Clinical and Diagnostic Features of West Nile Virus Neuroinvasive Disease in New York City.

West Nile virus (WNV) neuroinvasive disease (WNND) occurs in approximately 1 percent of WNV-infected patients and typically presents as encephalitis, meningitis, or acute flaccid paralysis (AFP). WNND remains a difficult inpatient diagnosis, creating significant challenges for prognostication and therapy selection. We characterized the clinical and diagnostic features of WNND cases at two major academic medical centers in New York City in routine clinical practice. We retrospectively reviewed the charts of thirty-six patients with WNND, including twenty-six encephalitis, four meningitis, and six AFP cases. The most common presenting symptoms were fever (86.1%) and gastrointestinal symptoms (38.9%) in addition to altered mental status (72.2%), lethargy (63.9%), gait disturbances (46.2%), and headache (44.4%). Fourteen (48.3%) patients displayed acute magnetic resonance imaging (MRI) findings, particularly T2 hyperintensities in the bilateral thalami, brainstem, and deep white matter. New York State Department of Health WNV CSF IgM testing was utilized for diagnosis in 58.3% of patients; however, just 38.1% had the result by discharge, compared to 85.6% of those who underwent serum IgM testing. The median length of stay was 13.5 days, 38.9% were intubated, and three patients (8.9%) died during acute hospitalization. Our findings underscore the morbidity, mortality, and diagnostic challenges of WNND, suggesting the potential utility of serum IgM testing in combination with confirmatory CSF testing to expedite diagnosis in the acute setting.

Development of a novel serological assay for the detection of mpox infection in vaccinated populations.

In 2022 the World Health Organization declared a Public Health Emergency for an outbreak of mpox, the zoonotic Orthopoxvirus (OPV) affecting at least 104 nonendemic locations worldwide. Serologic detection of mpox infection is problematic, however, due to considerable antigenic and serologic cross-reactivity among OPVs and smallpox-vaccinated individuals. In this report, we developed a high-throughput multiplex microsphere immunoassay using a combination of mpox-specific peptides and cross-reactive OPV proteins that results in the specific serologic detection of mpox infection with 93% sensitivity and 98% specificity. The New York State Non-Vaccinia Orthopoxvirus Microsphere Immunoassay is an important tool to detect subclinical mpox infection and understand the extent of mpox spread in the community through retrospective analysis.

Development of a Novel Serological Assay for the Detection of Mpox Infection in Vaccinated Populations.

In 2022 the World Health Organization declared a Public Health Emergency for an outbreak of mpox, the zoonotic Orthopoxvirus (OPV) affecting at least 103 non-endemic locations world-wide. Serologic detection of mpox infection is problematic, however, due to considerable antigenic and serologic cross-reactivity among OPVs and smallpox-vaccinated individuals. In this report, we developed a high-throughput multiplex microsphere immunoassay (MIA) using a combination of mpox-specific peptides and cross-reactive OPV proteins that results in the specific serologic detection of mpox infection with 93% sensitivity and 98% specificity. The New York State Non-Vaccinia Orthopoxvirus Microsphere Immunoassay is an important diagnostic tool to detect subclinical mpox infection and understand the extent of mpox spread in the community through retrospective analysis.

Harmonization of Multiple SARS-CoV-2 Reference Materials Using the WHO IS (NIBSC 20/136): Results and Implications.

Background: There is an urgent need for harmonization between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serology platforms and assays prior to defining appropriate correlates of protection and as well inform the development of new rapid diagnostic tests that can be used for serosurveillance as new variants of concern (VOC) emerge. We compared multiple SARS-CoV-2 serology reference materials to the WHO International Standard (WHO IS) to determine their utility as secondary standards, using an international network of laboratories with high-throughput quantitative serology assays. This enabled the comparison of quantitative results between multiple serology platforms. Methods: Between April and December 2020, 13 well-characterized and validated SARS-CoV-2 serology reference materials were recruited from six different providers to qualify as secondary standards to the WHO IS. All the samples were tested in parallel with the National Institute for Biological Standards and Control (NIBSC) 20/136 and parallel-line assays were used to calculate the relevant potency and binding antibody units. Results: All the samples saw varying levels of concordance between diagnostic methods at specific antigen-antibody combinations. Seven of the 12 candidate materials had high concordance for the spike-immunoglobulin G (IgG) analyte [percent coefficient of variation (%CV) between 5 and 44%]. Conclusion: Despite some concordance between laboratories, qualification of secondary materials to the WHO IS using arbitrary international units or binding antibody units per milliliter (BAU/ml) does not provide any benefit to the reference materials overall, due to the lack of consistent agreeable international unit (IU) or BAU/ml conversions between laboratories. Secondary standards should be qualified to well-characterized reference materials, such as the WHO IS, using serology assays that are similar to the ones used for the original characterization of the WHO IS.

Evaluation of measles IgM antibody detection assays during the 2018-2019 outbreak in New York State.

Although measles was eliminated in the United States in 2000, a severe outbreak occurred between October 2018 and September 2019. New York was especially hard hit. Serology played an integral role in determining immune status (IgG) and identifying, along with molecular analyses, acute measles infections (IgM). Although an indirect immunofluorescence assay (IFA) was historically used by the New York State Department of Health for measles IgM detection, a higher throughput assay was needed to address the increased specimen numbers. Four commercial enzyme-linked immunosorbent assays (ELISAs) were evaluated for sensitivity and specificity in detecting measles IgM. Two ELISA formats were compared, indirect ELISA and IgM antibody capture. Both formats had comparable specificity as determined by cross-reactivity to non-measles specimens. Overall, the sensitivity of the capture ELISAs was greater than the indirect ELISAs and comparable to the indirect immunofluorescence assay with benefits regarding capacity, cost, and turnaround time.

Congenital Measles in a Premature 25-week Gestation Infant.

We describe a premature infant with congenital measles. Laboratory testing confirmed measles in the mother (polymerase chain reaction- and IgM-positive) and congenital measles in the infant (polymerase chain reaction-positive, culture-positive and IgM-positive). The infant never developed a rash, pneumonia, or neurologic complications. This case supports using compatible laboratory findings to diagnose congenital measles in infants without clinical manifestations of measles.

Serological analysis reveals an imbalanced IgG subclass composition associated with COVID-19 disease severity.

Coronavirus disease 2019 (COVID-19) is associated with a wide spectrum of disease presentation, ranging from asymptomatic infection to acute respiratory distress syndrome (ARDS). Paradoxically, a direct relationship has been suggested between COVID-19 disease severity and the levels of circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibodies, including virus-neutralizing titers. A serological analysis of 536 convalescent healthcare workers reveals that SARS-CoV-2-specific and virus-neutralizing antibody levels are elevated in individuals that experience severe disease. The severity-associated increase in SARS-CoV-2-specific antibody is dominated by immunoglobulin G (IgG), with an IgG subclass ratio skewed toward elevated receptor binding domain (RBD)- and S1-specific IgG3. In addition, individuals that experience severe disease show elevated SARS-CoV-2-specific antibody binding to the inflammatory receptor FcÉ£RIIIa. Based on these correlational studies, we propose that spike-specific IgG subclass utilization may contribute to COVID-19 disease severity through potent Fc-mediated effector functions. These results may have significant implications for SARS-CoV-2 vaccine design and convalescent plasma therapy.

Neutralizing Antibody Responses in COVID-19 Convalescent Sera.

Passive transfer of antibodies from COVID-19 convalescent patients is being used as an experimental treatment for eligible patients with SARS-CoV-2 infections. The United States Food and Drug Administration's (FDA) guidelines for convalescent plasma initially recommended target antibody titers of 160. We evaluated SARS-CoV-2 neutralizing antibodies in sera from recovered COVID-19 patients using plaque reduction neutralization tests (PRNT) at moderate (PRNT50) and high (PRNT90) stringency thresholds. We found that neutralizing activity significantly increased with time post symptom onset (PSO), reaching a peak at 31-35 days PSO. At this point, the number of sera having neutralizing titers of at least 160 was approximately 93% (PRNT50) and approximately 54% (PRNT90). Sera with high SARS-CoV-2 antibody levels (>960 enzyme-linked immunosorbent assay titers) showed maximal activity, but not all high-titer sera contained neutralizing antibody at FDA recommended levels, particularly at high stringency. These results underscore the value of serum characterization for neutralization activity.

Epidemiological Characteristics and Laboratory Findings of Zika Virus Cases in New York City, January 1, 2016-June 30, 2017.

BACKGROUND: An outbreak of Zika virus (ZIKV) began in May 2015 in Brazil and rapidly spread throughout the Americas; New York City (NYC) has a diverse population with ∼1.8 million residents who were born in ZIKV-affected areas. Before July 24, 2017, the Centers for Disease Control and Prevention (CDC) ZIKV testing recommendations included nucleic acid amplification-based tests for serum and urine specimens collected ≤14 days of illness onset or last potential exposure, and ZIKV immunoglobulin M (IgM) assay when ZIKV RNA is not detected or for specimens collected within 2-12 weeks of illness onset or last potential exposure, followed by a plaque reduction neutralization test (PRNT). However, the New York public health laboratories and commercial laboratories tested specimens collected beyond these time frames. METHODS: We analyzed 1080 noncongenital ZIKV cases in NYC residents who met the Council for State and Territorial Epidemiologist's ZIKV case definitions. RESULTS: Among cases, 98% were travel associated, 1% were sexually transmitted, and 1% had unknown exposures; 412 (38%) cases were pregnant women. Of 672 patients with ZIKV RNA detected in serum or urine specimens, 48 (7%) tested positive >14 days after either symptom onset or last potential exposure date (range 15-99 days). Of 390 patients diagnosed based on serology alone (i.e., not tested or not detectable for ZIKV RNA), 60 (15%) had a positive ZIKV IgM and PRNT >12 weeks after symptom onset or last potential exposure date (range 85-273 days). CONCLUSION: Our findings correspond with CDC's updated guidance to test symptomatic pregnant women up to 12 weeks past onset of symptoms. ZIKV IgM antibody testing may also be warranted for pregnant women regardless of symptoms if their exposure occurred during their pregnancy or periconception period. Providers should understand the scope of diagnostic testing and its limitations to appropriately counsel patients, especially pregnant women.

Development of Zika Virus Serological Testing Strategies in New York State.

The recent outbreak of Zika virus (ZIKV) in the Americas has challenged diagnostic laboratory testing strategies. At the Wadsworth Center, ZIKV serological testing was performed for over 10,000 specimens, using a combination of an enzyme-linked immunosorbent assay (ELISA) for IgM antibodies (Abs) to ZIKV, a polyvalent microsphere immunoassay (MIA) to detect Abs broadly reactive with flaviviruses, and a plaque reduction neutralization test (PRNT) for further testing. Overall, 42% of patients showed serological evidence of flavivirus infection (primarily past dengue virus [DENV] infection), while 7% possessed IgM Abs to ZIKV and/or DENV. ZIKV IgM Abs typically arose within 3 to 4 days, with only one instance of duration beyond 100 days after reported symptoms. PRNT analysis of 826 IgM-positive specimens showed 7% positive neutralization to ZIKV alone, 9% to DENV alone, and 85% to both ZIKV and DENV. Thus, the extensive Ab cross-reactivity among flaviviruses significantly reduced the value of performing PRNT analysis, especially when a traditional paired serum algorithm with viral neutralization titering was used. Nevertheless, the finding of a negative ZIKV result by PRNT was invaluable for reassuring both physicians and patients. The MIA detected both IgM and IgG, which enabled us to identify patients who presented without IgM anti-ZIKV Abs but still had ZIKV-specific neutralizing Abs. On the basis of these results, a new algorithm, which included an IgM Ab capture (MAC)-ELISA to detect recent infection, a flavivirus MIA to identify patients no longer producing IgM, and a single-dilution PRNT for ZIKV exclusion and occasional discrimination of ZIKV and DENV, was implemented.

Powassan meningoencephalitis, New York, New York, USA.

Disease caused by Powassan virus (POWV), a tick-borne flavivirus, ranges from asymptomatic to severe neurologic compromise and death. Two cases of POWV meningoencephalitis in New York, USA, highlight diagnostic techniques, neurologic outcomes, and the effect of POWV on communities to which it is endemic.

Intraocular bevacizumab levels 24 hours after intravitreal injection in a normal eye.

BACKGROUND: To determine intraocular levels of bevacizumab in a normal eye 24 hours after the injection of bevacizumab into the vitreous cavity. METHODS: Fluid and tissue samples were analyzed for unbound bevacizumab levels using microsphere immunoassays. RESULTS: After a loading dose of 2.5 mg (0.1 mL) of bevacizumab, levels of unbound drug could be detected in all tissue extracts 24 hours after its injection. At least 60.2% of the injected bevacizumab could be accounted for in this study. Levels of 0.06 mg (2.4%) of bevacizumab were recorded in the choroid. CONCLUSION: At 24 hours, unbound bevacizumab levels can be detected at the level of the choroid. Therefore, bevacizumab can reach the site where choroidal neovascularization develops. This explains its reported therapeutic effect.

Multiplex immunoassay for Lyme disease using VlsE1-IgG and pepC10-IgM antibodies: improving test performance through bioinformatics.

The Centers for Disease Control and Prevention currently recommends a 2-tier serologic approach to Lyme disease laboratory diagnosis, comprised of an initial serum enzyme immunoassay (EIA) for antibody to Borrelia burgdorferi followed by supplementary IgG and IgM Western blotting of EIA-positive or -equivocal samples. Western blot accuracy is limited by subjective interpretation of weakly positive bands, false-positive IgM immunoblots, and low sensitivity for detection of early disease. We developed an objective alternative second-tier immunoassay using a multiplex microsphere system that measures VlsE1-IgG and pepC10-IgM antibodies simultaneously in the same sample. Our study population comprised 79 patients with early acute Lyme disease, 82 patients with early-convalescent-phase disease, 47 patients with stage II and III disease, 34 patients post-antibiotic treatment, and 794 controls. A bioinformatic technique called partial receiver-operator characteristic (ROC) regression was used to combine individual antibody levels into a single diagnostic score with a single cutoff; this technique enhances test performance when a high specificity is required (e.g., ≥ 95%). Compared to Western blotting, the multiplex assay was equally specific (95.6%) but 20.7% more sensitive for early-convalescent-phase disease (89.0% versus 68.3%, respectively; 95% confidence interval [95% CI] for difference, 12.1% to 30.9%) and 12.5% more sensitive overall (75.0% versus 62.5%, respectively; 95% CI for difference, 8.1% to 17.1%). As a second-tier test, a multiplex assay for VlsE1-IgG and pepC10-IgM antibodies performed as well as or better than Western blotting for Lyme disease diagnosis. Prospective validation studies appear to be warranted.

Development of an 8-plex Luminex assay to detect swine cytokines for vaccine development: assessment of immunity after porcine reproductive and respiratory syndrome virus (PRRSV) vaccination.

A Luminex (Luminex Corp., Austin, TX) multiplex swine cytokine assay was developed to measure 8 cytokines simultaneously in pig serum for use in assessment of vaccine candidates. The fluorescent microsphere immunoassay (FMIA) was tested on archived sera in a porcine reproductive and respiratory syndrome virus (PRRSV) vaccine/challenge study. This FMIA simultaneously detects innate (IL-1 beta, IL-8, IFN-alpha, TNF-alpha, IL-12), regulatory (IL-10), Th1 (IFN-gamma) and Th2 (IL-4) cytokines. These proteins were measured to evaluate serum cytokine levels associated with vaccination strategies that provided for different levels of protective immunity against PRRSV. Pigs were vaccinated with a modified-live virus (MLV) vaccine and subsequently challenged with a non-identical PRRSV isolate (93% identity in the glycoprotein (GP5) gene). Protection (as defined by no serum viremia) was observed in the MLV vaccinated pigs after PRRSV challenge but not those vaccinated with killed virus vaccine with adjuvant (KV/ADJ) (99% identity in the GP5 gene to the challenge strain) or non-vaccinates. Significantly elevated levels of IL-12 were observed in the KV/ADJ group compared to MLV vaccinated and control groups. However, this significant increase in serum IL-12 did not correlate with protection against PRRSV viremia. Additional studies using this assay to measure the local cytokine tissue responses may help in defining a protective cytokine response and would be useful for the targeted design of efficacious vaccines, not only for PRRSV, but also for other swine pathogens.

Infliximab stability after reconstitution, dilution, and storage under refrigeration.

PURPOSE: The purpose of this study was to investigate the stability of reconstituted infliximab solutions and determine whether infliximab is suitable for compounding for potential intravitreal use. METHODS: Infliximab was reconstituted, and the solution was aliquoted and stored refrigerated. On each day of testing, an aliquot was serially diluted to concentrations ranging from 50,000 pg/mL to 69 pg/mL. Each dilution was assayed by microsphere immunoassay daily for 5 days and weekly for a total of 6 weeks. The outcome measure was median fluorescence intensity measured by dual laser flow analysis of fluorochrome-labeled secondary antibodies to infliximab bound to tumor necrosis factor-alpha-coated microspheres. RESULTS: There was an increasing median fluorescence intensity for increasing infliximab concentration in a sigmoidal dose-response curve with a variable slope that was equivalent for each time point. Each respective concentration of infliximab showed nearly equivalent median fluorescence intensity for every time point over the 6-week period. CONCLUSION: The authors found that the immunoreactivity of 2 different concentrations of infliximab stored at 4 degrees C over a 6-week period remained stable. Infliximab is suitable for compounding and could be a cost-effective intravitreal medication for use in clinical practice if further study supports its safety and efficacy.