COVID-19-neutralizing antibodies predict disease severity and survival.

Coronavirus disease 2019 (COVID-19) exhibits variable symptom severity ranging from asymptomatic to life-threatening, yet the relationship between severity and the humoral immune response is poorly understood. We examined antibody responses in 113 COVID-19 patients and found that severe cases resulting in intubation or death exhibited increased inflammatory markers, lymphopenia, pro-inflammatory cytokines, and high anti-receptor binding domain (RBD) antibody levels. Although anti-RBD immunoglobulin G (IgG) levels generally correlated with neutralization titer, quantitation of neutralization potency revealed that high potency was a predictor of survival. In addition to neutralization of wild-type SARS-CoV-2, patient sera were also able to neutralize the recently emerged SARS-CoV-2 mutant D614G, suggesting cross-protection from reinfection by either strain. However, SARS-CoV-2 sera generally lacked cross-neutralization to a highly homologous pre-emergent bat coronavirus, WIV1-CoV, which has not yet crossed the species barrier. These results highlight the importance of neutralizing humoral immunity on disease progression and the need to develop broadly protective interventions to prevent future coronavirus pandemics.

Compromised Humoral Functional Evolution Tracks with SARS-CoV-2 Mortality.

The urgent need for an effective SARS-CoV-2 vaccine has forced development to progress in the absence of well-defined correlates of immunity. While neutralization has been linked to protection against other pathogens, whether neutralization alone will be sufficient to drive protection against SARS-CoV-2 in the broader population remains unclear. Therefore, to fully define protective humoral immunity, we dissected the early evolution of the humoral response in 193 hospitalized individuals ranging from moderate to severe. Although robust IgM and IgA responses evolved in both survivors and non-survivors with severe disease, non-survivors showed attenuated IgG responses, accompanied by compromised Fcɣ receptor binding and Fc effector activity, pointing to deficient humoral development rather than disease-enhancing humoral immunity. In contrast, individuals with moderate disease exhibited delayed responses that ultimately matured. These data highlight distinct humoral trajectories associated with resolution of SARS-CoV-2 infection and the need for early functional humoral immunity.

Distinct Early Serological Signatures Track with SARS-CoV-2 Survival.

As SARS-CoV-2 infections and death counts continue to rise, it remains unclear why some individuals recover from infection, whereas others rapidly progress and die. Although the immunological mechanisms that underlie different clinical trajectories remain poorly defined, pathogen-specific antibodies often point to immunological mechanisms of protection. Here, we profiled SARS-CoV-2-specific humoral responses in a cohort of 22 hospitalized individuals. Despite inter-individual heterogeneity, distinct antibody signatures resolved individuals with different outcomes. Although no differences in SARS-CoV-2-specific IgG levels were observed, spike-specific humoral responses were enriched among convalescent individuals, whereas functional antibody responses to the nucleocapsid were elevated in deceased individuals. Furthermore, this enriched immunodominant spike-specific antibody profile in convalescents was confirmed in a larger validation cohort. These results demonstrate that early antigen-specific and qualitative features of SARS-CoV-2-specific antibodies point to differences in disease trajectory, highlighting the potential importance of functional antigen-specific humoral immunity to guide patient care and vaccine development.

The Identification of Enteric Fever-Specific Antigens for Population-Based Serosurveillance.

BACKGROUND: Enteric fever, caused by Salmonella enterica serovars Typhi and Paratyphi A, is a major public health problem in low- and middle-income countries. Moderate sensitivity and scalability of current methods likely underestimate enteric fever burden. Determining the serological responses to organism-specific antigens may improve incidence measures. METHODS: Plasma samples were collected from blood culture-confirmed enteric fever patients, blood culture-negative febrile patients over the course of 3 months, and afebrile community controls. A panel of 17 Salmonella Typhi and Paratyphi A antigens was purified and used to determine antigen-specific antibody responses by indirect ELISAs. RESULTS: The antigen-specific longitudinal antibody responses were comparable between enteric fever patients, patients with blood culture-negative febrile controls, and afebrile community controls for most antigens. However, we found that IgG responses against STY1479 (YncE), STY1886 (CdtB), STY1498 (HlyE), and the serovar-specific O2 and O9 antigens were greatly elevated over a 3-month follow up period in S. Typhi/S. Paratyphi A patients compared to controls, suggesting seroconversion. CONCLUSIONS: We identified a set of antigens as good candidates to demonstrate enteric fever exposure. These targets can be used in combination to develop more sensitive and scalable approaches to enteric fever surveillance and generate invaluable epidemiological data for informing vaccine policies. CLINICAL TRIAL REGISTRATION: ISRCTN63006567.

Evaluation of a point-of-care multiplex immunochromatographic assay for the diagnosis of typhoid: results from a retrospective diagnostic accuracy study.

There is a clear medical need for an accurate diagnostic test for typhoid that can be performed at point of care. Two antigens (lipopolysaccharide [LPS] and hemolysin E [HlyE]) have recently been identified that can distinguish typhoid from other bacterial infections. Here, we present the results of a diagnostic accuracy study of the Dual Path Platform (DPP) Typhoid assay (Chembio) that detects IgA to both LPS and HlyE using blood culture as the reference standard. This was a retrospective, observational, laboratory study conducted at the Aga Khan University research laboratory, Pakistan, to evaluate the sensitivity and specificity of the DPP Typhoid assay, using archived frozen serum samples collected during a previous typhoid diagnostic accuracy study (NCT04801602). The sensitivity, specificity, and accuracy (area under the receptor operating characteristics curve [AUC]) were then assessed using the manufacturer's and Youden's optimal thresholds. In total, 385 samples were included in the analysis. Using the manufacturer's thresholds, the sensitivity, specificity, and AUC were 97.8% (95% confidence interval [CI] 94.6-99.2), 65.3% (95% CI 58.5-71.6), and 81.5% (95% CI 75.5-85.3), respectively. At Youden's optimal threshold, the overall sensitivity of the DPP Typhoid assay was 89.7% and the specificity was 82.2%. In latent class modeling compared with other nine rapid diagnostic tests evaluated from the same cohort sample, the DPP Typhoid assay demonstrated the highest balanced accuracy (89.2%). The DPP Typhoid assay demonstrated a high diagnostic accuracy for typhoid fever. However, further adjustment to new thresholds is recommended to enhance its performance capabilities. IMPORTANCE: Currently available diagnostic tests for typhoid have several limitations, including low sensitivity and specificity. Dual Path Platform Typhoid assay is a multiplex rapid test that detects IgA antibodies to lipopolysaccharide and hemolysin E antigen. It is considered to have high sensitivity and specificity, and its results were found to be highly correlated with ELISA results. However, very few studies have been conducted to evaluate this test and limited information about the accuracy of this test is present. Hence, this study evaluated the new typhoid test.

Distinguishing Severe Acute Respiratory Syndrome Coronavirus 2 Persistence and Reinfection: A Retrospective Cohort Study.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reinfection is poorly understood, partly because few studies have systematically applied genomic analysis to distinguish reinfection from persistent RNA detection related to initial infection. We aimed to evaluate the characteristics of SARS-CoV-2 reinfection and persistent RNA detection using independent genomic, clinical, and laboratory assessments. METHODS: All individuals at a large academic medical center who underwent a SARS-CoV-2 nucleic acid amplification test (NAAT) ≥45 days after an initial positive test, with both tests between 14 March and 30 December 2020, were analyzed for potential reinfection. Inclusion criteria required having ≥2 positive NAATs collected ≥45 days apart with a cycle threshold (Ct) value <35 at repeat testing. For each included subject, likelihood of reinfection was assessed by viral genomic analysis of all available specimens with a Ct value <35, structured Ct trajectory criteria, and case-by-case review by infectious diseases physicians. RESULTS: Among 1569 individuals with repeat SARS-CoV-2 testing ≥45 days after an initial positive NAAT, 65 (4%) met cohort inclusion criteria. Viral genomic analysis characterized mutations present and was successful for 14/65 (22%) subjects. Six subjects had genomically supported reinfection, and 8 subjects had genomically supported persistent RNA detection. Compared to viral genomic analysis, clinical and laboratory assessments correctly distinguished reinfection from persistent RNA detection in 12/14 (86%) subjects but missed 2/6 (33%) genomically supported reinfections. CONCLUSIONS: Despite good overall concordance with viral genomic analysis, clinical and Ct value-based assessments failed to identify 33% of genomically supported reinfections. Scaling-up genomic analysis for clinical use would improve detection of SARS-CoV-2 reinfections.

Seroprevalence of Vibrio cholerae in Adults, Haiti, 2017.

In Haiti in 2017, the prevalence of serum vibriocidal antibody titers against Vibrio cholerae serogroup O1 among adults was 12.4% in Cerca-la-Source and 9.54% in Mirebalais, suggesting a high recent prevalence of infection. Improved surveillance programs to monitor cholera and guide public health interventions in Haiti are necessary.

Seroincidence of Enteric Fever, Juba, South Sudan.

We applied a new serosurveillance tool to estimate typhoidal Salmonella burden using samples collected during 2020 from a population in Juba, South Sudan. By using dried blood spot testing, we found an enteric fever seroincidence rate of 30/100 person-years and cumulative incidence of 74% over a 4-year period.

Proteomic analysis of cardiometabolic biomarkers and predictive modeling of severe outcomes in patients hospitalized with COVID-19.

BACKGROUND: The high heterogeneity in the symptoms and severity of COVID-19 makes it challenging to identify high-risk patients early in the disease. Cardiometabolic comorbidities have shown strong associations with COVID-19 severity in epidemiologic studies. Cardiometabolic protein biomarkers, therefore, may provide predictive insight regarding which patients are most susceptible to severe illness from COVID-19. METHODS: In plasma samples collected from 343 patients hospitalized with COVID-19 during the first wave of the pandemic, we measured 92 circulating protein biomarkers previously implicated in cardiometabolic disease. We performed proteomic analysis and developed predictive models for severe outcomes. We then used these models to predict the outcomes of out-of-sample patients hospitalized with COVID-19 later in the surge (N = 194). RESULTS: We identified a set of seven protein biomarkers predictive of admission to the intensive care unit and/or death (ICU/death) within 28 days of presentation to care. Two of the biomarkers, ADAMTS13 and VEGFD, were associated with a lower risk of ICU/death. The remaining biomarkers, ACE2, IL-1RA, IL6, KIM1, and CTSL1, were associated with higher risk. When used to predict the outcomes of the future, out-of-sample patients, the predictive models built with these protein biomarkers outperformed all models built from standard clinical data, including known COVID-19 risk factors. CONCLUSIONS: These findings suggest that proteomic profiling can inform the early clinical impression of a patient's likelihood of developing severe COVID-19 outcomes and, ultimately, accelerate the recognition and treatment of high-risk patients.

Seroprevalence of Severe Acute Respiratory Syndrome Coronavirus 2 IgG in Juba, South Sudan, 20201.

Relatively few coronavirus disease cases and deaths have been reported from sub-Saharan Africa, although the extent of its spread remains unclear. During August 10-September 11, 2020, we recruited 2,214 participants for a representative household-based cross-sectional serosurvey in Juba, South Sudan. We found 22.3% of participants had severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor binding domain IgG titers above prepandemic levels. After accounting for waning antibody levels, age, and sex, we estimated that 38.3% (95% credible interval 31.8%-46.5%) of the population had been infected with SARS-CoV-2. At this rate, for each PCR-confirmed SARS-CoV-2 infection reported by the Ministry of Health, 103 (95% credible interval 86-126) infections would have been unreported, meaning SARS-CoV-2 has likely spread extensively within Juba. We also found differences in background reactivity in Juba compared with Boston, Massachusetts, USA, where the immunoassay was validated. Our findings underscore the need to validate serologic tests in sub-Saharan Africa populations.

Clinical sensitivity and interpretation of PCR and serological COVID-19 diagnostics for patients presenting to the hospital.

The diagnosis of COVID-19 requires integration of clinical and laboratory data. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnostic assays play a central role in diagnosis and have fixed technical performance metrics. Interpretation becomes challenging because the clinical sensitivity changes as the virus clears and the immune response emerges. Our goal was to examine the clinical sensitivity of two most common SARS-CoV-2 diagnostic test modalities, polymerase chain reaction (PCR) and serology, over the disease course to provide insight into their clinical interpretation in patients presenting to the hospital. We conducted a single-center, retrospective study. To derive clinical sensitivity of PCR, we identified 209 PCR-positive SARS-CoV-2 patients with multiple PCR test results (624 total PCR tests) and calculated daily sensitivity from date of symptom onset or first positive test. Clinical sensitivity of PCR decreased with days post symptom onset with >90% clinical sensitivity during the first 5 days after symptom onset, 70%-71% from Days 9 to 11, and 30% at Day 21. To calculate daily clinical sensitivity by serology, we utilized 157 PCR-positive patients with a total of 197 specimens tested by enzyme-linked immunosorbent assay for IgM, IgG, and IgA anti-SARS-CoV-2 antibodies. In contrast to PCR, serological sensitivity increased with days post symptom onset with >50% of patients seropositive by at least one antibody isotype after Day 7, >80% after Day 12, and 100% by Day 21. Taken together, PCR and serology are complimentary modalities that require time-dependent interpretation. Superimposition of sensitivities over time indicate that serology can function as a reliable diagnostic aid indicating recent or prior infection.

Ultra-Sensitive Serial Profiling of SARS-CoV-2 Antigens and Antibodies in Plasma to Understand Disease Progression in COVID-19 Patients with Severe Disease.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected over 21 million people worldwide since August 16, 2020. Compared to PCR and serology tests, SARS-CoV-2 antigen assays are underdeveloped, despite their potential to identify active infection and monitor disease progression. METHODS: We used Single Molecule Array (Simoa) assays to quantitatively detect SARS-CoV-2 spike, S1 subunit, and nucleocapsid antigens in the plasma of patients with coronavirus disease (COVID-19). We studied plasma from 64 patients who were COVID-19 positive, 17 who were COVID-19 negative, and 34 prepandemic patients. Combined with Simoa anti-SARS-CoV-2 serological assays, we quantified changes in 31 SARS-CoV-2 biomarkers in 272 longitudinal plasma samples obtained for 39 patients with COVID-19. Data were analyzed by hierarchical clustering and were compared to longitudinal RT-PCR test results and clinical outcomes. RESULTS: SARS-CoV-2 S1 and N antigens were detectable in 41 out of 64 COVID-19 positive patients. In these patients, full antigen clearance in plasma was observed a mean ± 95% CI of 5 ± 1 days after seroconversion and nasopharyngeal RT-PCR tests reported positive results for 15 ± 5 days after viral-antigen clearance. Correlation between patients with high concentrations of S1 antigen and ICU admission (77%) and time to intubation (within 1 day) was statistically significant. CONCLUSIONS: The reported SARS-CoV-2 Simoa antigen assay is the first to detect viral antigens in the plasma of patients who were COVID-19 positive to date. These data show that SARS-CoV-2 viral antigens in the blood are associated with disease progression, such as respiratory failure, in COVID-19 cases with severe disease.

Plasma Immunoglobulin A Responses Against 2 Salmonella Typhi Antigens Identify Patients With Typhoid Fever.

BACKGROUND: There is a need for a reliable, simple diagnostic assay for typhoid fever. Available commercial serologic assays for typhoid fever have limited sensitivity and specificity. Using high-throughput immunoscreening technologies, we previously identified several immunoreactive Salmonella Typhi antigens that seem promising for possible inclusion in a new diagnostic assay: hemolysin E (HlyE), cytolethal distending toxin, S. Typhi lipopolysaccharide (LPS), and S. Typhi membrane preparation. METHODS: We assessed plasma antibody responses (immunoglobulin [Ig] M, IgA, and IgG) to these antigens by means of enzyme-linked immunosorbent assay in patients with suspected enteric fever, controls with other febrile illnesses, and healthy controls in Dhaka, Bangladesh and performed Tubex and Typhidot tests, the Widal assay, and the typhoid/paratyphoid test (TPTest) in each patient. Using machine learning methods, we identified a parsimonious serology signature to distinguish acute typhoid cases from controls and then validated our findings in an independent test cohort from Nepal of patients with culture-confirmed S. Typhi and controls with other bacteremic illnesses. RESULTS: We demonstrated that the use of 2 antigens (HlyE and LPS) with 1 antibody isotype (IgA) could distinguish typhoid from other invasive bacterial infections (area under the receiver operating characteristic curve [AUC], 0.95; sensitivity, 90%, specificity, 92%). Use of a single antigen (HlyE) and isotype (IgA) had an AUC of 0.93. CONCLUSION: Our results suggest that development of a diagnostic assay for acute typhoid fever focused on detecting IgA responses against HlyE, with or without LPS, is warranted.

Analysis of the Human Mucosal Response to Cholera Reveals Sustained Activation of Innate Immune Signaling Pathways.

To better understand the innate immune response to Vibrio cholerae infection, we tracked gene expression in the duodenal mucosa of 11 Bangladeshi adults with cholera, using biopsy specimens obtained immediately after rehydration and 30 and 180 days later. We identified differentially expressed genes and performed an analysis to predict differentially regulated pathways and upstream regulators. During acute cholera, there was a broad increase in the expression of genes associated with innate immunity, including activation of the NF-κB, mitogen-activated protein kinase (MAPK), and Toll-like receptor (TLR)-mediated signaling pathways, which, unexpectedly, persisted even 30 days after infection. Focusing on early differences in gene expression, we identified 37 genes that were differentially expressed on days 2 and 30 across the 11 participants. These genes included the endosomal Toll-like receptor gene TLR8, which was expressed in lamina propria cells. Underscoring a potential role for endosomal TLR-mediated signaling in vivo, our pathway analysis found that interferon regulatory factor 7 and beta 1 and alpha 2 interferons were among the top upstream regulators activated during cholera. Among the innate immune effectors, we found that the gene for DUOX2, an NADPH oxidase involved in the maintenance of intestinal homeostasis, was upregulated in intestinal epithelial cells during cholera. Notably, the observed increases in DUOX2 and TLR8 expression were also modeled in vitro when Caco-2 or THP-1 cells, respectively, were stimulated with live V. cholerae but not with heat-killed organisms or cholera toxin alone. These previously unidentified features of the innate immune response to V. cholerae extend our understanding of the mucosal immune signaling pathways and effectors activated in vivo following cholera.

Plasma and Mucosal Immunoglobulin M, Immunoglobulin A, and Immunoglobulin G Responses to the Vibrio cholerae O1 Protein Immunome in Adults With Cholera in Bangladesh.

Background: Cholera is a severe dehydrating illness of humans caused by toxigenic strains of Vibrio cholerae O1 or O139. Identification of immunogenic V. cholerae antigens could lead to a better understanding of protective immunity in human cholera. Methods: We probed microarrays containing 3652 V. cholerae antigens with plasma and antibody-in-lymphocyte supernatant (ALS, a surrogate marker of mucosal immune responses) from patients with severe cholera caused by V. cholerae O1 in Bangladesh and age-, sex-, and ABO-matched Bangladeshi controls. We validated a subset of identified antigens using enzyme-linked immunosorbent assay. Results: Overall, we identified 608 immunoreactive V. cholerae antigens in our screening, 59 of which had higher immunoreactivity in convalescent compared with acute-stage or healthy control samples (34 in plasma, 39 in mucosal ALS; 13 in both sample sets). Identified antigens included cholera toxin B and A subunits, V. cholerae O-specific polysaccharide and lipopolysaccharide, toxin coregulated pilus A, sialidase, hemolysin A, flagellins (FlaB, FlaC, and FlaD), phosphoenolpyruvate-protein phosphotransferase, and diaminobutyrate-2-oxoglutarate aminotransferase. Conclusions: This study is the first antibody profiling of the mucosal and systemic antibody responses to the nearly complete V. cholerae O1 protein immunome; it has identified antigens that may aid in the development of an improved cholera vaccine.

Immunogenicity of the Bivalent Oral Cholera Vaccine Shanchol in Haitian Adults With HIV Infection.

We evaluated immune responses following bivalent oral cholera vaccination (Shanchol [Shantha Biotechnics]; BivWC) in a cohort of 25 human immunodeficiency virus (HIV)-infected adults in Haiti. Compared with adults without HIV infection, vaccination in HIV-infected individuals resulted in lower vibriocidal responses against Vibrio cholerae O1, and there was a positive relationship between the CD4(+) T-cell count and vibriocidal responses following vaccination. Nevertheless, seroconversion occurred at a rate of 65% against the Ogawa serotype and 74% against the Inaba serotype in adults with HIV infection. These results suggest that the vaccine retains substantial immunogenicity in adults with HIV infection and may benefit this population by protecting against cholera.

Fluorescence Polarization Based Nucleic Acid Testing for Rapid and Cost-Effective Diagnosis of Infectious Disease.

A new nucleic acid detection method was developed for a rapid and cost-effective diagnosis of infectious disease. This approach relies on the three unique elements: 1) detection probes that regulate DNA polymerase activity in response to the complementary target DNA; 2) universal reporters conjugated with a single fluorophore; and 3) fluorescence polarization (FP) detection. As a proof-of-concept, the assay was used to detect and sub-type Salmonella bacteria with sensitivities down to a single bacterium in less than three hours.

The origin of the Haitian cholera outbreak strain.

BACKGROUND: Although cholera has been present in Latin America since 1991, it had not been epidemic in Haiti for at least 100 years. Recently, however, there has been a severe outbreak of cholera in Haiti. METHODS: We used third-generation single-molecule real-time DNA sequencing to determine the genome sequences of 2 clinical Vibrio cholerae isolates from the current outbreak in Haiti, 1 strain that caused cholera in Latin America in 1991, and 2 strains isolated in South Asia in 2002 and 2008. Using primary sequence data, we compared the genomes of these 5 strains and a set of previously obtained partial genomic sequences of 23 diverse strains of V. cholerae to assess the likely origin of the cholera outbreak in Haiti. RESULTS: Both single-nucleotide variations and the presence and structure of hypervariable chromosomal elements indicate that there is a close relationship between the Haitian isolates and variant V. cholerae El Tor O1 strains isolated in Bangladesh in 2002 and 2008. In contrast, analysis of genomic variation of the Haitian isolates reveals a more distant relationship with circulating South American isolates. CONCLUSIONS: The Haitian epidemic is probably the result of the introduction, through human activity, of a V. cholerae strain from a distant geographic source. (Funded by the National Institute of Allergy and Infectious Diseases and the Howard Hughes Medical Institute.).