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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.

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.

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.

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.

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 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.

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., 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.

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.

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.

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.

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., Highlights • A high-throughput assay has been developed to measure neutralizing titers of patient specimens using a reporter ZIKV. • The new assay matches the “gold standard” of the traditional labor-intensive plaque assay when testing patient specimens. • The new assay has increased the diagnostic dynamic range and shortened the turnaround time to

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

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.

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.

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., Author summary Inflammation of the brain and spinal cord, known as encephalomyelitis, is a dangerous condition that can be caused by a wide range of pathogens, such as viruses and bacteria, and other medical conditions including autoimmunity or drug intoxications. Given the many possible causes, it is often difficult for clinicians treating patients with encephalomyelitis to identify the underlying cause, which in turn determines the appropriate treatment. Infections and other diseases causing neurological inflammation work by distinct biological mechanisms and, consequently, can cause unique biochemical changes that can be observed in cerebrospinal fluid of affected individuals. The researchers used a metabolomics technique to measure a range of small molecules in cerebrospinal fluid and examine biochemical differences in patients with encephalomyelitis caused by Lyme disease, West Nile Virus, multiple sclerosis, rabies, or fungal infection. The researchers found distinct differences in the biochemical profiles of patients whose encephalomyelitis was caused by infections versus patients with no infection, and also identified different patterns among the individual diseases. This study showed that metabolomics may be useful in improving diagnosis and treatment of diseases affecting the central nervous system by enhancing understanding of their unique effects on metabolism.

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., 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.

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.

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 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.

Infections with West Nile virus (WNV) are typically asymptomatic, but some patients experience severe neurological disease and even death. Over 1500 fatalities have resulted from the more than 37,000 WNV cases in the USA between 1999 and 2012. While it is clear that age is a significant risk factor, markers of immune status associated with susceptibility to severe infections are incompletely defined. We have taken advantage of stable characteristics of individual status to profile immune markers from a stratified cohort of healthy subjects with a history of asymptomatic or severe infection with WNV. We characterized individual variations in antibody and serum cytokine levels and genome-wide transcriptional profiles of peripheral blood cells (PBMCs). While antibody levels were not significantly different between cohorts, we found that subjects with a history of severe infection had significantly lower levels of serum IL-4, and that these changes in IL-4 levels were associated with altered gene expression patterns in PBMCs. In addition, we identified a signature of 105 genes that displayed altered expression levels when comparing subjects with a history of asymptomatic or severe infection. These results suggest that systems-level analysis of immune system status can be used to identify factors relevant for susceptibility to severe infections, and specifically point to an important contribution for IL-4 in resistance to WNV infection.

TOC Summary: The epidemiologic pattern and limited laboratory testing indicate that this virus lineage might account for most of these illnesses., Powassan virus, a member of the tick-borne encephalitis group of flaviviruses, encompasses 2 lineages with separate enzootic cycles. The prototype lineage of Powassan virus (POWV) is principally maintained between Ixodes cookei ticks and the groundhog (Marmota momax) or striped skunk (Mephitis mephitis), whereas the deer tick virus (DTV) lineage is believed to be maintained between Ixodes scapularis ticks and the white-footed mouse (Peromyscus leucopus). We report 14 cases of Powassan encephalitis from New York during 2004–2012. Ten (72%) of the patients were residents of the Lower Hudson Valley, a Lyme disease–endemic area in which I. scapularis ticks account for most human tick bites. This finding suggests that many of these cases were caused by DTV rather than POWV. In 2 patients, DTV infection was confirmed by genetic sequencing. As molecular testing becomes increasingly available, more cases of Powassan encephalitis may be determined to be attributable to the DTV lineage.

The antemortem diagnosis of rabies in humans employs techniques that require accuracy, speed, and sensitivity. A combination of histochemical analysis, in vitro virus isolation, immunological methods, and molecular amplification procedures are utilized in efforts to diagnose the disease. Modern medicine now offers potentially life-saving treatment for a disease that was considered invariably fatal once clinical signs develop. However, medical intervention efforts require a rapid and accurate diagnosis as early in the course of clinical disease as possible. Indirect fluorescent-antibody (IFA) testing on cerebrospinal fluid and serum specimens provides rapid results, but the specificity of the assay has not been well studied. Because false-positive IFA results could significantly affect patient treatment and outcomes, it is critical to understand the specificity of this assay. In this study, IFA testing was performed on 135 cerebrospinal fluid and serum specimens taken from patients with viral encephalitis or a presumed viral infection involving an agent other than rabies virus. Results indicate that false-positive results can occur in interpreting the rabies IFA test. Staining patterns morphologically similar to antirabies staining were observed in 7 of the 135 cerebrospinal fluid specimens examined. In addition, a majority of the cerebrospinal fluid specimens tested from patients with encephalitis presented immunoglobulin that bound to antigens present in the cell culture substrate. Of marked concern was the frequent presence of cross-reactive antibodies in encephalitis cases associated with West Nile and Powassan flaviviruses. Because IFA testing for rabies on human specimens may result in false-positive results, it should not be used as the sole basis for initiating antirabies treatment.

Cache Valley virus was initially isolated from mosquitoes and had been linked to central nervous system-associated diseases. A case of Cache Valley virus infection is described. The virus was cultured from a patient's cerebrospinal fluid and identified with real-time reverse transcription-PCR and sequencing, which also yielded the complete viral coding sequences.

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.

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.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.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.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.

The development and validation of a microsphere immunoassay (MIA) to detect equine antibodies to the major structural proteins of equine arteritis virus (EAV) are described. The assay development process was based on the cloning and expression of genes for full-length individual major structural proteins (GP5 amino acids 1 to 255 [GP5 1-255 ], M 1-162 , and N 1-110 ), as well as partial sequences of these structural proteins (GP5 1-116 , GP5 75-112 , GP5 55-98 , M 88-162 , and N 1-69 ) that constituted putative antigenic regions. Purified recombinant viral proteins expressed in Escherichia coli were covalently bound to fluorescent polystyrene microspheres and analyzed with the Luminex xMap 100 instrument. Of the eight recombinant proteins, the highest concordance with the virus neutralization test (VNT) results was obtained with the partial GP5 55-98 protein. The MIA was validated by testing a total of 2,500 equine serum samples previously characterized by the VNT. With the use of an optimal median fluorescence intensity cutoff value of 992, the sensitivity and specificity of the assay were 92.6% and 92.9%, respectively. The GP5 55-98 MIA and VNT outcomes correlated significantly ( r = 0.84; P < 0.0001). Although the GP5 55-98 MIA is less sensitive than the standard VNT, it has the potential to provide a rapid, convenient, and more economical test for screening equine sera for the presence of antibodies to EAV, with the VNT then being used as a confirmatory assay.

West Nile virus (WNV) causes severe neurological disease in less than 1% of infections. However, meningoencephalitis may be more common in immunosuppressed transplant patients. In 2002, a WNV outbreak occurred in our region. To determine the spectrum of disease of community acquired WNV infection and assess public health behavior patterns in transplant recipients, we carried out a seroprevalence study. Patients were enrolled from outpatient transplant clinics in October 2002 and sera were screened for WNV. Questionnaires about WNV were provided to patients. Eight hundred sixteen organ transplant patients were enrolled. The seroprevalence of WNV IgM was 2/816 (0.25%; 95% CI 0.03-0.88%). By extrapolation to our entire transplant population of 2360 patients, and using data from hospital-based surveillance, the risk of meningoencephalitis in a transplant patient infected with WNV is estimated to be 40% (95% CI 16-80%). With regards to knowledge and behavior, 56% patients knew of and 47% used at least one protective measure against WNV. Only 33% used insect repellent. The risk of meningoencephalitis in transplant recipients is much higher than in the general population. There is incomplete knowledge and poor rates of compliance amongst patients with regards to WNV prevention.

The epidemic of West Nile virus (WNV) in the USA in 2002 represents the largest outbreak of meningoencephalitis in the Western Hemisphere ever reported. Besides natural transmission by mosquitoes, five new modes of WNV transmission to humans have been reported: blood transfusion, organ transplantation, transplacental transmission, breastfeeding and laboratory-acquired infection. The recognition of these new transmission routes has made the development of rapid and accurate serological diagnosis of WNV infection a public health priority. In this article, the current serologic assays for WNV diagnosis are reviewed, including immunoglobulin M antibody-capture ELISA, immunoglobulin G ELISA, indirect fluorescent antibody tests, hemagglutination inhibition tests and plaque reduction neutralization tests. The recently developed immunoassays that use purified recombinant envelope and nonstructural protein 5 of WNV as antigens are also reviewed. The nonstructural protein 5 protein-based assay can reliably discriminate between WNV and dengue or St. Louis encephalitis virus, as well as between natural WNV infection and flavivirus vaccination.

We describe a 54-year-old spleen-intact man with transfusion-associated Babesia microti infection after a heart transplant. Adult respiratory distress syndrome developed in the patient, and he required mechanical ventilation. Our experiences with this patient suggest that babesiosis should be considered in the differential diagnosis of transplant patients who have fever and hemolytic anemia.

West Nile (WN) virus is a mosquito-borne flavivirus that emerged in the United States in 1999 and can cause fatal encephalitis. Envelope (E) protein cDNA from a WN virus isolate recovered from Culex pipiens in Connecticut was expressed in Escherichia coli. The recombinant E protein was purified and used as Ag in immunoblot assays and immunization experiments. Patients with WN virus infection had Abs that recognized the recombinant E protein. C3H/HeN mice immunized with E protein developed E protein Abs and were protected from infection with WN virus. Passive administration of E protein antisera was also sufficient to afford immunity. E protein is a candidate vaccine to prevent WN virus infection.

The major antigenic protein 2 (MAP2) homolog of Ehrlichia chaffeensis was cloned and expressed. The recombinant protein was characterized and tested in an enzyme-linked immunosorbent assay (ELISA) format for potential application in the serodiagnosis of human monocytic ehrlichiosis. The recombinant protein, which contained a C-terminal polyhistidine tag, had a molecular mass of approximately 26 kDa. The antigen was clearly identified by Western immunoblotting using antihistidine antibody. However, immune sera failed to react with the recombinant on immunoblots when the antigen was denatured by heat or reduced using β-mercaptoethanol. The recombinant MAP2 (rMAP2) was used in an ELISA format with 60 blinded serum samples. Twenty of the serum samples were previously demonstrated to contain antibodies reactive with E. chaffeensis by indirect immunofluorescence assays (IFAs). The remaining 40 samples were seronegative. All samples negative by IFA were also found to be negative for antibodies against the rMAP2 of E. chaffeensis by using the ELISA. Only 1 of 20 IFA-positive samples tested negative in the rMAP2 ELISA. There was 100% agreement using IFA-negative samples and 95% agreement using IFA-positive samples, resulting in a 97.5% overall agreement between the two assays. These data suggest that the rMAP2 homolog of E. chaffeensis may have potential as a test antigen for the serodiagnosis of human monocytic ehrlichiosis. To our knowledge, this recombinant is unique because it is thus far the only E. chaffeensis recombinant antigen that has been shown to work in an ELISA format.

purpose: To describe a nosocomial outbreak of Legionella micdadei pneumonia in transplant patients and to characterize the source of the outbreak and the control measures utilized. subjects and methods: We performed retrospective Legionella micdadei serologic testing to enhance case finding in transplant patients with pneumonia that lacked a documented microbial etiology, as well as prospective environmental surveillance of water sites and testing for Legionella in clinical specimens. results: During a 3-month period, 12 cases of Legionella micdadei pneumonia were identified either by culture or serologic testing among 38 renal and cardiac transplant patients. Legionella micdadei isolates from hot water sources were found by pulsed-field gel electrophoresis to have a DNA banding pattern that was identical to the isolates from the first 3 culture-positive cases and from 2 cases that occurred 16 months later. conclusions: Hospitals caring for organ transplant recipients and other immunosuppressed patients must be aware of the possibility of environmental sources of outbreaks of Legionella infection. A first-line screen with the Legionella urine antigen test will identify Legionella pneumophila serogroup 1. However, specific cultures in outbreak situations should be considered to identify other Legionella pneumophila serotypes and the nonpneumophila Legionella species.

Human granulocytic ehrlichiosis was first described in the United States, in the northern Midwest, in 1994.1 Human granulocytic ehrlichiosis is caused by an organism, still referred to as the agent of human granulocytic ehrlichiosis, that is similar to two animal pathogens, Ehrlichia phagocytophila and E. equi. 2–4 Transmission of human granulocytic ehrlichiosis occurs through the bites of ixodes ticks, which are the arthropod vectors for Borrelia burgdorferi and Babesia microti. 5,6 Human granulocytic ehrlichiosis is an acute, febrile, nonspecific illness that may be severe enough to cause hospitalization and even death, particularly in the elderly.1,7,8 We describe a . . .

Human blood collected from two patients from Westchester County, New York with human granulocytic ehrlichia (HGE) infection was inoculated into two ponies. Inoculated ponies developed clinical signs similar to a previous report (Madigan et al., 1995). Histopathological changes involved follicular hyperplasia of lymphoid tissues. HGE DNA was detected by PCR in muscle, fascia, peritoneum, and adrenal gland after the ponies produced a high level of antibodies to HGE. We suggest that HGE may reside in poorly vascularized connective tissues, where the antibodies may have some difficulties to penetrate, resulting in persistent infection. Since HGE and E. equi cause very similar diseases in both humans and horses, they may be the same organism with minor genetic differences.

Serological testing at the New York State Department of Health for human granulocytic ehrlichiosis in the residents of Westchester County, N.Y., was performed with specimens from 176 patients by the indirect fluorescent-antibody (IFA) technique with Ehrlichia equi MRK-infected neutrophils. To understand whether human monocytotropic ehrlichiosis also occurs in this northeastern geographic region, specimens were also tested for antibodies to Ehrlichia chaffeensis Arkansas. Screening tests and immunoblots for Lyme disease (Borrelia burgdorferi infection) were also performed. Thirty-two patients had antibodies only to E. equi and 21 patients had antibodies to both E. equi and E. chaffeensis, whereas 12 patients had only E. chaffeensis antibodies by the IFA technique. The remaining patients did not have antibodies to either ehrlichia. Eighteen serum samples from 13 of these patients were coded and sent to the Ehrlichia Research Laboratory (Baltimore, Md.) for repeat analysis by the IFA test and for E. equi and E. chaffeensis immunoblots. Immunoblot analysis for E. equi in samples with positive IFA test results confirmed the results for eight of the nine specimens. Immunoblot analyses for E. chaffeensis were negative for all 18 serum samples. Borrelia-reactive antibodies were found in sera both from patients with granulocytic ehrlichiosis and from patients with monocytotropic ehrlichiosis from New York State. Our results suggest that E. equi antigen is an appropriate substrate for identifying human granulocytic ehrlichiosis. E. chaffeensis antigen lacks appropriate sensitivity to serve as a surrogate substrate for the detection of human granulocytic ehrlichiosis and should be used solely for the diagnosis of human monocytotropic ehrlichiosis. Heat shock proteins may, in some cases, cause cross-reactivity between B. burgdorferi and ehrlichiae.

Background Deer tick virus (DTV) is a tick-borne flavivirus that has only recently been appreciated as a cause of viral encephalitis. We describe the clinical presentation of a patient who had DTV encephalitis diagnosed before death and survived for 8 months despite severe neurologic dysfunction. Methods Diagnosis was made from a cerebrospinal fluid specimen, using a flavivirus-specific polymerase chain-reaction assay followed by sequence confirmation, and the phylogeny was analyzed. Serologic testing, including plaque reduction neutralization testing, was also performed. Results Molecular analysis indicated that the virus was closely related to 2 strains of DTV that had been detected in Ixodes scapularis ticks from Massachusetts in 1996 and in the brain of a patient from New York in 2007. Conclusions DTV encephalitis should be considered in the differential diagnosis of encephalitis in geographic areas that are endemic for Lyme disease.

Babesiosis is usually acquired from a tick bite or through a blood transfusion. We report a case of babesiosis in an infant for whom vertical transmission was suggested by evidence of Babesia spp. antibodies in the heel-stick blood sample and confirmed by detection of Babesia spp. DNA in placenta tissue.

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. Lyme disease (LD) is the most common vector-borne disease in the United States, with a reported incidence of nearly 35,000 new cases annually (10, 21). There are three disease stages: stage I is the early acute phase, characterized by a rash (erythema migrans [EM]) that occurs in at least 70% of patients; stage II represents early disseminated infection, including lymphocytic meningitis, cranial neuropathy, radiculopathy, and Lyme carditis; and stage III represents late disseminated

West Nile virus (WNV; Flavivirus, Flaviviridae) is a spherical enveloped virion containing single-stranded, positive-sense RNA, approximately 11 kb in length. The virus is the most widely distributed flavivirus in the world. Genetic analysis reveals two major lineages of virus, I and II, and several possible newly recognized lineages. Lineage I strains are most commonly associated with outbreaks of neurologic disease, although lineage II virus has led to large epidemics of fever, as in South Africa in 1974. Infection with WNV leads to a wide range of diseases from mildly febrile to severely neurologic, but asymptomatic -infections occur most frequently. Approximately one in 140 infected individuals develop neurologic -disease. The virus is maintained in an enzootic cycle, where it is transmitted between ornithophilic mosquitoes of the Culex genus and predominantly passeriform birds. Equines and humans are considered incidental hosts since they do not mount high enough viremia for mosquitoes to become infected -following feeding. Laboratory diagnosis of WNV infection is predominantly serological, although -caution is advised because of the high degree of cross-reactivity among flaviviruses. Field specimens, especially mosquitoes and dead birds, collected as part of surveillance programs, are tested for the presence of viral nucleic acid, viral antigen, or infectious virus. Rapid test protocols have been developed in response to the expansion of WNV in the United States. Since WNV is classified as a Biosafety Level-3 (BSL-3) agent by CDC, it is recommended that once this virus is identified in a diagnostic specimen, all infectious virus should be handled in a BSL-3 laboratory in Class II biosafety cabinets by laboratory staff who are trained to work at this level of containment. Assay protocols are described and the necessary equipment and supplies listed.