Clofazimine, a Promising Drug for the Treatment of Babesia microti Infection in Severely Immunocompromised Hosts.

BACKGROUND: Persistent and relapsing babesiosis caused by Babesia microti often occurs in immunocompromised patients, and has been associated with resistance to antimicrobial agents such as atovaquone. Given the rising incidence of babesiosis in the United States, novel drugs are urgently needed. In the current study, we tested whether clofazimine (CFZ), an antibiotic used to treat leprosy and drug-resistant tuberculosis, is effective against B. microti. METHODS: Mice with severe combined immunodeficiency were infected with 107B. microti-infected erythrocytes. Parasites were detected by means of microscopic examination of Giemsa-stained blood smears or nested polymerase chain reaction. CFZ was administered orally. RESULTS: Uninterrupted monotherapy with CFZ curtailed the rise of parasitemia and achieved radical cure. B. microti parasites and B. microti DNA were cleared by days 10 and 50 of therapy, respectively. A 7-day administration of CFZ delayed the rise of parasitemia by 22 days. This rise was caused by B. microti isolates that did not carry mutations in the cytochrome b gene. Accordingly, a 14-day administration of CFZ was sufficient to resolve high-grade parasitemia caused by atovaquone-resistant B. microti parasites. CONCLUSIONS: Clofazimine is effective against B. microti infection in the immunocompromised host. Additional preclinical studies are required to identify the minimal dose and dosage of CFZ for babesiosis.

Post-Babesiosis Warm Autoimmune Hemolytic Anemia.

Background Babesiosis, a tickborne zoonotic disease caused by intraerythrocytic protozoa of the genus babesia, is characterized by nonimmune hemolytic anemia that resolves with antimicrobial treatment and clearance of parasitemia. The development of warm-antibody autoimmune hemolytic anemia (also known as warm autoimmune hemolytic anemia [WAHA]) in patients with babesiosis has not previously been well described. Methods After the observation of sporadic cases of WAHA that occurred after treatment of patients for babesiosis, we conducted a retrospective cohort study of all the patients with babesiosis who were cared for at our center from January 2009 through June 2016. Data on covariates of interest were extracted from the medical records, including any hematologic complications that occurred within 3 months after the diagnosis and treatment of babesiosis. Results A total of 86 patients received a diagnosis of babesiosis during the 7.5-year study period; 18 of these patients were asplenic. WAHA developed in 6 patients 2 to 4 weeks after the diagnosis of babesiosis, by which time all the patients had had clinical and laboratory responses to antimicrobial treatment of babesiosis, including clearance of Babesia microti parasitemia. All 6 patients were asplenic (P<0.001) and had positive direct antiglobulin tests for IgG and complement component 3; warm autoantibodies were identified in all these patients. No alternative explanation for clinical hemolysis was found. WAHA required immunosuppressive treatment in 4 of the 6 patients. Conclusions We documented post-babesiosis WAHA in patients who did not have a history of autoimmunity; asplenic patients appeared to be particularly at risk.

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 .).

Elimination of Babesia microti Is Dependent on Intraerythrocytic Killing and CD4+ T Cells.

Babesiosis is a tick-borne zoonosis caused by protozoans of the genus Babesia, apicomplexan parasites that replicate within erythrocytes. However, unlike related Plasmodium species, the pathogenesis of Babesia infection remains poorly understood. The primary etiological agent of babesiosis in the United States is B. microti. In healthy individuals, tick-transmitted infection with Babesia causes no specific clinical manifestations, with many having no symptoms at all. However, even in asymptomatic people, a Babesia carriage state can be established that can last up to a year or more. Current blood bank screening methods do not identify infected donors, and Babesia parasites survive blood-banking procedures and storage. Thus, Babesia can also be transmitted by infected blood, and it is currently the number one cause of reportable transfusion-transmitted infection in the United States. Despite a significant impact on human health, B. microti remains understudied. In this study, we evaluated the course of Babesia infection in three strains of mice, C57BL/6J, BALB/cJ, and C3H-HeJ, and examined the contribution of multiple immune parameters, including TLRs, B cells, CD4+ cells, IFN-γ, and NO, on the level of parasitemia and parasite clearance during acute babesiosis. We found that B. microti reaches high parasitemia levels during the first week of infection in all three mice strains before resolving spontaneously. Our results indicate that resolution of babesiosis requires CD4 T cells and a novel mechanism of parasite killing within infected erythrocytes.

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

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

Analysis of antigen-antibody cross-reactivity among lineages and sublineages of Babesia microti parasites using human babesiosis specimens.

BACKGROUND: Human babesiosis is caused mainly by Babesia microti and has recently become a public health concern due to an increase in transfusion-transmitted infection. Thus, the development of an antibody detection method with high specificity and sensitivity is a priority. Seroreactivity against B. microti has been reported to be highly specific not only to B. microti lineages but also to sublineages. This study aimed to elucidate the human antibody reactivity against various lineages, including US, Kobe, and Hobetsu, and sublineages (North America and East Asia) in the US lineage. STUDY DESIGN AND METHODS: Twenty samples obtained from individuals infected with B. microti in the United States were tested for the presence of anti-B. microti antibodies using indirect immunofluorescence assay (IFA) and Western blotting (WB) to indicate antigens of each (sub-)lineage. RESULTS: By IFA, 20 samples showed reactivity to the North America sublineage (titer range, 64-4096), 16 to the East Asia sublineage (64-512), 10 to the Kobe (64-128), and five to the Hobetsu (64). Antibody titers to the East Asia sublineage, Kobe, and Hobetsu were significantly lower than those to the North America sublineage (p < 0.01). By WB, in parallel with the IFA results, 18 samples showed strong reactions to the North America sublineage, weak reactions to the East Asia sublineage, and near-zero reactions to the Kobe and Hobetsu. CONCLUSION: Human antibodies induced by B. microti infection are highly specific against B. microti lineages and sublineages with low cross-reactivity. Developing a precise antibody detection method may require specific antigens based on B. microti lineages and sublineages.

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.

A pilot serosurvey of Babesia microti in Chinese blood donors.

BACKGROUND AND OBJECTIVES: Babesia spp. are tick-borne, intraerythrocytic protozoan parasites, several of which are transfusion-transmissible. Transfusion-transmitted babesiosis poses serious risk to a diverse patient population, including neonates, patients aged >50 years, the asplenic and the immunocompromised that are over-represented among transfusion recipients. Despite reports of B. microti and B. venatorum in People's Republic of China (PRC), no surveillance of blood donors for Babesia has previously been undertaken. We sought to determine the rates of B. microti seroreactivity in a sample of blood donors in the PRC. MATERIALS AND METHODS: A pilot serosurvey was conducted of community blood donors (n = 1000) who donated July-August 2016 at Mudanjiang Blood Center (Heilongjiang Province) using indirect fluorescent antibody testing for antibodies against B. microti. The slides were prepared using B. microti-infected hamster blood. Samples that were initially positive to a titre of 64 were subjected to repeat IFA testing. Final seroreactivity was based on repeat reactivity to ≥64. RESULTS: A total of 1000 individual donor samples were evaluated, comprising 888 whole blood and 112 platelet donations. Thirteen of 1000 (1·3%) donors were seroreactive for B. microti [8 (0·8%) and five (0·05%) at titres of 64 and 128, respectively]. CONCLUSION: Our preliminary findings support the need for expanded Babesia surveillance in Chinese blood donors, replete with molecular evaluation, to evaluate the risk to the blood supply.

Babesia microti: from Mice to Ticks to an Increasing Number of Highly Susceptible Humans.

Babesia microti, a zoonotic intraerythrocytic parasite, is the primary etiological agent of human babesiosis in the United States. Human infections range from subclinical illness to severe disease resulting in death, with symptoms being related to host immune status. Despite advances in our understanding and management of B. microti, the incidence of infection in the United States has increased. Therefore, research focused on eradicating disease and optimizing clinical management is essential. Here we review this remarkable organism, with emphasis on the clinical, diagnostic, and therapeutic aspects of human disease.

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.

Expression of truncated Babesia microti apical membrane protein 1 and rhoptry neck protein 2 and evaluation of their protective efficacy.

In this study, we evaluated the protective effect of recombinant Babesia microti apical membrane protein 1 (rBmAMA1) and rhoptry neck protein 2 (rBmRON2) against B. microti infection using a hamster model. The genes encoding the predicted domains I and II of BmAMA1 and the gene encoding the predicted transmembrane regions 2 and 3 of BmRON2 were expressed as His fusion recombinant proteins in Escherichia coli. Three groups with 5 hamsters in each group were immunized with rBmAMA1, rBmRON2 and rBmAMA1+rBmRON2, then challenged with B. microti. The result showed that only the group immunized with rBmAMA1+rBmRON2 exhibited limited protection against B. microti challenge infection, characterized by significant decreased of parasitemia and higher hematocrit values from day 6-10 post challenge infection. However, there was no significant difference in the groups immunized with rBmAMA1 or rBmRON2 alone. The absence of a significant difference in the total amount of antibodies against rBmAMA1 and rBmRON2 between the group immunized with single and combined proteins. This result suggests that the protection cannot be solely attributed to the quantity of antibodies produced, but also to their ability to target important epitopes from both antigens. These results suggest that combined immunization with rBmAMA1 and rBmRON2 is a promising strategy against B. microti.

Serological and Molecular Detection of Toxoplasma gondii and Babesia microti in the Blood of Rescued Wild Animals in Gangwon-do (Province), Korea.

Infections of Toxoplasma gondii and Babesia microti are reported in many wild animals worldwide, but information on their incidence and molecular detection in Korean wild fields is limited. In this study, the prevalence of T. gondii and B. microti infection in blood samples of 5 animal species (37 Chinese water deer, 23 raccoon dogs, 6 roe deer, 1 wild boar, and 3 Eurasian badgers) was examined during 2008-2009 in Gangwon-do (Province), the Republic of Korea (=Korea) by using serological and molecular tests. The overall seropositivity of T. gondii was 8.6% (6/70); 10.8% in Chinese water deer, 4.3% in raccoon dogs, and 16.7% in roe deer. PCR revealed only 1 case of T. gondii infection in Chinese water deer, and phylogenic analysis showed that the positive isolate was practically identical to the highly pathogenetic strain type I. In B. microti PCR, the positive rate was 5.7% (4/70), including 2 Chinese water deer and 2 Eurasian badgers. Phylogenetic analysis results of 18S rRNA and the ß-tubulin gene showed that all positive isolates were US-type B. microti. To our knowledge, this is the first report of B. microti detected in Chinese water deer and Eurasian badger from Korea. These results indicate a potentially high prevalence of T. gondii and B. microti in wild animals of Gangwon-do, Korea. Furthermore, Chinese water deer might act as a reservoir for parasite infections of domestic animals.

Transmission of Babesia microti Parasites by Solid Organ Transplantation.

Babesia microti, an intraerythrocytic parasite, is tickborne in nature. In contrast to transmission by blood transfusion, which has been well documented, transmission associated with solid organ transplantation has not been reported. We describe parasitologically confirmed cases of babesiosis diagnosed ≈8 weeks posttransplantation in 2 recipients of renal allografts from an organ donor who was multiply transfused on the day he died from traumatic injuries. The organ donor and recipients had no identified risk factors for tickborne infection. Antibodies against B. microti parasites were not detected by serologic testing of archived pretransplant specimens. However, 1 of the organ donor's blood donors was seropositive when tested postdonation and had risk factors for tick exposure. The organ donor probably served as a conduit of Babesia parasites from the seropositive blood donor to both kidney recipients. Babesiosis should be included in the differential diagnosis of unexplained fever and hemolytic anemia after blood transfusion or organ transplantation.

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.

Primary Babesia rodhaini infection followed by recovery confers protective immunity against B. rodhaini reinfection and Babesia microti challenge infection in mice.

In the present study, we investigated the protective immunity against challenge infections with Babesia rodhaini and Babesia microti in the mice recovered from B. rodhaini infection. Six groups with 5 test mice in each group were used in this study, and were intraperitoneally immunized with alive and dead B. rodhaini. The challenge infections with B. rodhaini or B. microti were performed using different time courses. Our results showed that the mice recovered from primary B. rodhaini infection exhibited low parasitemia and no mortalities after the challenge infections, whereas mock mice which had received no primary infection showed a rapid increase of parasitemia and died within 7 days after the challenge with B. rodhaini. Mice immunized with dead B. rodhaini were not protected against either B. rodhaini or B. microti challenge infections, although high titers of antibody response were induced. These results indicate that only mice immunized with alive B. rodhaini could acquire protective immunity against B. rodhaini or B. microti challenge infection. Moreover, the test mice produced high levels of antibody response and low levels of cytokines (INF-γ, IL-4, IL-12, IL-10) against B. rodhaini or B. microti after challenge infection. Mock mice, however, showed rapid increases of these cytokines, which means disordered cytokines secretion occurred during the acute stage of challenge infection. The above results proved that mice immunized with alive B. rodhaini could acquire protective immunity against B. rodhaini and B. microti infections.

Recombinant methionine aminopeptidase protein of Babesia microti: immunobiochemical characterization as a vaccine candidate against human babesiosis.

Human babesiosis is the most important zoonotic protozoan infection in the world. This is the first report of the cloning, expression, purification, and immunobiochemical characterization of a methionine aminopeptidase 1 (MetAP1) protein from Babesia microti (B. microti). The gene encodes a MetAP1 protein of B. microti (BmMetAP1) of approximately 66.8 kDa that includes glutathione S-transferase (GST) tag and shows MetAP activity. BmMetAP1 was detected in a lysate of B. microti and further localized in cytoplasm of the B. microti merozoite. rBmMetAP1 was found to be immunogenic, eliciting a high antibody titer in mice. Moreover, rBmMetAP1 stimulated the production of IFN-γ and IL-12 but not IL-4. Finally, rBmMetAP1 was able to provide considerable protection to mice against a B. microti challenge infection based on a reduction in peak parasitemia levels and earlier clearance of the parasite as compared with control mice. Taken together, these results suggest that rBmMetAP1 confers significant protection against experimental B. microti infection and might be considered a potential vaccine target against human babesiosis.

The incidence of Borrelia burgdorferi, Anaplasma phagocytophilum and Babesia microti coinfections among foresters and farmers in eastern Poland.

BACKGROUND & OBJECTIVES: Lyme borreliosis is the most common tick-borne disease in Europe and the USA. However, a great variety of pathogens are transmitted by ticks, which results in mixed infections, with Lyme borreliosis. The aim of the present study was to show the incidence of Borrelia burgdorferi, Anaplasma phagocytophilum, and Babesia microti coinfections among the population of foresters and farmers, as these people, due to their profession, are particularly exposed to tick contact. METHODS: The study was carried out in eastern Poland (the northern part of the Lublin Province) in 2013. The study was performed in a group of 93 individuals occupationally exposed to tick bites (foresters and farmers), whose blood serum showed the presence of IgG anti-B. burgdorferi antibodies. Blood serum in this group were evaluated for IgG anti-A. phagocytophilum and IgG anti-B. microti antibodies by means of IFA IgG indirect immunofluorescence tests. Information related to age, sex, number of tick bite episodes, presence of various symptoms related to the tick bites, and antibiotic therapy applied as treatment for diagnosed Lyme borreliosis were obtained from the subjects through a structured questionnaire. The results were analyzed in Statistica v. 7.1 statistical analysis software. RESULTS: The presence of IgG antibodies against the analyzed pathogens revealed B. burgdorferi and A. phagocytophilum coinfection in 26 (28%) of the examined subjects and B. burgdorferi and B. microti coinfection in one person (1.1%). No coinfection with all the three pathogens was observed in any individual. The co-occurrence of headache plus bone, joint and muscle pain was noted significantly more often among individuals diagnosed with B. burgdorferi and A. phagocytophilum coinfection. INTERPRETATION & CONCLUSION: Foresters and farmers are exposed to B. burgdorferi and A. phagocytophilum coinfection in the study area. Therefore, it is probable that these pathogens may severely interfere with the clinical course of Lyme borreliosis.

Macrophages are the determinant of resistance to and outcome of nonlethal Babesia microti infection in mice.

In the present study, we examined the contributions of macrophages to the outcome of infection with Babesia microti, the etiological agent of human and rodent babesiosis, in BALB/c mice. Mice were treated with clodronate liposome at different times during the course of B. microti infection in order to deplete the macrophages. Notably, a depletion of host macrophages at the early and acute phases of infection caused a significant elevation of parasitemia associated with remarkable mortality in the mice. The depletion of macrophages at the resolving and latent phases of infection resulted in an immediate and temporal exacerbation of parasitemia coupled with mortality in mice. Reconstituting clodronate liposome-treated mice at the acute phase of infection with macrophages from naive mice resulted in a slight reduction in parasitemia with improved survival compared to that of mice that received the drug alone. These results indicate that macrophages play a crucial role in the control of and resistance to B. microti infection in mice. Moreover, analyses of host immune responses revealed that macrophage-depleted mice diminished their production of Th1 cell cytokines, including gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α). Furthermore, depletion of macrophages at different times exaggerated the pathogenesis of the infection in deficient IFN-γ(-/-) and severe combined immunodeficiency (SCID) mice. Collectively, our data provide important clues about the role of macrophages in the resistance and control of B. microti and imply that the severity of the infection in immunocompromised patients might be due to impairment of macrophage function.

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.

Determination of Babesia microti seroprevalence in blood donor populations using an investigational enzyme immunoassay.

BACKGROUND: Transfusion-transmitted babesiosis caused by Babesia microti has emerged as a significant risk to the US blood supply. This study estimated the prevalence of B. microti antibodies in blood donors using an investigational enzyme immunoassay (EIA). STUDY DESIGN AND METHODS: A peptide-based EIA that detects both immunoglobulin (Ig)G and IgM antibodies to B. microti was developed and validated. Donor samples randomly selected from areas defined as high-risk endemic, lower-risk endemic, and nonendemic for B. microti were deidentified and tested using the investigational EIA. Samples that were EIA repeat reactive were further tested by B. microti immunofluorescent assay (IFA), polymerase chain reaction (PCR) on red blood cell lysates, and peripheral blood smear examination. A random subset of 1272 samples from high-risk endemic areas was tested by IFA, PCR, and peripheral blood smear in parallel with EIA. RESULTS: Among 15,000 donations tested with the investigational B. microti EIA, EIA repeat-reactive rates were 1.08% (54/5000) in a high-risk endemic area, 0.74% (37/5000) in a lower-risk area, and 0.40% (20/5000) in a nonendemic area. After application of a revised cutoff, these values were reduced to 0.92%, (46/5000), 0.54% (27/5000), and 0.16% (8/5000). Overall concordance between EIA and IFA among donor samples was 99.34%. One seropositive sample was positive by PCR. CONCLUSION: The seroprevalence of B. microti in blood donors in a high-risk area measured by an investigational EIA was approximately 1%. The EIA shows promise as an efficient high-throughput blood donor screening assay for B. microti.