Combination of Nonstructural Protein 1-Based Enzyme-Linked Immunosorbent Assays Can Detect and Distinguish Various Dengue Virus and Zika Virus Infections.

The recent outbreaks of Zika virus (ZIKV) and associated birth defects in regions of dengue virus (DENV) endemicity emphasize the need for sensitive and specific serodiagnostic tests. We reported previously that enzyme-linked immunosorbent assays (ELISAs) based on the nonstructural protein 1 (NS1) of DENV serotype 1 (DENV1) and ZIKV can distinguish primary DENV1, secondary DENV, and ZIKV infections. Whether ELISAs based on NS1 proteins of other DENV serotypes can discriminate various DENV and ZIKV infections remains unknown. We herein developed DENV2, DENV3, and DENV4 NS1 IgG ELISAs to test convalescent- and postconvalescent-phase samples from reverse transcription-PCR-confirmed cases, including 25 primary DENV1, 24 primary DENV2, 10 primary DENV3, 67 secondary DENV, 36 primary West Nile virus, 38 primary ZIKV, and 35 ZIKV with previous DENV infections as well as 55 flavivirus-naive samples. Each ELISA detected primary DENV infection with a sensitivity of 100% for the same serotype and 23.8% to 100% for different serotypes. IgG ELISA using a mixture of DENV1-4 NS1 proteins detected different primary and secondary DENV infections with a sensitivity of 95.6% and specificity of 89.5%. The ZIKV NS1 IgG ELISA detected ZIKV infection with a sensitivity of 100% and specificity of 82.9%. On the basis of the relative optical density ratio, the combination of DENV1-4 and ZIKV NS1 IgG ELISAs distinguished ZIKV with previous DENV and secondary DENV infections with a sensitivity of 91.7% to 94.1% and specificity of 87.0% to 95.0%. These findings have important applications to serodiagnosis, serosurveillance, and monitoring of both DENV and ZIKV infections in regions of endemicity.

Use of Urea Wash ELISA to Distinguish Zika and Dengue Virus Infections.

Serologic testing remains crucial for Zika virus diagnosis. We found that urea wash in a Zika virus nonstructural protein 1 IgG ELISA distinguishes secondary dengue virus infection from Zika virus infection with previous dengue (sensitivity 87.5%, specificity 93.8%). This test will aid serodiagnosis, serosurveillance, and monitoring of Zika complications in dengue-endemic regions.

Distinguishing Secondary Dengue Virus Infection From Zika Virus Infection With Previous Dengue by a Combination of 3 Simple Serological Tests.

BACKGROUND: The explosive spread of Zika virus (ZIKV) and associated microcephaly present an urgent need for sensitive and specific serodiagnostic tests, particularly for pregnant women in dengue virus (DENV)-endemic regions. Recent reports of enhanced ZIKV replication by dengue-immune sera have raised concerns about the role of previous DENV infection on the risk and severity of microcephaly and other ZIKV complications. METHODS: Enzyme-linked immunosorbent assays (ELISAs) based on ZIKV and DENV nonstructural protein 1 (NS1) were established to test acute, convalescent phase, and post-convalescent phase serum/plasma samples from reverse-transcription polymerase chain reaction-confirmed cases including 20 primary ZIKV, 25 ZIKV with previous DENV, 58 secondary DENV, and 16 primary DENV1 infections. RESULTS: ZIKV-NS1 immunoglobulin M (IgM) and immunoglobulin G (IgG) ELISAs combined can detect ZIKV infection with a sensitivity of 95% and specificity of 66.7%. The ZIKV-NS1 IgG cross-reactivity by samples from secondary DENV infection cases ranged from 66.7% to 28.1% (within 1 month to 1-2 years post-illness, respectively). Addition of DENV1-NS1 IgG ELISA can distinguish primary ZIKV infection; the ratio of absorbance of ZIKV-NS1 to DENV1-NS1 IgG ELISA can distinguish ZIKV with previous DENV and secondary DENV infections with a sensitivity of 87.5% and specificity of 81.3%. These findings were supported by analysis of sequential samples. CONCLUSIONS: An algorithm for ZIKV serodiagnosis based on 3 simple ELISAs is proposed to distinguish primary ZIKV, ZIKV with previous DENV, and secondary DENV infections; this could be applied to serodiagnosis for ZIKV, serosurveillance, and monitoring ZIKV infection during pregnancy to understand the epidemiology, pathogenesis, and complications of ZIKV in dengue-endemic regions.

Availability of Pharmacist-Prescribed Contraception in Hawai'i.

In 2017, the state of Hawai'i passed Act 067 which allows trained pharmacists to prescribe hormonal contraceptives in an effort to expand access to contraception. The extent to which this policy has been implemented is not known. This study aimed to determine the proportion of Hawai'i pharmacies that currently provide pharmacist-prescribed hormonal contraceptives. In June 2020, a list of retail pharmacies was compiled using Google and Google Maps searches and pharmacy directories from major health insurance providers. Between June 23, 2020, and July 2, 2020, two trained interviewers called pharmacies and inquired about the availability of pharmacist-prescribed contraceptives using a "secret shopper" technique. Of the 175 pharmacies included in our analysis, 54 (31%) offered pharmacist-prescribed contraceptives. Kaua'i (40%) had the highest proportion of pharmacies offering pharmacist-prescribed contraceptives, followed by Maui (35%), O'ahu (30%), and Hawai'i Island (29%) (P=.88). Among pharmacies located in rural communities, 20 of 63 (32%) prescribed contraceptives, compared to 34 of 112 (30 %) pharmacies located in urban communities (P=.85). Of the 118 pharmacies that did not prescribe contraceptives and provided a reason for not doing so 33% cited lack of training and 28% lack of knowledge about Act 067. Thirty-one pharmacies provided information on the cost of pharmacist-prescribed contraceptives with 71% (22) stating that the patient would have to pay extra for the consultation (mean cost = $34.6, range $30-$45). Findings from this study can help inform future public health policies and implementation strategies aimed at improving contraceptive access in Hawai'i.

Expanding Access to Contraception: Identifying Accessibility Gaps Across Hawai'i Communities.

In 2019, Hawai'i ended its Title X program resulting in a loss of federal family planning funds. Additionally, physician shortages have decreased family planning resources available to patients. The objective of this study was to assess contraception availability by determining the number and location of healthcare providers in Hawai'i that prescribed at least one form of contraception. A list of healthcare providers was compiled using Google searches, major health insurance, and hospital provider directories. Providers were organized by physical location (ie, address). Each location was contacted to inquire about each provider's ability to prescribe different forms of contraception (eg, intrauterine device, implant, injection, pill, patch, or ring). Of the 1,020 locations contacted, 274 prescribed at least one form of contraception. Of the 1,810 providers surveyed at these locations, 744 prescribed at least one form of contraception. In regard to insurance, 201 locations and 609 providers accepted at least one form of Medicaid. Most prescribing providers were located on the island of O'ahu. The majority of providers across the state prescribed the pill, patch, or ring. There are many additional barriers that were not addressed in this study, including factors that affect physician prescribing practices. Identifying these barriers is important to further address gaps in contraceptive accessibility. Consideration of improved support for training in specialties such as Family Medicine, Internal Medicine, and Pediatrics can expand access to contraception within primary care settings.

Provision of medication abortion in Hawai'i during COVID-19: Practical experience with multiple care delivery models.

OBJECTIVE: To demonstrate the effectiveness of medication abortion with the implementation of telemedicine and a no-test protocol in response to the COVID-19 pandemic. STUDY DESIGN: This is a retrospective cohort study of patients who had a medication abortion up to 77 days gestation at the University of Hawai'i between April and November 2020. Patients had the option of traditional in clinic care or telemedicine with either in clinic pickup or mailing of medications. During this time, a no-test protocol for medication abortion without prior labs or ultrasound was in place for eligible patients. The primary outcome was the rate of successful medication abortion without surgical intervention. Secondary outcomes included abortion-related complications. RESULTS: A total of 334 patients were dispensed mifepristone and misoprostol, 149 (44.6%) with telemedicine with in-person pickup of medications, 75 (22.5%) via telemedicine with medications mailed, and 110 (32.9%) via traditional in person visits. The overall rate of complete medication abortion without surgical intervention was 95.8%, with success rates of 96.8, 97.1, and 93.6% for the clinic pickup, mail, and clinic visit groups, respectively. Success for those without an ultrasound performed prior to the procedure was 96.6%, compared to 95.5% for those with ultrasound. We obtained follow-up data for 87.8% of participants. CONCLUSIONS: Medication abortion was safe and effective while offering multiple modes of care delivery including telemedicine visits without an ultrasound performed prior to dispensing medications. IMPLICATIONS: Incorporating telemedicine and a no-test protocol for medication abortion is safe and has the potential to expand access to abortion care. All care models had low rates of adverse events, which contradicts the idea that the Risk Evaluation and Mitigation Strategyincreases the safety of medication abortion.

The role of extracellular vesicles and PD-L1 in glioblastoma-mediated immunosuppressive monocyte induction.

BACKGROUND: Immunosuppression in glioblastoma (GBM) is an obstacle to effective immunotherapy. GBM-derived immunosuppressive monocytes are central to this. Programmed cell death ligand 1 (PD-L1) is an immune checkpoint molecule, expressed by GBM cells and GBM extracellular vesicles (EVs). We sought to determine the role of EV-associated PD-L1 in the formation of immunosuppressive monocytes. METHODS: Monocytes collected from healthy donors were conditioned with GBM-derived EVs to induce the formation of immunosuppressive monocytes, which were quantified via flow cytometry. Donor-matched T cells were subsequently co-cultured with EV-conditioned monocytes in order to assess effects on T-cell proliferation. PD-L1 constitutive overexpression or short hairpin RNA-mediated knockdown was used to determined the role of altered PD-L1 expression. RESULTS: GBM EVs interact with both T cells and monocytes but do not directly inhibit T-cell activation. However, GBM EVs induce immunosuppressive monocytes, including myeloid-derived suppressor cells (MDSCs) and nonclassical monocytes (NCMs). MDSCs and NCMs inhibit T-cell proliferation in vitro and are found within GBM in situ. EV PD-L1 expression induces NCMs but not MDSCs, and does not affect EV-conditioned monocytes T-cell inhibition. CONCLUSION: These findings indicate that GBM EV-mediated immunosuppression occurs through induction of immunosuppressive monocytes rather than direct T-cell inhibition and that, while PD-L1 expression is important for the induction of specific immunosuppressive monocyte populations, immunosuppressive signaling mechanisms through EVs are complex and not limited to PD-L1.

Zika Virus: Relevance to the State of Hawai'i.

Zika virus (ZIKV) is spread among human populations primarily through the bite of Aedes mosquitoes. While most ZIKV infections are asymptomatic or cause self-limited symptoms, the major concerns are its association with Guillain-Barré Syndrome and fetal microcephaly together with other birth defects, known as congenital Zika syndrome (CZS). This article reviews the confirmed Zika cases in the continental United States (U.S.) and Hawai'i thus far, as well as literature of Zika research relevant to Hawai'i. The first case of CZS within the U.S. was reported in Hawai'i, highlighting the unique position of Hawai'i for emerging and re-emerging infectious diseases. Recent studies of the Zika outbreak in Florida demonstrate the key role of Ae. aegypti mosquito in transmission; continuous and proactive vector surveillance in Hawai'i is warranted. Additionally, an updated interim pregnancy guidance for pregnant women with possible ZIKV exposure was summarized. Due to recent decline of ZIKV transmission in the Americas, the risk of ZIKV importation to Hawai'i has been greatly reduced. However, given the presence of Aedes mosquitoes, climate condition, and status of Hawai'i as a travel destination and foreign import market, public health officials and healthcare providers should remain vigilant for a potential outbreak of mosquito-borne diseases in the future.

A high-throughput and multiplex microsphere immunoassay based on non-structural protein 1 can discriminate three flavivirus infections.

The explosive spread of Zika virus (ZIKV) and associated complications in flavivirus-endemic regions underscore the need for sensitive and specific serodiagnostic tests to distinguish ZIKV, dengue virus (DENV) and other flavivirus infections. Compared with traditional envelope protein-based assays, several nonstructural protein 1 (NS1)-based assays showed improved specificity, however, none can detect and discriminate three flaviviruses in a single assay. Moreover, secondary DENV infection and ZIKV infection with previous DENV infection, both common in endemic regions, cannot be discriminated. In this study, we developed a high-throughput and multiplex IgG microsphere immunoassay (MIA) using the NS1 proteins of DENV1-DENV4, ZIKV and West Nile virus (WNV) to test samples from reverse-transcription-polymerase-chain reaction-confirmed cases, including primary DENV1, DENV2, DENV3, WNV and ZIKV infections, secondary DENV infection, and ZIKV infection with previous DENV infection. Combination of four DENV NS1 IgG MIAs revealed a sensitivity of 94.3% and specificity of 97.2% to detect DENV infection. The ZIKV and WNV NS1 IgG MIAs had a sensitivity/specificity of 100%/87.9% and 86.1%/78.4%, respectively. A positive correlation was found between the readouts of enzyme-linked immunosorbent assay and MIA for different NS1 tested. Based on the ratio of relative median fluorescence intensity of ZIKV NS1 to DENV1 NS1, the IgG MIA can distinguish ZIKV infection with previous DENV infection and secondary DENV infection with a sensitivity of 88.9-90.0% and specificity of 91.7-100.0%. The multiplex and high-throughput assay could be applied to serodiagnosis and serosurveillance of DENV, ZIKV and WNV infections in endemic regions.

Seroprevalence of dengue virus in two districts of Kaohsiung City after the largest dengue outbreak in Taiwan since World War II.

Dengue virus (DENV) is the leading cause of arboviral diseases in humans worldwide. In this study, we investigated the seroprevalence of DENV infection in two districts of Kaohsiung City, a metropolis in southern Taiwan, where major dengue outbreaks have occurred in the past three decades. We enrolled 1,088 participants from the Sanmin and Nanzih districts after the dengue outbreak of 2015, the largest in Taiwan since World War II, and found an overall DENV seroprevalence of 12.4% (95% confidence interval: 10.5-13.4%) based on the InBios DENV IgG ELISA kit. The ratios of clinically inapparent to symptomatic infections were 2.86 and 4.76 in Sanmin and Nanzih districts, respectively. Consistent with higher case numbers during recent outbreaks, the DENV seroprevalence was higher in Sanmin district (16.4%) than in Nanzih district (6.9%), suggesting district differences in seroprevalence and highlighting the importance of screening the DENV immune status of each individual before using the currently available DENV vaccine, Dengvaxia. In the two districts, the seroprevalence rates increased from 2.1% (in the 30-39-year age group) to 17.1% (60-69) and 50% (70-79). The pattern of a sharp and significant increase in seroprevalence in the 70-79-year age group correlated with a dramatic increase in the proportion of clinically severe DENV infections among total dengue cases in that age group. This differed from observations in the Americas and Southeast Asia and suggested that a large proportion of monotypically immune individuals together with other risk factors may contribute to clinically severe dengue among the elderly in Taiwan.