Tick-borne encephalitis among US travellers, 2010-20.

BACKGROUND: Tick-borne encephalitis (TBE) is an arboviral disease that is focally endemic in parts of Europe and Asia. TBE cases among US travellers are rare, with previous reports of only six cases among civilian travellers through 2009 and nine military-related cases through 2020. A TBE vaccine was licenced in the USA in August 2021. Understanding TBE epidemiology and risks among US travellers can help with the counselling of travellers going to TBE-endemic areas. METHODS: Diagnostic testing for TBE in the USA is typically performed at the Centers for Disease Control and Prevention (CDC) because no commercial testing is available. Diagnostic testing for TBE at CDC since 2010 was reviewed. For individuals with evidence of TBE virus infection, information was gathered on demographics, clinical presentations and risk factors for infection. RESULTS: From 2010-20, six patients with TBE were identified. Cases occurred among both paediatric and adult travellers and all were male. Patients were diagnosed with meningitis (n = 2) or encephalitis (n = 4); none died. Cases had travelled to various countries in Europe or Russia. Three cases reported visiting friends or relatives. Activities reported included hiking, camping, trail running, or working outdoors, and two cases had a recognized tick bite. CONCLUSIONS: TBE cases among US travellers are uncommon, with these six cases being the only known TBE cases among civilian travellers during this 11-year period. Nonetheless, given potential disease severity, pre-travel counselling for travellers to TBE-endemic areas should include information on measures to reduce the risk for TBE and other tick-borne diseases, including possible TBE vaccine use if a traveller's itinerary puts them at higher risk for infection. Clinicians should consider the diagnosis of TBE in a patient with a neurologic or febrile illness recently returned from a TBE-endemic country, particularly if a tick bite or possible tick exposure is reported.

A fatal case report of antibody-dependent enhancement of dengue virus type 1 following remote Zika virus infection.

BACKGROUND: Dengue virus (DENV) is endemic in many parts of the world. Antibody dependent enhancement (ADE) in DENV infections occurs when a person with primary immunity is infected by a second, different DENV strain. Antibodies to Zika virus (ZIKV), which emerged in the Western Hemisphere in 2015, are cross reactive with DENV and theoretically could provoke ADE in a DENV naïve individual. CASE PRESENTATION: DENV infection was suspected in a child who had recently returned from a one-month stay in the Dominican Republic. The child presented with fever, vomiting, abdominal pain, and in hypovolemic shock. Volume and pressor resuscitation were unsuccessful, and the child died less than 24 h after hospitalization. Laboratory results suggested an early acute first DENV infection since serum, plasma, and spinal fluid had DENV1 detected by polymerase chain reaction (PCR), yet the serum lacked IgG antibodies to DENV nonstructural protein 1 (NS1) of all four DENV serotypes. This acute DENV infection occurred in the presence of a remote ZIKV infection as determined by antibodies to ZIKV NS1 envelope by multiplex microsphere immunoassay and an exceptionally high plaque reduction neutralization titer to ZIKV. ZIKV IgG avidity index was high, confirming a past infection. DENV1 RNA was detected in all ten organs and tissues examined by PCR. The severe and fatal complications reported here suggest that a remote ZIKV infection may provoke an exaggerated immune response leading to hypovolemic shock when primarily infected by DENV1. CONCLUSION: We report the first known patient in the United States with a rapidly progressive and fatal case of travel-associated DENV in which prior exposure to ZIKV likely played a role in triggering an ADE phenomenon. This association of prior ZIKV immunity and subsequent new dengue infection is a worrisome phenomenon and an important contribution to the body of knowledge on immunity to flaviviruses.

Performance evaluation of antibody-based point-of-care devices intended for the identification of immune responses to SARS-CoV-2.

The novel coronavirus outbreak caused by the severe acute respiratory syndrome coronavirus (SARS-CoV-2) was first identified in December of 2019 in Wuhan, China. The local outbreak quickly rose to pandemic level that has spread to more than 188 countries with more than 19 million cases and 732,467 deaths worldwide. The current recommendation for testing is RT-PCR based tests of nasopharyngeal or alternatively nasal- and/or oropharyngeal swabs that detects infection with SARS-CoV-2 to diagnose acute infection. However, there is an urgent need for a quick and accurate antibody-based point-of-care test method to quickly identify evidence of SARS-CoV-2 infection among people who might be missed through active case finding and surveillance efforts. Serology tests measure the presence of antibodies in serum after infection. Here we compared the performance characteristics of 6 commercially available antibody-based point-of-care devices and their potential for identification of individuals infected at some time by SARS-CoV-2.

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.

Serosurvey for dengue virus infection among pregnant women in the West Nile virus enzootic community of El Paso Texas.

All 4 dengue viruses (DENV) cause sporadic outbreaks of human disease in the Rio Grande Valley along the US-Mexico border. In addition, West Nile virus (WNV) is enzootic in most border communities, and is the only arbovirus known to cause human disease in the El Paso, Texas community. In an effort to determine if DENV were also endemic in the El Paso community, a serosurvey was conducted among mothers at the time of delivery of their babies in selected hospitals. Cord-blood plasma samples obtained from mothers were tested for DENV antibody by an enzyme-linked immuno-sorbent assay (ELISA), plaque reduction neutralization test (PRNT) and a multiplex microsphere immunoassay. All DENV antibody positive plasma samples were also tested for WNV antibody by the same assays to consider the possibility that DENV antibody positive samples reflected WNV cross reactive antibody. The results indicated that 0.74% (11/1,472) of the mothers had a previous DENV infection and that 3.3% (48/1,472) had a previous WNV infection. Of these mothers, 0.20% (3/1,472) had antibody to both DENV and WNV as evidence of infection by both viruses. The results indicated that 0.2% (3/1472) of the mothers were positive for antibody to only WNV envelope, thus suggesting an undetermined flavivirus infection. Although 6 of the 11 DENV antibody positive mothers did not have a history of travel to a DENV endemic country, the findings of this survey provided further evidence of local transmission of WNV and suggested the possibility of focal autochthonous transmission of DENV in the El Paso community.

The Role of Antibody Testing for SARS-CoV-2: Is There One?

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) brought with it rapid development of both molecular and serologic assays for identification of COVID-19 infections. While Food and Drug Administration (FDA) emergency use authorization (EUA) is required for clinical application of SARS-CoV-2 molecular tests, submission for EUA is currently a voluntary process for manufacturers of serologic assays. The absence of FDA oversight of serologic tests is concerning given that the commercially available serologic assays are highly variable, differing in their format, the antibody class detected, the targeted antigen, and the acceptable specimen types. An added complication is the lack of a clear understanding for how such assays should be utilized and what the reported results ultimately indicate or, perhaps more importantly, what they do not indicate. Here, we provide a brief summary of the performance of a number of serologic assays reported in the literature, comment on what we do and do not know regarding our immune response to SARS-CoV-2, and provide a number of scenarios for which serologic testing will play a role during our global response to this pandemic.

Integrated Arbovirus Surveillance Improves the Detection Onset of Zika Virus in Panama.

We tested 700 serum samples collected throughout Panama from 2015 to 2016 for detecting antibodies and RNA of arboviruses. In convalescent specimens, microsphere immunoassay detected an antibody prevalence of 59.3% for dengue virus (DENV) and 30.3% for Zika virus (ZIKV), which included samples that were collected before the Panamanian surveillance system reported the first case of Zika in the country. For acute sera, the most common arbovirus was DENV with 18 positive samples (6%), followed by four (1.3%) of ZIKV and one (0.6%) of chikungunya virus (CHIKV). Our results indicate a change in the chronology of when ZIKV was first detected in Panama and stress the importance of integrating various approaches to enable improved surveillance of both endemic and emerging arboviruses.

Frequency and magnitude of seroreactivity to Babesia microti in 245 patients diagnosed by PCR in New York State.

Multiple methodologies have been used to detect antibodies to Babesia microti. Use of an indirect immunofluorescence assay (IFA) has been the most widely used approach, but IFAs have varied as to which antibody class or classes are being detected and in regard to cutoff titers. In this study, 245 different patients with polymerase chain reaction (PCR)-confirmed B. microti infection were tested by a polyvalent IFA using serum collected within 3 days of the date the blood sample for PCR testing was obtained. Of the 245 patients, 243 (99.2%) had a positive serologic test result (i.e., ≥1:64). Of the 243 patients who were seropositive, 242 (99.6%) had a titer of ≥1:256, 236 (97.1%) had a titer of ≥1:512, and 210 (86.4%) had a titer of ≥1:1024. In conclusion, high titer seropositivity based on a polyvalent IFA is to be expected at the time of PCR confirmation of active babesiosis in clinical practice.

A Multiplexed Serologic Test for Diagnosis of Lyme Disease for Point-of-Care Use.

Single multiplexed assays could replace the standard 2-tiered (STT) algorithm recommended for the laboratory diagnosis of Lyme disease if they perform with a specificity and a sensitivity superior or equal to those of the STT algorithm. We used human serum rigorously characterized to be sera from patients with acute- and convalescent-phase early Lyme disease, Lyme arthritis, and posttreatment Lyme disease syndrome, as well as the necessary controls (n = 241 samples), to select the best of 12 Borrelia burgdorferi proteins to improve our microfluidic assay (mChip-Ld). We then evaluated its serodiagnostic performance in comparison to that of a first-tier enzyme immunoassay and the STT algorithm. We observed that more antigens became positive as Lyme disease progressed from early to late stages. We selected three antigens (3Ag) to include in the mChip-Ld: VlsE and a proprietary synthetic 33-mer peptide (PepVF) to capture sensitivity in all disease stages and OspC for early Lyme disease. With the specificity set at 95%, the sensitivity of the mChip-Ld with 3Ag ranged from 80% (95% confidence interval [CI], 56% to 94%) and 85% (95% CI, 74% to 96%) for two panels of serum from patients with early Lyme disease and was 100% (95% CI, 83% to 100%) for serum from patients with Lyme arthritis; the STT algorithm detected early Lyme disease in the same two panels of serum from patients with early Lyme disease with a sensitivity of 48.5% and 75% and Lyme arthritis in serum from patients with Lyme arthritis with a sensitivity of 100%, and the specificity was 97.5% to 100%. The mChip-Ld platform outperformed the STT algorithm according to sensitivity. These results open the door for the development of a single, rapid, multiplexed diagnostic test for point-of-care use that can be designed to identify the Lyme disease stage.

Co-infections in Persons with Early Lyme Disease, New York, USA.

In certain regions of New York state, USA, Ixodes scapularis ticks can potentially transmit 4 pathogens in addition to Borrelia burgdorferi: Anaplasma phagocytophilum, Babesia microti, Borrelia miyamotoi, and the deer tick virus subtype of Powassan virus. In a prospective study, we systematically evaluated 52 adult patients with erythema migrans, the most common clinical manifestation of B. burgdorferi infection (Lyme disease), who had not received treatment for Lyme disease. We used serologic testing to evaluate these patients for evidence of co-infection with any of the 4 other tickborne pathogens. Evidence of co-infection was found for B. microti only; 4-6 patients were co-infected with Babesia microti. Nearly 90% of the patients evaluated had no evidence of co-infection. Our finding of B. microti co-infection documents the increasing clinical relevance of this emerging infection.

Direct Diagnostic Tests for Lyme Disease.

Borrelia burgdorferi was discovered to be the cause of Lyme disease in 1983, leading to seroassays. The 1994 serodiagnostic testing guidelines predated a full understanding of key B. burgdorferi antigens and have a number of shortcomings. These serologic tests cannot distinguish active infection, past infection, or reinfection. Reliable direct-detection methods for active B. burgdorferi infection have been lacking in the past but are needed and appear achievable. New approaches have effectively been applied to other emerging infections and show promise in direct detection of B. burgdorferi infections.

NMR metabolomics of cerebrospinal fluid differentiates inflammatory diseases of the central nervous system.

BACKGROUND: Myriad infectious and noninfectious causes of encephalomyelitis (EM) have similar clinical manifestations, presenting serious challenges to diagnosis and treatment. Metabolomics of cerebrospinal fluid (CSF) was explored as a method of differentiating among neurological diseases causing EM using a single CSF sample. METHODOLOGY/PRINCIPAL FINDINGS: 1H NMR metabolomics was applied to CSF samples from 27 patients with a laboratory-confirmed disease, including Lyme disease or West Nile Virus meningoencephalitis, multiple sclerosis, rabies, or Histoplasma meningitis, and 25 controls. Cluster analyses distinguished samples by infection status and moderately by pathogen, with shared and differentiating metabolite patterns observed among diseases. CART analysis predicted infection status with 100% sensitivity and 93% specificity. CONCLUSIONS/SIGNIFICANCE: These preliminary results suggest the potential utility of CSF metabolomics as a rapid screening test to enhance diagnostic accuracies and improve patient outcomes.

Performance Evaluation of a Prototype Architect Antibody Assay for Babesia microti.

The tick-borne protozoan Babesia microti is responsible for more than 200 cases of transfusion-transmitted babesiosis (TTB) infection in the United States that have occurred over the last 30 years. Measures to mitigate the risk of TTB include nucleic acid testing (NAT) and B. microti antibody testing. A fully automated prototype B. microti antibody test was developed on the Architect instrument. The specificity was determined to be 99.98% in volunteer blood donors (n = 28,740) from areas considered to have low endemicity for B. microti The sensitivity of the prototype test was studied in experimentally infected macaques; a total of 128 samples were detected as positive whereas 125 were detected as positive with an indirect fluorescent antibody (IFA) test; additionally, 83 (89.2%) of the PCR-positive samples were detected in contrast to 81 (87.1%) using an IFA test. All PCR-positive samples that tested negative in the prototype antibody test were preseroconversion period samples. Following seroconversion, periods of intermittent parasitemia occurred; 17 PCR-negative samples drawn in between PCR-positive bleed dates tested positive both by the prototype test (robust reactivity) and IFA test (marginal reactivity) prior to the administration of therapeutic drugs, indicating that the PCR test failed to detect samples from persistently infected macaques. The prototype assay detected 56 of 58 (96.6%) human subjects diagnosed with clinical babesiosis by both PCR and IFA testing. Overall, the prototype anti-Babesia assay provides a highly sensitive and specific test for the diagnosis of B. microti infection. While PCR is preferred for detection of window-period parasitemia, antibody tests detect infected subjects during periods of low-level parasitemia.

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.

Advances in Serodiagnostic Testing for Lyme Disease Are at Hand.

The cause of Lyme disease, Borrelia burgdorferi, was discovered in 1983. A 2-tiered testing protocol was established for serodiagnosis in 1994, involving an enzyme immunoassay (EIA) or indirect fluorescence antibody, followed (if reactive) by immunoglobulin M and immunoglobulin G Western immunoblots. These assays were prepared from whole-cell cultured B. burgdorferi, lacking key in vivo expressed antigens and expressing antigens that can bind non-Borrelia antibodies. Additional drawbacks, particular to the Western immunoblot component, include low sensitivity in early infection, technical complexity, and subjective interpretation when scored by visual examination. Nevertheless, 2-tiered testing with immunoblotting remains the benchmark for evaluation of new methods or approaches. Next-generation serologic assays, prepared with recombinant proteins or synthetic peptides, and alternative testing protocols, can now overcome or circumvent many of these past drawbacks. This article describes next-generation serodiagnostic testing for Lyme disease, focusing on methods that are currently available or near-at-hand.

Evaluation of a Novel Reporter Virus Neutralization Test for Serological Diagnosis of Zika and Dengue Virus Infection.

Currently, the laboratory diagnosis of Zika virus (ZIKV) infection is primarily through the detection of ZIKV RNA or antibodies against ZIKV proteins. The detection of viral RNA is highly sensitive and specific, but periods of viremia and viruria are brief, limiting the utility of ZIKV RNA assays. Instead, most ZIKV infections are diagnosed serologically, using an IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) for screening, followed by a confirmatory plaque reduction neutralization test (PRNT). Typical turnaround times vary, due to assay incubation periods and a lack of clinical laboratories performing these tests. Recently, a novel luciferase-ZIKV- and -dengue virus (DENV)-based serological assay, which considerably improves the turnaround times and throughput for ZIKV diagnosis, was described. Using the traditional PRNT as a reference method, we evaluated the performance characteristics of the reporter virus neutralization test (RVNT) with 258 clinical serum specimens. The ZIKV RVNT produced primary ZIKV screening and secondary confirmation results in 4 days, with 100% reproducibility. As a screening assay, the ZIKV RVNT displayed excellent diagnostic accuracy, sensitivity, and specificity of 98.2%, 100%, and 98.1%, respectively. As a confirmatory assay, the ZIKV RVNT titers displayed 93.1% agreement with the traditional ZIKV PRNT titers. Overall, the RVNT accurately and reliably detects neutralizing antibodies in patient serum specimens, with improved turnaround times, and can be used for the serological detection of ZIKV infections. Due to the homogeneous 96-well format, the RVNT has also significantly improved the assay throughput to allow testing of a large number of specimens in a single run.

A Rapid Zika Diagnostic Assay to Measure Neutralizing Antibodies in Patients.

The potential association of microcephaly and other congenital abnormalities with Zika virus (ZIKV) infection during pregnancy underlines the critical need for a rapid and accurate diagnosis. Due to the short duration of ZIKV viremia in infected patients, a serologic assay that detects antibody responses to viral infection plays an essential role in diagnosing patient specimens. The current serologic diagnosis of ZIKV infection relies heavily on the labor-intensive Plaque Reduction Neutralization Test (PRNT) that requires more than one-week turnaround time and represents a major bottleneck for patient diagnosis. To overcome this limitation, we have developed a high-throughput assay for ZIKV and dengue virus (DENV) diagnosis that can attain the "gold standard" of the current PRNT assay. The new assay is homogeneous and utilizes luciferase viruses to quantify the neutralizing antibody titers in a 96-well format. Using 91 human specimens, we showed that the reporter diagnostic assay has a higher dynamic range and maintains the relative specificity of the traditional PRNT assay. Besides the improvement of assay throughput, the reporter virus technology has also shortened the turnaround time to less than two days. Collectively, our results suggest that, along with the viral RT-PCR assay, the reporter virus-based serologic assay could be potentially used as the first-line test for clinical diagnosis of ZIKV infection as well as for vaccine clinical trials.

A Multiplex Microsphere Immunoassay for Zika Virus Diagnosis.

Rapid and accurate diagnosis of infectious agents is essential for patient care, disease control, and countermeasure development. The present serologic diagnosis of Zika virus (ZIKV) infection relies mainly on IgM-capture ELISA which is confounded with the flaw of cross-reactivity among different flaviviruses. In this communication, we report a multiplex microsphere immunoassay (MIA) that captures the diagnostic power of viral envelope protein (that elicits robust, yet cross-reactive antibodies to other flaviviruses) and the differential power of viral nonstructural proteins NS1 and NS5 (that induce more virus-type specific antibodies). Using 153 patient specimens with known ZIKV and/or dengue virus (DENV; a closely related flavivirus) infections, we showed that (i) ZIKV envelope-based MIA is equivalent or more sensitive than IgM-capture ELISA in diagnosing ZIKV infection, (ii) antibody responses to NS1 and NS5 proteins are more ZIKV-specific than antibody response to envelope protein, (iii) inclusion of NS1 and NS5 in the MIA improves the diagnostic accuracy when compared with the MIA that uses envelope protein alone. The multiplex MIA achieves a rapid diagnosis (turnaround time<4h) and requires small specimen volume (10µl) in a single reaction. This serologic assay could be developed for use in clinical diagnosis of ZIKV infection and for monitoring immune responses in vaccine trials.

Zika Virus Testing Considerations: Lessons Learned from the First 80 Real-Time Reverse Transcription-PCR-Positive Cases Diagnosed in New York State.

The performance and interpretation of laboratory tests for Zika virus (ZKV) continue to be evaluated. Serology is cross-reactive, laborious, and frequently difficult to interpret, and serum was initially solely recommended for molecular diagnosis. ZKV testing was initiated in January 2016 in New York State for symptomatic patients, pregnant women, their infants, and patients with Guillain-Barré syndrome who had traveled to areas with ZKV transmission. Subsequently, eligibility was expanded to pregnant women with sexual partners with similar travel histories. Serum and urine collected within 4 weeks of symptom onset or within 6 weeks of travel were tested with real-time reverse transcription-PCR (RT-PCR) assays targeting the ZKV envelope and NS2B genes. In this review of lessons learned from the first 80 positive cases in NYS, ZKV RNA was detected in urine only in 50 patients, in serum only in 19 patients, and in both samples concurrently in 11 patients, with average viral loads in urine a log higher than those in serum. Among 93 positive samples from the 80 patients, 41 were positive on both gene assays, 52 were positive on the envelope only, and none were positive on the NS2B only. Of the 80 infected patients, test results for 74 (93%) would have defined their infection status as not detected or equivocal if the requirement for positive results from two assay targets (two-target-positive requirement) in the initial federal guidance to public health laboratories was enforced, if urine was not tested, or if the extended eligibility time for molecular testing was not implemented. These changes facilitated more extensive molecular diagnosis of ZKV, reducing reliance on time-consuming and potentially inconclusive serology.

Sylvatic typhus associated with flying squirrels (Glaucomys volans) in New York State, United States.

Sylvatic typhus is an infrequent, potentially life-threatening emerging zoonotic disease. In January of 2009, the New York State Department of Health was notified of a familial cluster of two suspected cases. Due to the paucity of typhus cases in New York, epidemiologic and environmental investigations were conducted to establish rickettsial etiology and determine potential sources of infection. Patients presented with symptoms consistent with typhus, and serologic testing of each patient confirmed infection with typhus group rickettsiae. Serologic analysis of blood obtained from southern flying squirrels (Glaucomys volans) captured from the attic crawlspace above an enclosed front porch of the cases' residence indicated evidence of infection with Rickettsia prowazekii, with 100% seroprevalence (n=11). Both patients reported spending significant time on the porch and hearing animal activity above the ceiling prior to onset of illness, implicating these flying squirrels as the likely source of infection.