Blood Smears Have Poor Sensitivity for Confirming Borrelia miyamotoi Disease.

Borrelia miyamotoi disease (BMD) is a newly recognized borreliosis that is cotransmitted by ticks wherever Lyme disease is zoonotic. Unlike Borrelia burgdorferisensu lato, the agent of Lyme disease, B. miyamotoi is closely related to relapsing fever spirochetes, such as Borrelia hermsii Some authors have suggested that the disease caused by B. miyamotoi should be considered a hard-tick-transmitted relapsing fever, and thus, the main mode of confirming a diagnosis for that infection, microscopy to analyze a blood smear, may have clinical utility. To determine whether blood smears may detect B. miyamotoi in the blood of acute BMD patients, we made standard malariological thick smears from anticoagulated blood samples that were previously determined to contain this agent (by PCR) and analyzed them for morphological evidence of spirochetes. Spirochetes were not detected in the blood smears from 20 PCR positive patient blood samples after examination of 100 thick smear fields and only 2 of 20 demonstrated spirochetes when the examination was extended to 300 thick smear fields. Inoculation of severe combined immunodeficient (SCID) mice yielded isolates from 5 of 5 samples, but 0 of 3 BALB/c mice became infected. We conclude that in strong contrast to the diagnosis of typical relapsing fever, microscopy of blood smears is not sensitive enough for confirming a diagnosis of BMD but that SCID mouse inoculation could be a useful complement to PCR.

Zoonotic Babesia microti in the northeastern U.S.: Evidence for the expansion of a specific parasite lineage.

The recent range expansion of human babesiosis in the northeastern United States, once found only in restricted coastal sites, is not well understood. This study sought to utilize a large number of samples to examine the population structure of the parasites on a fine scale to provide insights into the mode of emergence across the region. 228 B. microti samples collected in endemic northeastern U.S. sites were genotyped using published Variable number tandem repeat (VNTR) markers. The genetic diversity and population structure were analysed on a geographic scale using Phyloviz and TESS, programs that utilize two different methods to identify population membership without predefined population data. Three distinct populations were detected in northeastern US, each dominated by a single ancestral type. In contrast to the limited range of the Nantucket and Cape Cod populations, the mainland population dominated from New Jersey eastward to Boston. Ancestral populations of B. microti were sufficiently isolated to differentiate into distinct populations. Despite this, a single population was detected across a large geographic area of the northeast that historically had at least 3 distinct foci of transmission, central New Jersey, Long Island and southeastern Connecticut. We conclude that a single B. microti genotype has expanded across the northeastern U.S. The biological attributes associated with this parasite genotype that have contributed to such a selective sweep remain to be identified.

Description of 15 DNA-positive and antibody-negative "window-period" blood donations identified during prospective screening for Babesia microti.

BACKGROUND: Blood donation screening detecting only antibodies fails to identify donors in the earliest stage of infection, before a detectable immunologic response, that is, the "window period" (WP). We present data on WP donations identified during prospective screening for Babesia microti, a transfusion-transmissible parasite of increasing concern in the United States. STUDY DESIGN AND METHODS: Blood donations collected in Connecticut, Massachusetts, Minnesota, and Wisconsin were screened using polymerase chain reaction (PCR) and arrayed fluorescence immunoassay (AFIA) to detect B. microti DNA and antibodies, respectively. Parasite loads were estimated using quantitative PCR. Red blood cell (RBC) samples were inoculated into hamsters to assess infectivity. Donors screening reactive were indefinitely deferred, tested by supplemental methods, and followed to assess DNA and antibody clearance. Demographic data from WP donors (i.e., those screening PCR positive and AFIA negative) were compared to data from other positive donors. RESULTS: Of 220,479 donations screened from June 2012 to August 2016, a total of 700 were positive, of which 15 (2% of positive donations or 1 per 14,699 screened donations) were confirmed WP donations. The median estimated parasite load in WP donations was 350 parasites/mL, no different than AFIA-positive and PCR-positive donors. Parasite loads in RBC samples from WP units ranged from 14 to 11,022 parasites/mL; RBC samples from three of 10 (30%) WP donations infected hamsters. The mean age of WP donors was 48 years (range, 17-75 years); three (20%) were female. WP donor demographics did not differ significantly from demographics of other donors. CONCLUSIONS: We report one per 15,000 B. microti WP infections in blood donors in endemic areas, demonstrating the importance of nucleic acid testing to mitigate the risk of transfusion-transmitted babesiosis.

Screening for Babesia microti in the U.S. Blood Supply.

BACKGROUND: Babesia microti, a tickborne intraerythrocytic parasite that can be transmitted by means of blood transfusion, is responsible for the majority of cases of transfusion-transmitted babesiosis in the United States. However, no licensed test exists for screening for B. microti in donated blood. We assessed data from a large-scale, investigational product-release screening and donor follow-up program. METHODS: From June 2012 through September 2014, we performed arrayed fluorescence immunoassays (AFIAs) for B. microti antibodies and real-time polymerase-chain-reaction (PCR) assays for B. microti DNA on blood-donation samples obtained in Connecticut, Massachusetts, Minnesota, and Wisconsin. We determined parasite loads with the use of quantitative PCR testing and assessed infectivity by means of the inoculation of hamsters and the subsequent examination for parasitemia. Donors with test-reactive samples were followed. Using data on cases of transfusion-transmitted babesiosis, we compared the proportions of screened versus unscreened donations that were infectious. RESULTS: Of 89,153 blood-donation samples tested, 335 (0.38%) were confirmed to be positive, of which 67 (20%) were PCR-positive; 9 samples were antibody-negative (i.e., 1 antibody-negative sample per 9906 screened samples), representing 13% of all PCR-positive samples. PCR-positive samples were identified all through the year; antibody-negative infections occurred from June through September. Approximately one third of the red-cell samples from PCR-positive or high-titer AFIA-positive donations infected hamsters. Follow-up showed DNA clearance in 86% of the donors but antibody seroreversion in 8% after 1 year. In Connecticut and Massachusetts, no reported cases of transfusion-transmitted babesiosis were associated with screened donations (i.e., 0 cases per 75,331 screened donations), as compared with 14 cases per 253,031 unscreened donations (i.e., 1 case per 18,074 unscreened donations) (odds ratio, 8.6; 95% confidence interval, 0.51 to 144; P=0.05). Overall, 29 cases of transfusion-transmitted babesiosis were linked to blood from infected donors, including blood obtained from 10 donors whose samples tested positive on the PCR assay 2 to 7 months after the implicated donation. CONCLUSIONS: Blood-donation screening for antibodies to and DNA from B. microti was associated with a decrease in the risk of transfusion-transmitted babesiosis. (Funded by the American Red Cross and Imugen; ClinicalTrials.gov number, NCT01528449 .).

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.

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.

Investigational screening for Babesia microti in a large repository of blood donor samples from nonendemic and endemic areas of the United States.

BACKGROUND: Babesia microti, a transfusion-transmissible intraerythrocytic parasite, is increasing in frequency in the United States with no available FDA-licensed donor screening assay. We utilized investigational arrayed fluorescence immunoassay (AFIA) and polymerase chain reaction (PCR) to detect B. microti antibodies and DNA in blood donors. STUDY DESIGN AND METHODS: AFIA and real-time PCR were performed on frozen paired EDTA plasma (AFIA) and EDTA whole blood (PCR) samples collected from May to September 2010 to 2011 in nonendemic (Arizona [AZ] and Oklahoma [OK]), moderately endemic (Minnesota [MN] and Wisconsin [WI]), and highly endemic (Connecticut [CT] and Massachusetts [MA]) areas of the United States. AFIA utilized B. microti piroplasm as an antigen substrate; PCR primers and probes targeted the B. microti 18S ribosomal RNA gene. Data from AZ and OK were used to calculate specificity. All AFIA- or PCR-positive or -inconclusive donors were deferred, notified, and invited to participate in a follow-up study involving repeat testing and a demographic and risk-factor questionnaire. Recipient tracing was performed for any cellular component transfused at index, at subsequent donation, or within the prior 12 months. RESULTS: Testing of 13,269 paired samples included 4022 from AZ and OK, 4167 from MN and WI, and 5080 from CT and MA. B. microti antibody and/or DNA prevalences were 0.025% (95% confidence interval [CI], 0.00%-0.14%), 0.12% (95% CI, 0.04%-0.28%), and 0.75% (95% CI, 0.53%-1.03%) in the nonendemic, mid-endemic, and high-endemic regions, respectively. Specificities were 99.95% (95% CI, 99.82%-99.99%) at a 1-in-64 AFIA cutoff and 99.98% (95% CI, 99.86%-100.00%) at a 1-in-128 cutoff. CONCLUSIONS: B. microti prevalence followed expected geographical patterns. Screening was feasible with a performance comparable or superior to other infectious disease blood donor screening assays.

Probable transfusion-transmission of Anaplasma phagocytophilum by leukoreduced platelets.

BACKGROUND: Anaplasma phagocytophilum (AP), a tick-borne obligate intracellular bacterium, causes human granulocytic anaplasmosis (HGA) and has been implicated in seven transfusion-transmitted (TT)-HGA cases associated with red blood cells (RBCs). Here we report the first probable case of TT-HGA involving leukoreduced platelets (PLTs). CASE REPORT: A hospitalized male received 25 blood components (November 2012) before his death from trauma. Hospital testing confirmed HGA by peripheral blood smears; samples were also sent to IMUGEN, Inc. (Norwood, MA), for AP-polymerase chain reaction (PCR) and AP-immunoglobulin (Ig)M and IgG enzyme immunoassay. All 12 potentially transmitting donors provided follow-up samples. RESULTS: Recipient smears progressed from negative to predominantly positive 16 days posttransfusion; hospital-performed AP-PCR was positive on Day 22. IMUGEN sample testing was PCR positive and IgM and IgG negative 14 to 23 days posttransfusion. The recipient had no known AP risk factors. One of 12 donors of RBCs or PLTs (leukoreduced 5-day-old PLTs) provided six follow-up samples; all were strongly IgG positive and IgM negative; one was PCR-positive. The IgG-positive donor was a 52-year-old female from Hudson Valley, New York, an area endemic for AP. She reported tick bites in September to October 2012 with no travel outside New York. The donor remained asymptomatic and received no treatment. The cocomponent PLT unit was transfused to a 78-year-old male who died of causes unrelated to AP. CONCLUSIONS: This eighth case of probable TT-HGA indicates that leukoreduced PLTs may be infectious. An antibody- and PCR-positive donor having prior tick exposure living in an endemic area was identified. PCR positivity and elevated IgG levels, which continue to exceed the assay's detectible range even in the absence of IgM, indicate active donor infection.

Borrelia miyamotoi infection presenting as human granulocytic anaplasmosis: a case report.

BACKGROUND: The diverse tickborne infections of the northeastern United States can present as undifferentiated flu-like illnesses. In areas endemic for Lyme and other tickborne diseases, patients presenting with acute febrile illness with myalgia, headache, neutropenia, thrombocytopenia, and elevated hepatic aminotransferase levels are presumptively diagnosed as having human granulocytic anaplasmosis (HGA). OBJECTIVE: To assign a cause for illness experienced by 2 case patients who were initially diagnosed with HGA but did not rapidly defervesce with doxycycline treatment and had no laboratory evidence of Anaplasma phagocytophilum infection. DESIGN: Case report. SETTING: 2 primary care medical centers in Massachusetts and New Jersey. PATIENTS: 2 case patients acutely presenting with fever. MEASUREMENTS: Identification of the causative agent by polymerase chain reaction and DNA sequencing. RESULTS: Molecular diagnostic assays detected Borrelia miyamotoi in the peripheral blood of both patients. There was no evidence of infection with other tickborne pathogens commonly diagnosed in the referral areas. LIMITATION: One of the case patients may have had concurrent Lyme disease. CONCLUSION: The presence of B. miyamotoi DNA in the peripheral blood and the patients' eventual therapeutic response to doxycycline are consistent with the hypothesis that their illness was due to this newly recognized spirochete. Samples from tick-exposed patients acutely presenting with signs of HGA but who have a delayed response to doxycycline therapy or negative confirmatory test results for HGA should be analyzed carefully for evidence of B. miyamotoi infection.

Babesia microti real-time polymerase chain reaction testing of Connecticut blood donors: potential implications for screening algorithms.

BACKGROUND: Babesia microti, an intraerythrocytic parasite, has been implicated in transfusion transmission. B. microti seroprevalence in Connecticut (CT) blood donors is approximately 1%; however, it is not known what percentage of donors is parasitemic and poses a risk for transmitting infection. Therefore, we determined the prevalence of demonstrable B. microti DNA in donors from a highly endemic area of CT and compared observed rates with concurrent immunofluorescence assay (IFA) testing results. STUDY DESIGN AND METHODS: Blood samples from consenting donors in southeastern CT were collected from mid-August through early October 2009 and tested by IFA for immunoglobulin G antibodies and real-time polymerase chain reaction (PCR) for B. microti DNA. IFA specificity was determined using blood donor samples collected in northwestern Vermont (VT), an area nonendemic for Babesia. RESULTS: Of 1002 CT donors, 25 (2.5%) were IFA positive and three (0.3%) were real-time PCR positive. Among the three real-time PCR-positive donors, two were also IFA positive, while one was IFA negative and may represent a window period infection. The two IFA- and real-time PCR-positive donors appeared to subsequently clear infection. The other real-time PCR-positive donor did not provide follow-up samples. Of 1015 VT donors tested by IFA, only one (0.1%) was positive, but may have acquired infection during travel to an endemic area. CONCLUSION: We prospectively identified several real-time PCR-positive blood donors, including an IFA-negative real-time PCR-positive donor, in an area highly endemic for B. microti. These results suggest the need to include nucleic acid testing in planned mitigation strategies for B. microti.

Meningoencephalitis from Borrelia miyamotoi in an immunocompromised patient.

Ixodes ticks serve as vectors for Borrelia burgdorferi, the agent of Lyme disease. Globally, these ticks often concurrently harbor B. miyamotoi, a spirochete that is classified within the relapsing-fever group of spirochetes. Although humans presumably are exposed to B. miyamotoi, there are limited data suggesting disease attributable to it. We report a case of progressive mental deterioration in an older, immunocompromised patient, and even though Koch's postulates were not met, we posit B. miyamotoi as the cause, owing to its direct detection in cerebrospinal fluid (CSF) with the use of microscopy and a polymerase-chain-reaction (PCR) assay. It is likely that B. miyamotoi is an underrecognized cause of disease, especially in sites where Lyme disease is endemic.

Transfusion-transmitted anaplasmosis from leukoreduced red blood cells.

BACKGROUND: Human granulocytic anaplasmosis (HGA) is a tick-borne rickettsial infectious disease. To date four cases of transfusion-transmitted anaplasmosis (TTA) have been described in the literature, and only one from leukoreduced red blood cells (RBCs). CASE REPORT: A 64-year-old patient with acute gastrointestinal blood loss was admitted to the hospital and received 5 units of prestorage leukoreduced RBCs. He was stabilized and discharged. He developed headache, fever, and chills 2 days after discharge and was readmitted. On Day 5 of his second admission polymorphonuclear leukocytes containing morulae consistent with HGA were reported in the peripheral smear. RESULTS: Samples from the recipient tested positive by polymerase chain reaction (PCR) for Anaplasma phagocytophilum, the causative agent of HGA and a segment from one of the five donors tested positive by both serology and PCR. CONCLUSION: Leukoreduction theoretically reduces the risk of TTA but does not interdict all infections. TTA requires consideration in recipients of RBC transfusion with unexplained fever.

Preventing transfusion-transmitted babesiosis: preliminary experience of the first laboratory-based blood donor screening program.

BACKGROUND: Babesiosis is the most common transfusion-transmitted infection reported to the Food and Drug Administration (FDA). We developed and implemented the first laboratory-based blood donor screening program for Babesia microti to help reduce and prevent transfusion-transmitted babesiosis (TTB) and report results for the initial year. STUDY DESIGN AND METHODS: Selective B. microti donor screening was performed using real-time polymerase chain reaction (PCR) and indirect immunofluorescence assay (IFA) to reduce the incidence of TTB in neonates and pediatric sickle cell and thalassemia patients under an FDA-approved investigational new drug application. We compared the reports of TTB in these patients in the first 12 months of the study with those of patients who received unscreened blood from 2005 to 2010. RESULTS: There were 2113 units tested with 2086 negative results, 26 positive IFA results (1.23%), and one indeterminate PCR result (0.05%). No reported case of TTB occurred with any B. microti-screened unit transfused to the targeted patients (0/787 units) or to any patient who received the screened units (0/2086 units). Before screening, there were seven cases of TTB in neonates, sickle cell, and thalassemia patients from 6500 unscreened units (one case/929 units) and 24 cases in the total transfused population from 496,545 units distributed (one case/20,686 units). CONCLUSION: Implementation of B. microti IFA and PCR screening is compatible with blood center operations to provide tested units. While the results after 1 year are not powered to demonstrate a change in the rate of TTB after testing, they are encouraging.

Identification of Babesia microti-specific immunodominant epitopes and development of a peptide EIA for detection of antibodies in serum.

BACKGROUND: Babesia microti is a tick-borne agent that is increasingly implicated in transfusion-acquired infection, especially in immunocompromised and elderly recipients. To develop a test that can detect antibody responses to B. microti, peptide epitopes identified in two serocomplementary B. microti-specific antigens were used in a prototype EIA. STUDY DESIGN AND METHODS: A prototype peptide EIA was used to detect B. microti-specific antibodies in 15 sera taken before infection and 107 taken after infection from 59 individuals with known tick-borne infections previously confirmed by other methods. Three additional groups of samples were also tested: a proficiency panel of 18 sera positive for B. microti by IFA, 38 sera from blood donors confirmed positive by IFA, and 30 sera from random blood donors. RESULTS: The combination peptide detected 98 out of 107 sera taken after infection that were IgG blot positive (4 equivocal). This included all 12 samples that were PCR positive and six sera from smear-negative patients that were confirmed positive by PCR, immunoblot, or IFA. Of the IgG blot-positive specimens that were equivocal (four specimens) or did not react (nine specimens) by EIA, most had low IFA titers consistent with previous exposure. In a second evaluation, 15 out of 15 Babesia IFA-positive sera and 3 out of 3 Babesia-Ehrlichia IFA-positive sera were positive, whereas sera from 30 random donors were negative. Finally, of 38 IFA-positive blood-donor samples, 35 were positive by peptide EIA. The three EIA-negative sera were Western blot negative. CONCLUSION: Reactivity of the B. microti-specific peptide EIA shows a high correlation with IFA, PCR, and B. microti immunoblot in confirmed B. microti cases. The peptide EIA may be the most suitable B. microti infection test for adaptation to the blood bank environment if testing for B. microti is required in the future.