for Epstein Barr virus (EBV), followed by cytomegalovirus (CMV) with 70%, 5% hepatitis C. There was one case of a patient with positive HTLV-I/II serology (negative by confirmatory testing). Of the 283 patients, 24% reported being born outside the US. Of the171 reported gastrointestinal symptoms at the time of strAb testing, 94% were post-SOT. There were 20/387 (5.2%) positive strAb, with 5.3% in pre-SOT and 4.1% in post-SOT. There were 3 cases with negative pre-SOT that seroconverted after transplantation. All of the 93 patients who underwent microscopic stool examinations tested negative. 17/20 patients with a positive strAb received treatment with ivermectin. There was one case of Hyperinfection Syndrome. There was no statistical association between demographics and aforementioned risks, and seropositivity. Conclusion The Strongyloides seropositivity rate in our cohort is comparable to other reports. No associated risk factor was identified. Therefore, universal screening may be needed in similar transplant cohorts, as undetected cases can have devastating outcomes. Disclosures Tirdad Zangeneh, DO, AiCuris.: Grant/Research Support

Optimization of Conditions for Expression of Dengue Serotype 2 EDIII Protein in Escherichia coli and Immune Responses of Adjuvant-Free EDIII Ferritin Nanoparticles Against Dengue Virus in BALB/c Mice.

Self-assembling ferritin nanoparticle technology is a widely used vaccine development platform for enhancing the efficacy of subunit vaccines by displaying multiple antigens on nanocages. The dengue virus (DENV) envelope domain III (EDIII) protein, the most promising antigen for DENV, has been applied in vaccine development, and it is essential to evaluate the relative immunogenicity of the EDIII protein and EDIII-conjugated ferritin to show the efficiency of the ferritin delivery system compared with EDIII. In this study, we optimized the conditions for the expression of the EDIII protein in E. coli, protein purification, and refolding, and these optimization techniques were applied for the purification of EDIII ferritin nanoparticles. Thus, purified DENV2 EDIII and EDIII human ferritin heavy chain nanoparticles were immunized intramuscularly into BALB/c mice without an adjuvant, and the immunogenicity was analyzed using IgG ELISA and a serum-neutralizing assay. Purified, properly refolded, aggregate-free EDIII and EDIII ferritin proteins were obtained, and ferritin nanoparticles were identified using an electron microscope. By analyzing the immunogenicity of mouse serum, EDIII ferritin generated significantly higher IgG responses and neutralizing activity than EDIII-immunized mice. The IgG ELISA results confirmed that EDIII ferritin can induce a significantly higher IgG titer (O.D.:1.8) than EDIII (O.D.:0.05). Furthermore, EDIII ferritin produced a neutralizing titer of 1:68, whereas EDIII protein produced an average titer of 1:16, which is the serum dilution that inhibited 90% of the viruses. The longevity of the immune responses was analyzed using the serum obtained 2 months after the final immunization, and the results confirmed that EDIII ferritin induced constant immunity throughout the period.

Evaluating diagnostic performance: A comparative analysis of cell-free DNA and serological test in hepatic cystic Echinococcosis.

Cystic Echinococcosis (CE) is a zoonotic disease caused by Echinococcus granulosus sensu lato. Diagnosing CE primarily relies on imaging techniques, and there is a crucial need for an objective laboratory test to enhance the diagnostic process. Today, cell-free DNAs (cfDNAs) have gained importance regarding their biomarker potential. This study aims to investigate the diagnostic capabilities of different cfDNA targets (Echinococcus-specific repeat sequences (mgs-4 and mgs-12) and partial fragment of repetitive sequence (EG1 Hae III)) and evaluate their diagnostic effectiveness when compared to a frequently used commercial E.granulosus-specific IgG ELISA. Seventy-six confirmed hepatic CE patients and healthy controls were included in the study. The EG1 Hae III region was assessed using nested PCR, whereas real-time PCR was employed to investigate other cfDNA targets. Analysis of the cfDNA-targeted tests indicated that mgs-4 demonstrated the highest diagnostic efficacy in distinguishing CE patients from healthy controls, achieving a sensitivity of 60.5% (p = 0.002). Combining ELISA with the mgs-4 target led to an increased sensitivity of 72.4% for distinguishing between CE patients and the control group. The sensitivity rates for ELISA and the three cfDNA targets varied among the groups. Active CE patients showed sensitivity rates of 52.9%, 52.9%, 23.5%, and 52.9% for ELISA, mgs-4, mgs-12, and EG1 Hae III assays, respectively. In contrast, inactive cyst patients displayed sensitivity rates of 21.4%, 66.7%, 19%, and 42.9% for the corresponding assays. The mgs-4, either alone or in combination with ELISA, demonstrated notably higher sensitivity values for CE diagnosis in all group comparisons compared to serology.

Establishing a baseline titer for reporting immunoglobulin M seropositivity of diagnosis of scrub typhus in our region

Abstract Background: Clinical diagnosis of scrub typhus an agent of undifferentiated fever caused by Orientia tsutsugamushi often rests on serological (immunoglobulin [Ig] M enzyme-linked immunosorbent assay [ELISA]) test. This assay needs a baseline titer in the region to serve to be used as a reference value. Owing to the lack of studies in our area, the present study aims to bridge the gap of data of a baseline titer value for reporting IgM ELISA for O. tsutsugamushi. Materials and Methods: The study was conducted in a prospective manner in Bhubaneswar, Odisha, wherein a period of 1 year, 150 serum samples of patients who were hospitalized with a suspicion of scrub typhus and 200 serum samples from healthy blood donors were included. The IgM ELISA was performed by Scrub Typhus Detect™ IgM ELISA system, InBios International Inc. kit. IgG ELISA was performed on 100 healthy blood donor samples by Scrub Typhus Detect™ IgM ELISA system, InBios International Inc. kit. Statistical Analysis: The optical density (OD) obtained as the result of ELISA study was entered into MS Excel and cutoff was determined by SPSS software. Results: The cutoff value for IgM was 0.6 by receiver operating characteristic curve analysis with a sensitivity and specificity of 92.16% and 98.01%, respectively. Similarly, cutoff OD of 2.1 for IgG ELISA was determined in our area. Conclusion: Considering the need for a cost-effective serological method and unavailability of paired sera, these cutoffs can be used in our area and surrounding nearby areas of Eastern part of India as a reference value for reporting of scrub typhus.

Immunogenicity, safety, and reactogenicity of concomitant administration of the novavax vaccine against Omicron XBB.1.5 (NVX-CoV2601) and a 20-valent pneumococcal conjugate vaccine in adults aged ≥60 years: A randomised, double-blind, placebo-controlled, non-inferiority trial.

There is conflicting evidence as to whether the combined administration of two vaccines can lead to poorer immunogenicity and reactogenicity. The co-administration of the Omicron-adapted COVID-19 vaccine from Novavax (NVX-CoV2601) and a 20-valent pneumococcal conjugate vaccine (PCV20) has not been previously investigated. In this randomised, double-blind, placebo-controlled, non-inferiority trial, immunocompetent participants aged ≥60 years were randomised in a 1:1:1:1 ratio to four groups: NVX-CoV2601 plus PCV20 (combination group); NVX-CoV2601 plus placebo (NVX-only group); PCV20 plus placebo (PCV20-only group); or placebo plus placebo (placebo group). The primary outcome was Omicron-specific anti-spike protein IgG ELISA units at day 28 in the combination group compared with the NVX-only group. Non-inferiority was established if the lower limit of the two-sided 95% CI of the geometric mean titre ratio was above the non-inferiority margin of 0.67. Secondary outcomes included anti-pneumococcal capsular polysaccharide (PCP) IgG ELISA units. Solicited local and systemic adverse events were collected for 7 days after vaccination. This study was registered with ClinicalTrials.gov, number NCT05767606, and the EU Clinical Trials Register, EudraCT number 2022-004118-12. All 256 randomised participants completed the study. The baseline characteristics were similar in the four groups. Overall, the median age was 64 (IQR 61 to 69) and 105 (41%) of 256 were male. At day 28, the geometric mean anti-spike protein IgG ELISA units were 534U/mL (95% CI 432-660) in the combination group and 556U/mL (95% CI 460-672) in the NVX-only group, resulting in a geometric mean titre ratio of 0.96 (95% CI 0.73-1.27), thereby meeting the criteria for non-inferiority. Anti-PCP IgG ELISA units at day 28 were 507U/mL (95% CI 416-619) in the combination group and 592U/mL (95% CI 485-723) in the PCV20-only group. Local and systemic reactogenicity was similar in the three active treatment groups. No safety concerns or serious adverse events were observed. Immunogenicity following co-administration of NVX-CoV2601 with PCV20 was non-inferior to administration of NVX-CoV2601 alone. Given the similar safety and reactogenicity profile, our findings may help to overcome concerns about concomitant vaccination and pave the way for combination vaccines. Novavax.

Diagnosis of West Nile virus infections: Evaluation of different laboratory methods.

The diagnosis of West Nile virus (WNV) is challenging due to short-term and low-level viremia, flavivirus cross-reactivity, and long immunoglobulin M (IgM) persistence. To evaluate different methods for WNV detection [reverse transcription-polymerase chain reaction (RT-PCR), IgM/IgG antibodies, IgG avidity] in serum, cerebrospinal fluid (CSF), and urine samples of patients with confirmed WNV infection. The study included patients with confirmed WNV neuroinvasive infection (n = 62), asymptomatic WNV seropositive individuals (n = 22), and individuals with false-positive WNV IgM antibodies (n = 30). WNV RNA was detected using RT-PCR. A commercial ELISA was used to detect WNV IgM/IgG antibodies with confirmation of cross-reactive samples using a virus neutralization test (VNT). IgG-positive samples were tested for IgG avidity. The WNV-RNA detection rates were significantly higher in the urine (54.5%)/serum (46.4%) than in CSF (32.2%). According to the sampling time, the WNV-RNA detection rates in urine collected within 7 days/8-14/≥ 15 days were 29.4/66.6/62.5% (P = 0.042). However, these differences were not observed in the CSF. The median RT-PCR cycle threshold values were significantly lower in urine (32.5, IQR = 28-34) than in CSF (34.5, IQR = 33-36). The frequency of positive WNV IgM and IgG significantly differed according to the sampling time in serum but not in CSF. Positive IgM/IgG antibodies were detected in 84.3/9.3% of serum samples collected within 7 days, 100/71.1% of samples collected 8-14, and 100% samples collected after ≥ 15 days. Recent WNV infection was confirmed by low/borderline avidity index (AI) in 13.6% of asymptomatic individuals. A correlation between ELISA and AI was strong negative for IgM and strong positive for IgG. No significant correlation between ELISA IgG and VNT was found. The frequency of WNV RNA and antibody detection depends on the sampling time and type of clinical samples. IgG avidity could differentiate recent WNV infections from long-persisting IgM antibodies.

Recombinant Anti-PF4 Antibodies Derived from Patients with Vaccine-Induced Immune Thrombocytopenia and Thrombosis (VITT) Facilitate Research and Laboratory Diagnosis of VITT.

Adenoviral vector-based vaccines against COVID-19 rarely cause vaccine-induced immune thrombocytopenia and thrombosis (VITT), a severe adverse reaction caused by IgG antibodies against platelet factor 4 (PF4). To study VITT, patient samples are crucial but have become a scarce resource. Recombinant antibodies (rAbs) derived from VITT patient characteristic amino acid sequences of anti-PF4 IgG are an alternative to study VITT pathophysiology. Amino acid sequences of the variable region of immunoglobulin light and heavy chain of anti-PF4 IgG derived from VITT patients were obtained by mass spectrometry sequencing and rAbs were synthetized by reverse-engineering. Six different rAbs were produced: CR23003, CR23004, and CR23005 (from a patient vaccinated with Jcovden, Johnson & Johnson-Janssen (Beerse, Belgium)), CR22046, and CR22050 and CR22066 (from two different patients vaccinated with Vaxzevria, AstraZeneca (Cambridge, UK)). These rAbs were further characterized using anti-PF4 and anti-PF4/heparin IgG ELISAs, rapid anti-PF4 and anti-PF4/polyanion chemiluminescence assays, and PF4-induced platelet activation assay (PIPA) and their capacity to induce procoagulant platelets. rAbs bound to PF4 alone, but not to PF4/polyanion complexes in rapid chemiluminescence assays. Chemiluminescence assays and both anti-PF4 IgG and anti-PF4 IgG/heparin ELISA showed concentration-dependent PF4 binding of all six rAbs, however, with different reactivities among them. PIPA showed a similar, concentration-dependent platelet activation pattern. rAbs varied in their reactivity and the majority of the tested rAbs were able to induce procoagulant platelets. The six rAbs derived from VITT patients reflect VITT-typical binding capacities and the ability to activate platelets. Therefore, these rAbs offer an attractive new option to study VITT pathophysiology.

Technical evaluation of the InBios Strongy Detect IgG ELISA assay for the diagnosis of Strongyloides stercoralis infection

Abstract Background Strongyloidiasis is a neglected tropical disease (NTD) caused by the soil-transmitted helminth Strongyloides stercoralis, recently included in the 2030 targets of the World Health Organization for the control of STHs. Assessment of infection prevalence is fundamental for decision-making about the implementation of control programs, but diagnostic assays to be applied in such context require evaluation. Seroassays based on recombinant antigens, which could be produced in a standardized and scalable manner, are particularly appealing for use in control programs. In this study, we performed a technical evaluation of the InBios Strongy Detect IgG ELISA, based on recombinant antigens NIE and SsIR, which has shown promising for field use. Methods A total of 46 plasma samples from Ethiopian children were used for this technical evaluation. Repeatability was evaluated on duplicate samples per plate, on four plates per day for 3 days using Bland–Altman plots, analysis of residuals, and variance components analysis. Three samples were selected for evaluation of the uniformity of test results within a single plate (border effect) by two-sided t-test. Correlation between samples and internal ELISA positive controls was analyzed using Spearman’s rank correlation coefficient applied on the results of 777 samples analyzed with the assay in a previous field-based study. Results Within and between plate residuals ranged from −0.05 to + 0.05 and −0.1 to + 0.1, respectively. Total variance was estimated at 0.327; 99.6% of variation could be attributed to the samples. There was no systematic border effect and a negligible correlation between positive internal control and samples results (R2 = 0.213; p < 0.001). Conclusion The results obtained in this study, in highly controlled conditions, point toward the InBios Strongy Detect IgG ELISA assay being reproducible, with no systematic border effect. These results encourage further assay’s development and evaluation for use in practice, including determination of preset cutoff values for positivity, which is currently not provided. Graphical Abstract

Modeling and optimization of culture media for recombinant Helicobacter pylori vaccine antigen HpaA.

H. pylori (Helicobacter pylori) infection represents a significant global health concern, exacerbated by the emergence of drug-resistant strains resulting from conventional antibiotic treatments. Consequently, the development of vaccines with both preventive and therapeutic properties has become crucial in addressing H. pylori infections. The H. pylori adhesin protein HpaA has demonstrated strong immunogenicity across various adjuvants and dosage forms, positioning it as a key candidate antigen for recombinant subunit vaccines against H. pylori. Optimizing fermentation culture conditions is an effective strategy to enhance product yield and lower production costs. However, to date, there has been no systematic investigation into methods for improving the fermentation yield of HpaA. Enhancing the fermentation medium to increase HpaA yield holds significant potential for application and economic benefits in the prevention and detection of H. pylori infection. To achieve a stable and high-yielding H. pylori vaccine antigen HpaA, this study constructed recombinant Escherichia coli expressing HpaA. The impact of fermentation medium components on the rHpaA yield was assessed using a one-factor-at-a-time approach alongside Plackett-Burman factorial experiments. Optimal conditions were effectively identified through response surface methodology (RSM) and artificial neural network (ANN) statistical computational models. The antigenicity and immunogenicity of the purified rHpaA were validated through immunization of mice, followed by Western Blot analysis and serum IgG ELISA quantification. Glucose, yeast extract, yeast peptone, NH4Cl and CaCl2 all contributed to the production of rHpaA, with glucose, yeast extract, and NH4Cl demonstrating particularly significant effects. The artificial neural network linked genetic algorithm (ANN-GA) model exhibited superior predictive accuracy, achieving a rHpaA yield of 0.61g/L, which represents a 93.2% increase compared to the initial medium. Animal immunization experiments confirmed that rHpaA possesses good antigenicity and immunogenicity. This study pioneers the statistical optimization of culture media to enhance rHpaA production, thereby supporting its large-scale application in H. pylori vaccines. Additionally, it highlights the advantages of the ANN-GA approach in bioprocess optimization.

Exposure to Dengue Virus During Pregnancy: Incidence and Impact on Maternal and Child Outcomes.

Dengue virus (DENV) is the most common arbovirus globally, with its incidence growing dramatically in recent decades. Although the effects of DENV infection during pregnancy are unclear, reported associations with adverse health outcomes include miscarriage, prematurity, and low birth weight. In this study, we used an IgG ELISA to identify mothers exposed to DENV during pregnancy by testing samples obtained from a previous study that followed a cohort of pregnant women in Kenya to investigate parasitic infections during pregnancy. We compared adverse pregnancy and infant health outcomes between seronegative mothers and those who seroconverted. Of the 289 participants tested for DENV exposure during pregnancy, we estimated that ∼12 women (4%) would have been exposed to DENV during their gestation period. However, we found that 34 mothers (11.8%) had been exposed to DENV during pregnancy. None of these mothers were hospitalized during pregnancy because of severe DENV infection, suggesting that many may have undergone asymptomatic seroconversion. The demographic risk factors of socioeconomic status, education level, bed net use, and maternal age were not associated with mild or asymptomatic DENV in pregnancy. Although mild or asymptomatic DENV during pregnancy was not associated with late prematurity, reduced postnatal childhood developmental measures, or adverse maternal pregnancy outcomes, we observed an increased risk of low birth weight. The larger-than-expected burden of DENV in pregnancy in this coastal Kenyan cohort and the observed potential risk of low birth weight provide evidence that a more comprehensive study is warranted to fully understand DENV infection during pregnancy.

Serological Evidence of Hantavirus in Bats from the Brazilian Atlantic Forest: An Investigation of Seroreactivity and Cross-Reactivity of Neotropical Bat Samples Using Nucleoproteins of Rodent- and Bat-Borne Hantaviruses.

Hantaviruses are zoonotic pathogens associated with severe human diseases such as hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome. Despite the extensive study of rodent-borne hantaviruses, research on bat-associated hantaviruses remains limited. This study aimed to investigate the seroprevalence and cross-reactivity of neotropical bat samples with rodent- and bat-associated recombinant hantavirus nucleoproteins (rNPs) to improve hantavirus surveillance in the Brazilian Atlantic Forest. The studied bat population consisted of 336 blood samples collected over nearly a decade in five Brazilian states (Bahia, Rio de Janeiro, Santa Catarina, Paraná, and Minas Gerais). Antibodies were detected using IgG ELISA assays with rNPs from bat-borne Mobatvirus xuansonense (XSV) and Loanvirus brunaense (BRNV) and the rodent-borne hantaviruses Orthohantavirus andesense (ANDV) and Orthohantavirus seoulense (SEOV). Results indicated a higher seroprevalence for the BRNV rNP (36.6%) compared to ANDV (7.4%), SEOV (5.7%), and XSV (0.6%). The high sensitivity of the BRNV rNP and the cross-reactivity observed with the ANDV rNP, the main protein used for serological tests in the Americas, indicates that BRNV rNP is a better antigen for the accurate detection of antibodies against hantaviruses in Brazilian bats. These findings underscore the presence of unknown hantaviruses antigenically similar to BRNV in Brazilian bat populations and highlight the urgent need for identifying better antigens for comprehensive hantavirus monitoring in bats.

Chronic Strongyloides stercoralis infection in Fijian migrants to the UK.

Introduction. Strongyloides stercoralis, the human threadworm, is a parasitic nematode with global distribution, estimated to infect over 600 million people. Chronic infection is often asymptomatic, but hyperinfection and dissemination syndromes can occur in the immunosuppressed with high case fatality rates. Whilst strongyloidiasis is endemic in Fiji, its prevalence in Fijian migrant groups in the UK is unknown.Gap Statement. No previous studies have been conducted on the prevalence of Strongyloides and other gastrointestinal parasites (GIPs) in Fijian migrants to the UK.Aim. We conducted a cross-sectional study of the prevalence of GIPs in a Fijian migrant population.Methodology. Participants completed a questionnaire on residence, travel and clinical symptoms and were asked to provide a serum sample for S. stercoralis IgG ELISA, venous blood samples for eosinophil count and a faecal sample for charcoal culture, multiplex real-time PCR (rtPCR) and microscopy after formalin-ethyl acetate concentration. Sequencing was performed on pooled Strongyloides larvae for nuclear 18S rRNA hyper-variable regions (HVRs) I and IV.Results. A total of 250 participants (94% male) with median (range) age 37 (20-51) years entered the study, 15 (1-24) years since leaving Fiji. S. stercoralis IgG ELISA was positive in 87/248 (35.1 %) and 14/74 (18.9 %) had a GIP detected in faeces. This included 7/74 (9.5 %) with Strongyloides and 5/74 (6.8 %) with hookworms. Dermatological symptoms were more common in those with Strongyloides, and eosinophilia (>0.5×109 cells per litre) was present in 55.6% of those with positive S. stercoralis IgG. rtPCR was the most sensitive faecal diagnostic test for Strongyloides and hookworms in faeces. Sequences of nuclear 18S rRNA for HVRs I and IV confirmed the presence of S. stercoralis.Conclusion. This first cross-sectional study in Fijian migrants found a high rate of chronic infection with GIPs, particularly S. stercoralis. Faecal microscopy was insensitive compared to charcoal culture, rtPCR or serology, demonstrating the importance of specialist parasitological tests when investigating people with a suspected chronic infection. Our study highlights an overlooked burden of strongyloidiasis in the UK and has implications for screening and treatment programmes in Fiji and for migrants from Fiji.

Seroepidemiological study of Epstein-Barr virus infection in reproductive age women in Bulgaria

Abstract Background At the present stage, studies on the involvement of Epstein-Barr virus (EBV) in neonatal pathology are rare. Establishing seroprevalence among women of reproductive age has important epidemiological significance in order to determine the risk of infection during pregnancy and intrauterine infection of the fetus. The aim of the study is to determine the seroepidemiological status of EBV in reproductive age women. Methods We conducted a prospective seroepidemiological study in which 96 healthy women of reproductive age (18 to 49 years) were tested during the period December 2023 - January 2024 in Medical Center “Clinical Institute for Reproductive Medicine”- Pleven, Bulgaria. Participants were surveyed on a voluntary basis. The exclusion criteria were: presence of autoimmune disorder, immunosuppressive states, malignancy and populations at risk such as persons under 18 years of age and pregnant women. The presence of specific antibodies were detected using a standardized Anti-EBV VCA IgM, Anti-EBV VCA IgG and Anti-EBV EBNA-1 IgG ELISA kits. The demographic and anamnestic data were collected for each participant in Case Report Form. Results The average age of the women was 34.41±5.44 years. Dominating were women with higher education (76.04%) living in urban areas (91.67%). The results of the seroepidemiological study of markers for EBV show that 98.96% of women of reproductive age are seropositive. There was no significant correlation between the presence of antibodies and socio-demographic indicators (living area, education and number of family members). Conclusions The results of this study imply that only 1.04% of the reproductive age women in Pleven region are susceptible to infection with EBV. Although interpretation of the serological status indicates a low risk of primary infection, active monitoring of high-risk groups pregnant women is necessary because of the possibility of EBV reactivation. Key messages • On-going monitoring of the seroepidemiological EBV status in reproductive age women is necessary to determining the possibility of EBV reactivation and to reduce the teratogenic risk of the infection. • Seroepidemiological surveillance of markers for EBV infection in reproductive age women is part of effective preventive measures at the public health level.

A comparative study of traditional and molecular diagnostic methods for detection of gastrointestinal parasites in Nepalese migrants to the UK

We evaluated the results of examining a single faecal sample for gastrointestinal parasites (GIP) using a combination of traditional methods with multiplex qPCR for helminths and protozoa, compared to a reference standard of examining three faecal samples from each person using traditional diagnostic methods alone. Three faecal samples were collected at weekly intervals from 596 healthy Nepalese men. Each sample underwent formalin-ethyl acetate (FEA) concentration and light microscopy, and charcoal culture. The combined results of these investigations for all three stool samples were designated the reference standard. The first sample was also analysed using a multiplex TaqMan™ qPCR assay, screening for five helminths and three protozoa. We compared sensitivity and specificity of analysing the first faecal sample with qPCR alone, or a hybrid approach combining qPCR with traditional methods, to the reference standard. Additionally, a serum sample was taken from each participant for Strongyloides stercoralis IgG ELISA. The reference standard identified 139 GIP infections in 133 (22.3%) participants. Use of qPCR alone in one stool identified 176 infections in 147 (24.8%) participants, rising to 187 infections in 156 (26.3%) when combined with FEA microscopy and charcoal culture. The sensitivity of this latter hybrid approach was 100% for Strongyloides spp., 90.9% for Trichuris trichiura, 86.8% for hookworm species and 75% for Giardia duodenalis compared to the reference standard. The hybrid approach increased the detected prevalence of G. duodenalis by 4.5% (27 cases) overall, T. trichiura by 2.9% (17 cases), Strongyloides spp. by 1% (6 cases), and hookworm by 0.5% (3 cases), compared to the reference standard. Examination of a single faecal sample using qPCR alone showed superior or equivalent sensitivity to traditional methods for most GIP infections when both were compared to the reference standard. Combining molecular and traditional methods to analyse a single stool improved the detection rate for most studied parasites. This approach has value in settings where repeated sampling and/or faecal culture for helminths is impractical, but molecular diagnostics are available.

Molecular Diagnosis and Antibodies Tests for Brucellosis Detected in Aborted and Apparently Healthy Sheep and Goats in and around Al Bayda City, Libya

The goal of the current research was to evaluate antigen and antibody reaction’s method in comparison with real time PCR for detection of brucella agent in sheep and goat. Therefore, seven hundreds and twenty-eight blood and milk samples were isolated from sheep and goats apparently healthy and suspected brucellosis brought to the Veterinary Clinical Complex from areas in and around Al Bayda, Libya. Blood samples were subjected to serological tests including Rose Bengal Plate Technique (RBPT), Enzyme-linked immunosorbent test (ELISA) for detection of IgA, IgM and also milk ring test to detect the antibodies of brucella agent. Also, the samples subjected by Real Time PCR to determine the presence of bcsp31 gene (Brucella gen). Out of the total examined samples (133) 18.26%, (139) 19.09%, (3) 0.41% and (126) 17.3% were tested positive by RBPT, ELISA IgG, IgM and Real Time PCR respectively. specificity and Sensitivity of RBPT compared with real- time PCR were 98.9% and 79.59% respectively. While, sensitivity and specificity of ELISA for IgG with Real Time PCR were 79.59% and 100% respectively, while, for ELISA IgM were 6.12% and 100% respectively. In conclusion, we suggest using Real Time PCR as a supplementary test in the diagnosis of brucella disease and as a confirmatory test in suspicious cases.

Anti-slit diaphragm antibodies on kidney biopsy identify pediatric patients with steroid-resistant nephrotic syndrome responsive to second-line immunosuppressants

Podocytopathies represent a group of glomerular disorders associated with minimal changes (MC) or focal segmental glomerulosclerosis (FSGS) lesion patterns at biopsy and heterogeneous responses to steroids. Anti-nephrin antibodies were previously found in such patients, suggesting an autoimmune form of podocytopathy. High resolution confocal microscopy on kidney biopsies of a cohort of 128 pediatric patients revealed localization of IgG along the slit diaphragm in 30% of patients with MC and 25% of those with FSGS, but not in other lesion patterns. Anti-nephrin IgG ELISA assay in the serum and stimulated emission depletion microscopy of kidney biopsies showed IgG-nephrin co-localization only in 77.8% of cases. Similar observations were obtained in a cohort of 48 adult patients with MC or FSGS at kidney biopsy, where IgG-nephrin colocalization was only 44.4%, suggesting the existence of autoantibodies binding to other slit proteins. Patients with anti-slit antibodies showed nephrotic syndrome at onset in 94.4% of cases. Patients with primary steroid-resistance had anti-slit antibodies in 27%, while those with secondary steroid-resistance in 87.5% of cases, irrespective of the histopathological lesion pattern. Steroid-resistant patients with anti-slit antibodies responded to second-line immunosuppressants in 92.3% vs. only 20% of patients that were anti-slit negative. No patient with anti-slit antibodies developed kidney failure vs. 51.7% of those negative for antibodies (66.7% with a genetic cause and 41.2% with a non-genetic cause). Thus, the detection of anti-slit antibodies can identify patients with an autoimmune podocytopathy responsive to treatment with second-line immunosuppressants, irrespective of the histopathological lesion pattern at biopsy.