Semicentennial of Human Babesiosis, Nantucket Island.

Fifty years ago, the index case of human babesiosis due to Babesia microti was diagnosed in a summer resident of Nantucket Island. Human babesiosis, once called "Nantucket fever" due to its seeming restriction to Nantucket and the terminal moraine islands of southern New England, has emerged across the northeastern United States to commonly infect people wherever Lyme disease is endemic. We review the history of babesiosis on Nantucket, analyze its epidemiology and ecology there, provide summaries of the first case histories, and comment on its future public health burden.

Detection of Borrelia burgdorferi Cell-free DNA in Human Plasma Samples for Improved Diagnosis of Early Lyme Borreliosis.

BACKGROUND: Laboratory confirmation of early Lyme borreliosis (LB) is challenging. Serology is insensitive during the first days to weeks of infection, and blood polymerase chain reaction (PCR) offers similarly poor performance. Here, we demonstrate that detection of Borrelia burgdorferi (B.b.) cell-free DNA (cfDNA) in plasma can improve diagnosis of early LB. METHODS: B.b. detection in plasma samples using unbiased metagenomic cfDNA sequencing performed by a commercial laboratory (Karius Inc) was compared with serology and blood PCR in 40 patients with physician-diagnosed erythema migrans (EM), 28 of whom were confirmed to have LB by skin biopsy culture (n = 18), seroconversion (n = 2), or both (n = 8). B.b. sequence analysis was performed using investigational detection thresholds, different from Karius' clinical test. RESULTS: B.b. cfDNA was detected in 18 of 28 patients (64%) with laboratory-confirmed EM. In comparison, sensitivity of acute-phase serology using modified 2-tiered testing (MTTT) was 50% (P = .45); sensitivity of blood PCR was 7% (P = .0002). Combining B.b. cfDNA detection and MTTT increased diagnostic sensitivity to 86%, significantly higher than either approach alone (P ≤ .04). B.b. cfDNA sequences matched precisely with strain-specific sequence generated from the same individual's cultured B.b. isolate. B.b. cfDNA was not observed at any level in plasma from 684 asymptomatic ambulatory individuals. Among 3000 hospitalized patients tested as part of clinical care, B.b. cfDNA was detected in only 2 individuals, both of whom had clinical presentations consistent with LB. CONCLUSIONS: This is the first report of B.b. cfDNA detection in early LB and a demonstration of potential diagnostic utility. The combination of B.b. cfDNA detection and acute-phase MTTT improves clinical sensitivity for diagnosis of early LB.

Antigen Discovery, Bioinformatics and Biological Characterization of Novel Immunodominant Babesia microti Antigens.

Babesia microti is an intraerythrocytic parasite and the primary causative agent of human babesiosis. It is transmitted by Ixodes ticks, transfusion of blood and blood products, organ donation, and perinatally. Despite its global public health impact, limited progress has been made to identify and characterize immunodominant B. microti antigens for diagnostic and vaccine use. Using genome-wide immunoscreening, we identified 56 B. microti antigens, including some previously uncharacterized antigens. Thirty of the most immunodominant B. microti antigens were expressed as recombinant proteins in E. coli. Among these, the combined use of two novel antigens and one previously described antigen provided 96% sensitivity and 100% specificity in identifying B. microti antibody containing sera in an ELISA. Using extensive computational sequence and bioinformatics analyses and cellular localization studies, we have clarified the domain architectures, potential biological functions, and evolutionary relationships of the most immunodominant B. microti antigens. Notably, we found that the BMN-family antigens are not monophyletic as currently annotated, but rather can be categorized into two evolutionary unrelated groups of BMN proteins respectively defined by two structurally distinct classes of extracellular domains. Our studies have enhanced the repertoire of immunodominant B. microti antigens, and assigned potential biological function to these antigens, which can be evaluated to develop novel assays and candidate vaccines.

Superior real-time polymerase chain reaction detection of Babesia microti parasites in whole blood utilizing high-copy BMN antigens as amplification targets.

BACKGROUND: Babesiosis is a zoonotic disease transmitted to humans by the bite of infected ticks and caused by apicomplexan parasites, most commonly Babesia microti. Additionally, blood and blood products collected from asymptomatically infected blood donors may cause transfusion-transmitted infections in recipients. Highly sensitive molecular assays that detect parasite nucleic acid are needed for laboratory diagnosis and to identify and defer clinically silent but parasitemic blood donors. STUDY DESIGN AND METHODS: Here we report the development and analytical and clinical characterization of a real-time polymerase chain reaction (RT-PCR)-based assay for the detection of B. microti genomic DNA in whole blood. We evaluate the detection of Babesia parasites using two separate targets, the traditional18S ribosomal subunit gene (Bm18S) and members of the abundant BMN family of seroreactive antigens (BmBMN). RESULTS: Analytical sensitivity determination using a probit analysis demonstrated an analytical sensitivity of 30.9 parasites/mL for 18S amplification and 10.0 parasites/mL for BMN amplification The BMN primer set also demonstrates superior sensitivity for serial dilution panels prepared from clinically diagnosed Babesia-infected blood samples, generally detecting 10-fold more dilute nucleic acid. CONCLUSIONS: Cumulatively, our data demonstrate that RT-PCR detection of the BMN family of seroreactive antigens reflects a sensitive and superior assay for the detection of B. microti in whole blood samples.

Evaluation of Modified 2-Tiered Serodiagnostic Testing Algorithms for Early Lyme Disease.

Background: The conventional 2-tiered serologic testing protocol for Lyme disease (LD), an enzyme immunoassay (EIA) followed by immunoglobulin M and immunoglobulin G Western blots, performs well in late-stage LD but is insensitive in patients with erythema migrans (EM), the most common manifestation of the illness. Western blots are also complex, difficult to interpret, and relatively expensive. In an effort to improve test performance and simplify testing in early LD, we evaluated several modified 2-tiered testing (MTTT) protocols, which use 2 assays designed as first-tier tests sequentially, without the need of Western blots. Methods: The MTTT protocols included (1) a whole-cell sonicate (WCS) EIA followed by a C6 EIA; (2) a WCS EIA followed by a VlsE chemiluminescence immunoassay (CLIA); and (3) a variable major protein-like sequence, expressed (VlsE) CLIA followed by a C6 EIA. Sensitivity was determined using serum from 55 patients with erythema migrans; specificity was determined using serum from 50 patients with other illnesses and 1227 healthy subjects. Results: Sensitivity of the various MTTT protocols in patients with acute erythema migrans ranged from 36% (95% confidence interval [CI], 25%-50%) to 54% (95% CI, 42%-67%), compared with 25% (95% CI, 16%-38%) using the conventional protocol (P = .003-0.3). Among control subjects, the 3 MTTT protocols were similarly specific (99.3%-99.5%) compared with conventional 2-tiered testing (99.5% specificity; P = .6-1.0). Conclusions: Although there were minor differences in sensitivity and specificity among MTTT protocols, each provides comparable or greater sensitivity in acute EM, and similar specificity compared with conventional 2-tiered testing, obviating the need for Western blots.

Babesia microti from humans and ticks hold a genomic signature of strong population structure in the United States.

BACKGROUND: Babesia microti is an emerging tick-borne apicomplexan parasite with increasing geographic range and incidence in the United States. The rapid expansion of B. microti into its current distribution in the northeastern USA has been due to the range expansion of the tick vector, Ixodes scapularis, upon which the causative agent is dependent for transmission to humans. RESULTS: To reconstruct the history of B. microti in the continental USA and clarify the evolutionary origin of human strains, we used multiplexed hybrid capture of 25 B. microti isolates obtained from I. scapularis and human blood. Despite low genomic variation compared with other Apicomplexa, B. microti was strongly structured into three highly differentiated genetic clusters in the northeastern USA. Bayesian analyses of the apicoplast genomes suggest that the origin of the current diversity of B. microti in northeastern USA dates back 46 thousand years with a signature of recent population expansion in the last 1000 years. Human-derived samples belonged to two rarely intermixing clusters, raising the possibility of highly divergent infectious phenotypes in humans. CONCLUSIONS: Our results validate the multiplexed hybrid capture strategy for characterizing genome-wide diversity and relatedness of B. microti from ticks and humans. We find strong population structure in B. microti samples from the Northeast indicating potential barriers to gene flow.

A targeted immunomic approach identifies diagnostic antigens in the human pathogen Babesia microti.

BACKGROUND: Babesia microti is a protozoan parasite responsible for the majority of reported cases of human babesiosis and a major risk to the blood supply. Laboratory screening of blood donors may help prevent transfusion-transmitted babesiosis but there is no Food and Drug Administration-approved screening method yet available. Development of a sensitive, specific, and highly automated B. microti antibody assay for diagnosis of acute babesiosis and blood screening could have an important impact on decreasing the health burden of B. microti infection. STUDY DESIGN AND METHODS: Herein, we take advantage of recent advances in B. microti genomic analyses, field surveys of the reservoir host, and human studies in endemic areas to apply a targeted immunomic approach to the discovery of B. microti antigens that serve as signatures of active or past babesiosis infections. Of 19 glycosylphosphatidylinositol (GPI)-anchored protein candidates (BmGPI1-19) identified in the B. microti proteome, 17 were successfully expressed, printed on a microarray chip, and used to screen sera from uninfected and B. microti-infected mice and humans to determine immune responses that are associated with active and past infection. RESULTS: Antibody responses to various B. microti BmGPI antigens were detected and BmGPI12 was identified as the best biomarker of infection that provided high sensitivity and specificity when used in a microarray antibody assay. CONCLUSION: BmGPI12 alone or in combination with other BmGPI proteins is a promising candidate biomarker for detection of B. microti antibodies that might be useful in blood screening to prevent transfusion-transmitted babesiosis.

Borrelia miyamotoi Disease: Neither Lyme Disease Nor Relapsing Fever.

Borrelia miyamotoi disease (BMD) is a newly recognized borreliosis globally transmitted by ticks of the Ixodes persulcatus species complex. Once considered to be a tick symbiont with no public health implications, B miyamotoi is increasingly recognized as the agent of a nonspecific febrile illness often misdiagnosed as acute Lyme disease without rash, or as ehrlichiosis. The frequency of its diagnosis in the northeastern United States is similar to that of human granulocytic ehrlichiosis. A diagnosis of BMD is confirmed by polymerase chain reaction analysis of acute blood samples, or by seroconversion using a recombinant glycerophosphodiester phosphodiesterase enzyme immunoassay. BMD is successfully treated with oral doxycycline or amoxicillin.

Expression, Purification, and Biological Characterization of Babesia microti Apical Membrane Antigen 1.

The intraerythrocytic apicomplexan Babesia microti, the primary causative agent of human babesiosis, is a major public health concern in the United States and elsewhere. Apicomplexans utilize a multiprotein complex that includes a type I membrane protein called apical membrane antigen 1 (AMA1) to invade host cells. We have isolated the full-length B. microti AMA1 (BmAMA1) gene and determined its nucleotide sequence, as well as the amino acid sequence of the AMA1 protein. This protein contains an N-terminal signal sequence, an extracellular region, a transmembrane region, and a short conserved cytoplasmic tail. It shows the same domain organization as the AMA1 orthologs from piroplasm, coccidian, and haemosporidian apicomplexans but differs from all other currently known piroplasmida, including other Babesia and Theileria species, in lacking two conserved cysteines in highly variable domain III of the extracellular region. Minimal polymorphism was detected in BmAMA1 gene sequences of parasite isolates from six babesiosis patients from Nantucket. Immunofluorescence microscopy studies showed that BmAMA1 is localized on the cell surface and cytoplasm near the apical end of the parasite. Native BmAMA1 from parasite lysate and refolded recombinant BmAMA1 (rBmAMA1) expressed in Escherichia coli reacted with a mouse anti-BmAMA1 antibody using Western blotting. In vitro binding studies showed that both native BmAMA1 and rBmAMA1 bind to human red blood cells (RBCs). This binding is trypsin and chymotrypsin treatment sensitive but neuraminidase independent. Incubation of B. microti parasites in human RBCs with a mouse anti-BmAMA1 antibody inhibited parasite growth by 80% in a 24-h assay. Based on its antigenically conserved nature and potential role in RBC invasion, BmAMA1 should be evaluated as a vaccine candidate.

Borrelia miyamotoi Disease in the Northeastern United States: A Case Series.

BACKGROUND: The first recognized cases of Borrelia miyamotoi disease (BMD) in North America were reported in the northeastern United States in 2013. OBJECTIVE: To further describe the clinical spectrum and laboratory findings for BMD. DESIGN: Case series. SETTING: Patients presenting to primary care offices, emergency departments, or urgent care clinics in 2013 and 2014. PARTICIPANTS: Acutely febrile patients from the northeastern United States in whom the treating health care providers suspected and ordered testing for tick-transmitted infections. MEASUREMENTS: Whole-blood polymerase chain reaction (PCR) testing was performed for the presence of specific DNA sequences of common tickborne infections (including BMD). Serologic testing for B. miyamotoi was performed using a recombinant glycerophosphodiester phosphodiesterase (rGlpQ) protein. Clinical records were analyzed to identify the major features of acute disease. RESULTS: Among 11,515 patients tested, 97 BMD cases were identified by PCR. Most of the 51 case patients on whom clinical histories were reviewed presented with high fever, chills, marked headache, and myalgia or arthralgia. Twenty-four percent were hospitalized. Elevated liver enzyme levels, neutropenia, and thrombocytopenia were common. At presentation, 16% of patients with BMD were seropositive for IgG and/or IgM antibody to B. miyamotoi rGlpQ. Most (78%) had seropositive convalescent specimens. Symptoms resolved after treatment with doxycycline, and no chronic sequelae or symptoms were observed. LIMITATION: Findings were based on specimens submitted for testing to a reference laboratory, and medical records of only 51 of the 97 case patients with BMD were reviewed. CONCLUSION: Patients with BMD presented with nonspecific symptoms, including fever, headache, chills, myalgia, and arthralgia. Laboratory confirmation of BMD was possible by PCR on blood from acutely symptomatic patients who were seronegative at presentation. Borrelia miyamotoi disease may be an emerging tickborne infection in the northeastern United States. PRIMARY FUNDING SOURCE: IMUGEN.

Borrelia miyamotoi sensu lato seroreactivity and seroprevalence in the northeastern United States.

Borrelia miyamotoi sensu lato, a relapsing fever Borrelia sp., is transmitted by the same ticks that transmit B. burgdorferi (the Lyme disease pathogen) and occurs in all Lyme disease-endemic areas of the United States. To determine the seroprevalence of IgG against B. miyamotoi sensu lato in the northeastern United States and assess whether serum from B. miyamotoi sensu lato-infected persons is reactive to B. burgdorferi antigens, we tested archived serum samples from area residents during 1991-2012. Of 639 samples from healthy persons, 25 were positive for B. miyamotoi sensu lato and 60 for B. burgdorferi. Samples from ≈10% of B. miyamotoi sensu lato-seropositive persons without a recent history of Lyme disease were seropositive for B. burgdorferi. Our results suggest that human B. miyamotoi sensu lato infection may be common in southern New England and that B. burgdorferi antibody testing is not an effective surrogate for detecting B. miyamotoi sensu lato infection.

Monitoring human babesiosis emergence through vector surveillance New England, USA.

Human babesiosis is an emerging tick-borne disease caused by the intraerythrocytic protozoan Babesia microti. Its geographic distribution is more limited than that of Lyme disease, despite sharing the same tick vector and reservoir hosts. The geographic range of babesiosis is expanding, but knowledge of its range is incomplete and relies exclusively on reports of human cases. We evaluated the utility of tick-based surveillance for monitoring disease expansion by comparing the ratios of the 2 infections in humans and ticks in areas with varying B. microti endemicity. We found a close association between human disease and tick infection ratios in long-established babesiosis-endemic areas but a lower than expected incidence of human babesiosis on the basis of tick infection rates in new disease-endemic areas. This finding suggests that babesiosis at emerging sites is underreported. Vector-based surveillance can provide an early warning system for the emergence of human babesiosis.

Quantitative PCR for detection of Babesia microti in Ixodes scapularis ticks and in human blood.

Babesia microti, the primary cause of human babesiosis in the United States, is transmitted by Ixodes scapularis ticks; transmission may also occur through blood transfusion and transplacentally. Most infected people experience a viral-like illness that resolves without complication, but those who are immunocompromised may develop a serious and prolonged illness that is sometimes fatal. The geographic expansion and increasing incidence of human babesiosis in the northeastern and midwestern United States highlight the need for high-throughput sensitive and specific assays to detect parasites in both ticks and humans with the goals of improving epidemiological surveillance, diagnosis of acute infections, and screening of the blood supply. Accordingly, we developed a B. microti-specific quantitative PCR (qPCR) assay (named BabMq18) designed to detect B. microti DNA in tick and human blood samples using a primer and probe combination that targets the 18S rRNA gene of B. microti. This qPCR assay was compared with two nonquantitative B. microti PCR assays by testing tick samples and was found to exhibit higher sensitivity for detection of B. microti DNA. The BabMq18 assay has a detection threshold of 10 copies per reaction and does not amplify DNA in I. scapularis ticks infected with Babesia odocoilei, Borrelia burgdorferi, Borrelia miyamotoi, or Anaplasma phagocytophilum. This highly sensitive and specific qPCR assay can be used for detection of B. microti DNA in both tick and human samples. Finally, we report the prevalence of B. microti infection in field-collected I. scapularis nymphs from three locations in southern New England that present disparate incidences of human babesiosis.

Development of a real-time polymerase chain reaction assay for sensitive detection and quantitation of Babesia microti infection.

BACKGROUND: Babesia microti, the most frequently implicated pathogen in transfusion-transmitted babesiosis, is widely endemic in the Northeast and upper Midwestern United States. High seroprevalence in endemic areas limits antibody-based donor screening. A high-performance molecular test is needed to identify donors in the preseroconversion window phase as well as to discriminate past serologic exposure with parasite clearance from continued parasitemia. STUDY DESIGN AND METHODS: Frozen Babesia-spiked whole blood was microcentrifuged, and the supernatant transferred and microcentrifuged again to concentrate the parasite. The DNA was extracted and amplified using real-time polymerase chain reaction (PCR) using Babesia-specific primers. The assay was employed in three series of experiments: 1) a validation and optimization spiking experiment, 2) a blinded serial dilution probit analysis to determine the limit of detection, and 3) evaluation of two blinded panels of clinical samples from possible babesiosis cases. RESULTS: At a decreasing inoculum of 445, 44.5, and 4.45 copies/mL, the assay had positive rates of 100, 97.5, and 81%, respectively. The blinded probit analysis demonstrated a detection rate of 95 and 50% at 12.92 and 1.52 parasites/2 mL of whole blood, respectively. Evaluation of clinical samples showed 13 of 21 samples to be positive, with a range of 85 to 4.8 million parasites/mL. There were no positives detected among 48 healthy donors CONCLUSION: We have developed a highly sensitive and specific, quantitative real-time PCR-based assay for detection of B. microti that could have a useful role in blood screening. It can also be employed broadly to understand Babesia epidemiology, disease pathogenesis, and host immunology.

Case Report: Successful non-operative management of spontaneous splenic rupture in a patient with babesiosis.

BACKGROUND: Babesiosis is a zoonotic disease transmitted by the Ixodes tick species. Infection often results in sub-clinical manifestations; however, patients with this disease can become critically ill. Splenic rupture has been a previously reported complication of babesiosis, but treatment has always led to splenectomy. Asplenia places a patient at greater risk for overwhelming post-splenectomy infection from encapsulated bacteria, Lyme disease, Ehrlichia as well as Babesia microti. Therefore, avoiding splenectomy in these patients must be considered by the physician; particularly, if the patient is at risk for re-infection by living in an endemic area. CASE PRESENTATION: A 54 year-old male from the northeast United States presented with left upper quadrant abdominal pain associated with fever, chills, night sweats and nausea. A full evaluation revealed active infection with Babesia microti and multiple splenic lacerations. This patient was successfully treated with appropriate pharmacological therapy and non-operative observation for the splenic injury. CONCLUSION: Patients diagnosed with Babesia microti infection are becoming more common, especially in endemic areas. Although clinical manifestations are usually minimal, this infection can present with significant injuries leading to critical illness. We present the successful non-operative treatment of a patient with splenic rupture due to babesiosis infection.

Early evening questing and oviposition activity by the Culex (Diptera: Culicidae) vectors of West Nile virus in northeastern North America.

To determine whether the Culex (Diptera: Culicidae) mosquitoes that transmit West Nile virus (family Flaviviridae, genus Flavivirus, WNV) in the northeastern United States seek hosts and oviposit contemporaneously, we recorded when these mosquitoes attacked caged birds and when they deposited eggs. They traversed oviposition sites most frequently approximately 2 h after astronomical sunset, and eggs generally were deposited at that time. Although they most frequently approached avian hosts approximately 2 h after sunset during midsummer, they are more opportunistic during mid- to late fall. Because the Culex mosquitoes that serve as the main vectors of West Nile virus in the northeastern United States quest for hosts and seek to oviposit well after sunset, insecticidal aerosols would be most effective when applied at that time.

Efficacy of resmethrin aerosols applied from the road for suppressing Culex vectors of West Nile virus.

We determined whether aerosol applications of resmethrin, delivered from the road, suppress the reproductive activity of Culex pipiens pipiens and Cx. restuans mosquitoes in suburban sites located near Boston. Oviposition implies a prior blood-feeding event and hence a potential West Nile virus (WNV) transmission-related event. Droplet size, rate of delivery and meteorological conditions were monitored. The target populations proved to be fully susceptible to the insecticide that was used. The roads in the test sites generally gave adequate opportunity for insecticidal coverage. We found that the aerosol plume may have failed to contact the target mosquitoes and conclude that such insecticidal aerosols, delivered from the road, may not effectively reduce the force of transmission of WNV in our test sites.

Fetuin-A, a hepatocyte-specific protein that binds Plasmodium berghei thrombospondin-related adhesive protein: a potential role in infectivity.

Malaria infection is initiated when the insect vector injects Plasmodium sporozoites into a susceptible vertebrate host. Sporozoites rapidly leave the circulatory system to invade hepatocytes, where further development generates the parasite form that invades and multiplies within erythrocytes. Previous experiments have shown that the thrombospondin-related adhesive protein (TRAP) plays an important role in sporozoite infectivity for hepatocytes. TRAP, a typical type-1 transmembrane protein, has a long extracellular region, which contains two adhesive domains, an A-domain and a thrombospondin repeat. We have generated recombinant proteins of the TRAP adhesive domains. These TRAP fragments show direct interaction with hepatocytes and inhibit sporozoite invasion in vitro. When the recombinant TRAP A-domain was used for immunoprecipitation against hepatocyte membrane fractions, it bound to alpha2-Heremans-Schmid glycoprotein/fetuin-A, a hepatocyte-specific protein associated with the extracellular matrix. When the soluble sporozoite protein fraction was immunoprecipitated on a fetuin-A-adsorbed protein A column, TRAP bound this ligand. Importantly, anti-fetuin-A antibodies inhibited invasion of hepatocytes by sporozoites. Further, onset of malaria infection was delayed in fetuin-A-deficient mice compared to that in wild-type C57BL/6 mice when they were challenged with Plasmodium berghei sporozoites. These data demonstrate that the extracellular region of TRAP interacts with fetuin-A on hepatocyte membranes and that this interaction enhances the parasite's ability to invade hepatocytes.