Rapid diagnostics for melioidosis: a comparative study of a novel lateral flow antigen detection assay.

The rapid diagnosis of septicaemic melioidosis will have an impact on reduction of mortality. Currently, this relies almost exclusively upon culture of the causative agent Burkholderia pseudomallei from clinical samples. In acute sepsis, blood is the preferred specimen for culture and therefore should be the target for a rapid diagnostic tool. A lateral flow immunoassay (LFI) for the detection of B. pseudomallei antigen has been developed. This was compared with molecular detection using the targets T3SS1 and IpxO. Forty-five clinical samples of EDTA blood, which were culture-positive, were tested using both modalities. The LFI had a sensitivity of 40 %, whilst molecular detection had a sensitivity of 20 %. The poor performance of molecular detection has been described previously and is largely related to the use of whole-blood specimens collected into blood tubes containing EDTA. Whilst suboptimal, the LFI would be an adjunct in the rapid diagnosis of melioidosis.

Immunoassay for Capsular Antigen of Bacillus anthracis Enables Rapid Diagnosis in a Rabbit Model of Inhalational Anthrax.

Inhalational anthrax is a serious biothreat. Effective antibiotic treatment of inhalational anthrax requires early diagnosis; the further the disease has progressed, the less the likelihood for cure. Current means for diagnosis such as blood culture require several days to a result and require advanced laboratory infrastructure. An alternative approach to diagnosis is detection of a Bacillus anthracis antigen that is shed into blood and can be detected by rapid immunoassay. The goal of the study was to evaluate detection of poly-γ-D-glutamic acid (PGA), the capsular antigen of B. anthracis, as a biomarker surrogate for blood culture in a rabbit model of inhalational anthrax. The mean time to a positive blood culture was 26 ± 5.7 h (mean ± standard deviation), whereas the mean time to a positive ELISA was 22 ± 4.2 h; P = 0.005 in comparison with blood culture. A lateral flow immunoassay was constructed for detection of PGA in plasma at concentrations of less than 1 ng PGA/ml. Use of the lateral flow immunoassay for detection of PGA in the rabbit model found that antigen was detected somewhat earlier than the earliest time point at which the blood culture became positive. The low cost, ease of use, and rapid time to result of the lateral flow immunoassay format make an immunoassay for PGA a viable surrogate for blood culture for detection of infection in individuals who have a likelihood of exposure to B. anthracis.

Melioidosis diagnostic workshop, 2013.

Melioidosis is a severe disease that can be difficult to diagnose because of its diverse clinical manifestations and a lack of adequate diagnostic capabilities for suspected cases. There is broad interest in improving detection and diagnosis of this disease not only in melioidosis-endemic regions but also outside these regions because melioidosis may be underreported and poses a potential bioterrorism challenge for public health authorities. Therefore, a workshop of academic, government, and private sector personnel from around the world was convened to discuss the current state of melioidosis diagnostics, diagnostic needs, and future directions.

Development of a prototype lateral flow immunoassay (LFI) for the rapid diagnosis of melioidosis.

Burkholderia pseudomallei is a soil-dwelling bacterium and the causative agent of melioidosis. Isolation of B. pseudomallei from clinical samples is the "gold standard" for the diagnosis of melioidosis; results can take 3-7 days to produce. Alternatively, antibody-based tests have low specificity due to a high percentage of seropositive individuals in endemic areas. There is a clear need to develop a rapid point-of-care antigen detection assay for the diagnosis of melioidosis. Previously, we employed In vivo Microbial Antigen Discovery (InMAD) to identify potential B. pseudomallei diagnostic biomarkers. The B. pseudomallei capsular polysaccharide (CPS) and numerous protein antigens were identified as potential candidates. Here, we describe the development of a diagnostic immunoassay based on the detection of CPS. Following production of a CPS-specific monoclonal antibody (mAb), an antigen-capture immunoassay was developed to determine the concentration of CPS within a panel of melioidosis patient serum and urine samples. The same mAb was used to produce a prototype Active Melioidosis Detect Lateral Flow Immunoassay (AMD LFI); the limit of detection of the LFI for CPS is comparable to the antigen-capture immunoassay (∼0.2 ng/ml). The analytical reactivity (inclusivity) of the AMD LFI was 98.7% (76/77) when tested against a large panel of B. pseudomallei isolates. Analytical specificity (cross-reactivity) testing determined that 97.2% of B. pseudomallei near neighbor species (35/36) were not reactive. The non-reactive B. pseudomallei strain and the reactive near neighbor strain can be explained through genetic sequence analysis. Importantly, we show the AMD LFI is capable of detecting CPS in a variety of patient samples. The LFI is currently being evaluated in Thailand and Australia; the focus is to optimize and validate testing procedures on melioidosis patient samples prior to initiation of a large, multisite pre-clinical evaluation.

Comparison of a new prototype immunochromatographic assay and a commercial enzyme-linked immunosorbent assay for the detection of serum antibodies against Toscana virus.

Toscana virus (TOSV) is an arthropod-borne virus, transmitted to humans by Phlebotomus spp. Sandflies, which causes neurological diseases such as aseptic meningitis and meningoencephalitis. The commercial enzyme-linked immunosorbent assay (ELISA) is used widely to detect anti-TOSV IgG and IgM antibodies and to allow for rapid diagnosis of infection (Diesse Diagnostica Senese, Siena, Italy). Recently, an immunochromatographic assay (ICA) was developed for human anti-TOSV IgG or IgM detection by InBios International (Seattle, WA, USA). A comparison of the two diagnostic assays was performed on one hundred serum samples collected from patients hospitalized with suspected TOSV meningitis. Both assays were in excellent agreement, for both IgG and IgM detection. For IgM, 64/65 ELISA positive samples were positive by ICA. One serum, positive for specific IgM by ELISA but negative by ICA, was confirmed by direct diagnosis, with TOSV RNA detection in the patient's cerebrospinal fluid by PCR. For IgG, 64 samples were positive by ICA out of 71 ELISA positive samples. The discordant sera were positive by immunofluorescence and neutralization tests. Three out of these seven samples were also positive by IgM ICA. The sensitivity of these new assays compared to ELISA, which is used routinely, was 98.5% for IgM and 90.1% for IgG, while specificity was 100% in both cases. This data shows that ICA could be a reliable alternative test for serological diagnosis of TOSV infection in humans.

Burkholderia pseudomallei proteins presented by monocyte-derived dendritic cells stimulate human memory T cells in vitro.

Melioidosis is a severe infectious disease caused by the saprophytic facultative intracellular pathogen Burkholderia pseudomallei. The disease is endemic in Southeast Asia and Northern Australia, and no effective vaccine exists. To describe human cell-mediated immune responses to B. pseudomallei and to identify candidate antigens for vaccine development, the ability of antigen-pulsed monocyte-derived dendritic cells (moDCs) to trigger autologous T-cell responses to B. pseudomallei and its products was tested. moDCs were prepared from healthy individuals exposed or not exposed to B. pseudomallei, based on serological evidence. These were pulsed with heat-killed B. pseudomallei or purified antigens, including ABC transporters (LolC, OppA, and PotF), Bsa type III secreted proteins (BipD and BopE), tandem repeat sequence-containing proteins (Rp1 and Rp2), flagellin, and heat shock proteins (Hsp60 and Hsp70), prior to being mixed with autologous T-cell populations. After pulsing of cells with either heat-killed B. pseudomallei, LolC, or Rp2, coculturing the antigen-pulsed moDCs with T cells elicited gamma interferon production from CD4(+) T cells from seropositive donors at levels greater than those for seronegative donors. These antigens also induced granzyme B (cytotoxic) responses from CD8(+) T cells. Activation of antigen-specific CD4(+) T cells required direct contact with moDCs and was therefore not dependent on soluble mediators. Rp peptide epitopes recognized by T cells in healthy individuals were identified. Our study provides valuable novel data on the induction of human cell-mediated immune responses to B. pseudomallei and its protein antigens that may be exploited in the rational development of vaccines to combat melioidosis.

Lateral flow immunoassay for diagnosis of Trypanosoma cruzi infection with high correlation to the radioimmunoprecipitation assay.

The incidence of blood donors seropositive for Trypanosoma cruzi in North America has increased with population migration and more rigorous surveillance. The United States, considered nonendemic for T. cruzi, could therefore be at risk to exposure to parasite transmission through blood or organ donations. Current tests show variable reactivity, especially with Central American sera. Here we describe the development of a lateral flow immunoassay for the rapid detection of T. cruzi infection that has a strong correlation to the radioimmunoprecipitation assay (RIPA) "gold standard" in the United States. Such a test could have utility in small blood banks for prescreening donors, as well as in cardiac transplantation evaluation. T. cruzi consensus and/or RIPA-positive sera from Central and South America were evaluated in enzyme immunoassays (EIAs). These included commercial panels from Boston Biomedica, Inc. (BBI) (n = 14), and HemaBio (n = 21). Other sources included RIPA-positive sera from the American Red Cross (ARC) (n = 42), as well as from Chile. Sera were tested with the multiepitope recombinant TcF. All but one of the BBI samples were positive and 7 of 21 HemaBio samples and 6 of 42 ARC samples were low positive or negative. This observation indicated the need for additional antigens. To complement TcF reactivity, we tested the sera with peptides 30, 36, SAPA, and 1.1, 1.2, and 1.3 His fragments of 85-kDa trans-sialidase. We identified a promising combination of the tested antigens and constructed a single recombinant protein, ITC6, that enhanced the relative sensitivity in U.S. blood donor sera compared to that of TcF. The data on its evaluation using RIPA-confirmed positive sera in EIA and lateral flow immunoassay studies are presented, along with an additional recombinant protein, ITC8.2, with two additional sequences for peptide 1 and Kmp-11. The latter, when evaluated in a dipstick assay with consensus positive sera, had a sensitivity of 99.2% and a specificity of 99.1%.

Detection of alpha-methylacyl-coenzyme-A racemase transcripts in blood and urine samples of prostate cancer patients.

BACKGROUND: Alpha-methylacyl-coenzyme-A racemase (AMACR) has been shown to be a highly specific marker for prostate cancer cells, even in the earliest stages of malignant progression. It is expressed at much higher levels than prostate-specific antigen (PSA) in malignant tissues, and is not expressed at appreciable levels in normal prostatic epithelium. In this study, we demonstrate the quantitative detection of AMACR transcripts in peripheral blood of prostate cancer patients using real-time RT-PCR. In addition, we have undertaken a pilot study to demonstrate the potential application of this technique for the detection of prostate tumor cells in urine samples from patients with prostate cancer. METHODS: A real-time RT-PCR assay was developed for detection of the expression of AMACR in prostate cancer patients. Blood samples from 163 patients were tested at various stages of disease progression, with or without therapy. Blood specimens from patients with benign prostate disorders and other types of cancer were also evaluated. RESULTS: In 28 of 58 samples from patients with known metastatic disease who were undergoing treatment, an AMACR expression signal above the cut-off value was detected, consistent with the presence of circulating tumor cells. In 39 of 88 patients with presumptive organ-confined disease, there was evidence of low levels of circulating tumor cells. Comparison of AMACR RT-PCR with known serum PSA values indicated that a combination of these parameters significantly increased the sensitivity for detection of progressive disease. In a pilot study analyzing urine samples from seven prostate cancer patients, elevated AMACR expression levels were detected in the urine sediments of four of six stage-T1 prostate cancer patients and in the one patient with stage-T2 prostate cancer. CONCLUSION: The data presented in this study indicates that AMACR real-time RT-PCR may aid in the detection and staging of prostate cancer.

Detection of circulating tumor cells in peripheral blood of breast cancer patients during or after therapy using a multigene real-time RT-PCR assay.

AIM: To evaluate the utility of a multigene real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay to detect circulating tumor cells in peripheral blood specimens of breast cancer patients during or after treatment. METHOD: Using this assay, peripheral blood samples were analyzed for expression levels of mammaglobin and three complementary transcribed breast cancer-specific genes: B305D, gamma-aminobutyrate type A receptor pi subunit (GABA pi; GABRP), and B726P. We examined 172 blood specimens from 82 breast cancer patients during or after therapy for the presence of circulating tumor cells using the multigene real-time RT-PCR assay. RESULTS: In 63.4% of the blood samples, a positive signal for mammaglobin and/or three breast cancer-associated gene transcripts was detected. Of breast cancer patients, 75.6% had at least one positive blood sample. Blood specimens from 51 of 53 healthy female volunteers tested negative in the assay whereas two samples had a low expression signal. In addition, three patients were monitored for more than a year during their adjuvant therapy treatment. CONCLUSION: This assay could be a valuable tool for monitoring breast cancer patients during and after therapy.

Multigene real-time PCR detection of circulating tumor cells in peripheral blood of lung cancer patients.

BACKGROUND: CLCA2, HMGB3, L587S and ASH1 were identified in lung cancer tissues using genetic subtraction, microarray and quantitative PCR, and found to be specific and complementary for detection of non-small cell lung carcinoma (NSCLC) and small cell lung carcinoma (SCLC). MATERIALS AND METHODS: A real-time RT-PCR assay, simultaneously detecting four genes, was developed and tested on lung cancer specimens. RESULTS: Twenty-two out of 24 adenocarcinomas, 18/18 squamous, 4/5 large cell, 2/2 small cell and 2/2 bronchoalveolar/neuroendocrine cancer tissue samples tested positive. Specificity was demonstrated by evaluation of 194 other tumor and corresponding normal tissues. Circulating tumor cells in the peripheral blood of 49/108 lung cancer patient samples tested positive, and general correlations of multigene expression signals to disease status were observed. Changes in multigene expression during treatment and disease recurrence in individual patients could be detected. CONCLUSION: These data indicate the diagnostic and prognostic utility of a multigene real-time RT-PCR assay to detect tumor cells in the peripheral blood of lung cancer patients.

Demonstrable parasitemia among Connecticut blood donors with antibodies to Babesia microti.

BACKGROUND: Reports of transfusion-transmitted Babesia microti have risen steadily during the past several years, reflecting a concurrent increase in US cases of human babesiosis. Although several studies have measured B. microti antibodies in blood donors, little is known about associated parasitemia and the inherent risk of transmitting the parasite by transfusion. STUDY DESIGN AND METHODS: Donations from blood donors located in Babesia-endemic and nonendemic areas of Connecticut were tested for B. microti antibodies from July through September. Subsequently, an additional blood sample was collected from selected seropositive donors and tested by nested polymerase chain reaction (PCR) for B. microti nucleic acids. RESULTS: A total of 3490 donations, 1745 each from endemic and nonendemic areas, were tested for B. microti antibodies; 30 (0.9%) were confirmed as positive and seroprevalence rates peaked in July. Significantly more seropositive donations were from endemic areas (24, 1.4%) than nonendemic areas (6, 0.3%). Ten (53%) of 19 seropositive donors subsequently tested by PCR were positive. CONCLUSION: B. microti seroprevalence was highest in those areas of Connecticut where the parasite is endemic. More than half of seropositive donors tested had demonstrable parasitemia, indicating that many are at risk for transmitting B. microti by blood transfusion. Three donors were identified as parasitemic in October, suggesting that donors may be at risk for transmitting the parasite outside of the peak period of community-acquired infection.

Evaluation of a panel of tumor markers for molecular detection of circulating cancer cells in women with suspected breast cancer.

PURPOSE: We examined the feasibility of using molecular characterization of circulating tumor cells as a method for early detection of breast cancer. RESEARCH DESIGN: Women without a prior history of cancer who had a breast abnormality detected on imaging followed by a breast biopsy were enrolled in this study. Density gradient centrifugation and immunomagnetic capture were used to enrich for epithelial cells from approximately 20 mL of blood. Real-time reverse transcription-PCR was used to quantitate the expression levels of the highly breast-specific genes, mammaglobin, gamma-aminobutyric acid type A receptor pi subunit (GABA A(pi)), B305D-C, and B726P in the epithelial cell-enriched samples. RESULTS: The assay was technically feasible in 154 of 199 accrued patients. From their clinical assessment, 100 patients had benign breast disease, 10 patients had ductal carcinoma in situ, and 44 patients had invasive breast cancer. We constructed a diagnostic test that classified patients with mammaglobin levels of at least 32.2 copies/pg beta-actin (units) in their circulating epithelial cells as positive for invasive breast cancer. This resulted in a sensitivity and specificity of 63.3% and 75.0%, respectively. A diagnostic test that classified patients as positive for invasive breast cancer when either mammaglobin levels were >46.3 units or B305D-C levels were >11.6 units increased the sensitivity and specificity to 70.5% and 81.0%, respectively. In the latter test, 12 of the 14 node-positive breast cancer patients were correctly identified. Including GABA A(pi) and B726P in the test did not increase its diagnostic potential. CONCLUSIONS: These results suggest that molecular characterization of circulating epithelial cells using mammaglobin and B305D-C offers potential for early detection of invasive breast cancer.

Mammaglobin as a novel breast cancer biomarker: multigene reverse transcription-PCR assay and sandwich ELISA.

BACKGROUND: The aim of this study was to examine the potential usefulness of a mammaglobin multigene reverse transcription-PCR (RT-PCR) assay and a mammaglobin sandwich ELISA as diagnostic tools in breast cancer. METHODS: We studied peripheral blood samples from 147 untreated Senegalese women with biopsy-confirmed breast cancer and gathered patient information regarding demographic, and clinical staging of disease. The samples were tested for mammaglobin and three breast cancer-associated gene transcripts by a multigene real-time RT-PCR assay and for serum mammaglobin protein by a sandwich ELISA assay. RESULTS: In 77% of the breast cancer blood samples, a positive signal was obtained in the multigene RT-PCR assay detecting mammaglobin and three complementary transcribed genes. Fifty samples from healthy female donors tested negative. Significant correlations were found between mammaglobin protein in serum, presence of mammaglobin mRNA-expressing cells in blood, stage of disease, and tumor size. Circulating mammaglobin protein was detected in 68% of the breast cancer sera, and was increased in 38% in comparison with a mixed control population. The RT-PCR assay and the ELISA for mammaglobin produced a combined sensitivity of 84% and specificity of 97%. CONCLUSION: The ELISA and RT-PCR for mammaglobin and mammaglobin-producing cells could be valuable tools for diagnosis and prognosis of breast cancer.

P704P, P712P, and P775P: A genomic cluster of prostate-specific genes.

BACKGROUND: Discovery of prostate cancer- and tissue-specific genes will lead to an increased understanding of the molecular events associated with the malignant transformation and tumorigenesis of prostate cells. Such understanding will likely result in the development of promising new markers for screening, diagnosis, and prognosis, as well as potential therapeutic approaches for combating this disease. METHODS: A PCR-based subtraction method was combined with a high-throughput microarray screening approach to identify prostate tissue- and/or cancer-specific genes. Northern blot and quantitative real-time PCR were used to confirm prostate specificity. Bioinformatics analysis was performed to determine gene localization and to identify the open reading frame of novel genes. RESULTS: Three novel cDNA clones, P704P, P712P, and P775P, were identified and characterized to be specific for normal and malignant prostate tissues. Furthermore, P712P mRNA expression was found to be androgen responsive in LNCaP cells. Sequences for all three cDNAs were localized to an 80 kb genomic region on chromosome 22. Attempts to identify full-length transcripts did not reveal any apparent open reading frames, indicating that P704P, P712P, and P775P may belong to a novel class of transcripts with specific patterns of gene expression that do not code for translated proteins. CONCLUSIONS: A genomic cluster of prostate-specific genes with no apparent open reading frame has been discovered using a high-throughput approach combining subtraction with microarray. This may represent an important genomic region having possible connections to prostate biology with potential applications in prostate diagnostics and therapy.

Potential serological use of a recombinant protein that is a replica of a Mycobacterium tuberculosis protein found in the urine of infected mice.

The recent availability of numerous well-characterized Mycobacterium tuberculosis recombinant proteins has revived interest in the serological diagnosis of tuberculosis. Several promising results have been reported, particularly when more than one antigen is used in the test. However, thus far these antigens have not been used in routine diagnostic tests because they lack sufficient sensitivity. In addition, with the exception of one antigen, most recombinant M. tuberculosis proteins do not identify the majority of tuberculosis patients coinfected with human immunodeficiency virus (HIV). Here, we report a newer M. tuberculosis protein that is a promising candidate for increasing the sensitivity of the serological tests, in particular for patients coinfected with HIV. The protein was found in the urine of mice during the early stages of infection with M. tuberculosis (10 to 14 days), thus suggesting that the antigen is abundantly released during the in vivo growth of the mycobacterium. Reverse genetics was used to produce the recombinant protein, which we named U1 (for urine protein 1). Using a conventional enzyme-linked immunosorbent assay (ELISA), antibody to U1 could be detected in 60% of patients with pulmonary tuberculosis with no signs of coinfection with HIV (n = 83). Conversely, anti-U1 antibody was detected in 87% of the sera from tuberculosis patients coinfected with HIV (n = 47). Out of 12 HIV-infected nontuberculosis patients' sera, 9 did not react with U1 and three sera gave borderline ELISA signals (signal/cutoff of < or =1.75). These results suggest that the high efficiency of U1 in identifying tuberculosis patients coinfected with HIV may be related to abundant release of this protein during the initial phase of the HIV coinfection. The immediate availability of the antigen at a time point in which the patient's immune system is still competent would lead to a secondary immune response to U1 that persists for months in the patient's serum.

Expression cloning.

Expression cloning involves the selection of specific polypeptides, generated from a cDNA or genomic DNA library, based on certain characteristics of the expressed proteins, such as antibody or ligand binding, recognition by T-cells, function, or complementation of cell defects. Here we describe the detailed construction of a genomic, random shear lambda expression library, adsorption of anti Escherichia coli antibody from antiserum, the screening of an expression library with specific antisera, and the cloning of genes with potential use in the diagnosis of infectious disease. This approach has been used successfully by our laboratory for the discovery of antigenic components of diagnostics and vaccines for several infectious agents including: Mycobacterium tuberculosis, Anaplasma phagocytophila (formerly Ehrlichia spp. or E. phagocytophila), Babesia microti, Trypanosoma cruzi, Leishmania chagasi, and Chlamydia spp.

Improved serodetection of Mycobacterium bovis infection in badgers (Meles meles) using multiantigen test formats.

Despite attempts to control bovine tuberculosis, the incidence of disease in Great Britain continues to rise. In GB, the European badger (Meles meles) is a reservoir of infection with Mycobacterium bovis. In an effort to improve the serodetection of badger tuberculosis, we examined sera from M. bovis culture-positive and culture-negative badgers for their ability to recognize M. bovis antigens, using a multi-antigen print immunoassay (MAPIA). Depending on the antigens used in the MAPIA, the assay had a sensitivity of 49-59% and a specificity of 84-88% Results from the MAPIA were used to select antigens for the development of a lateral-flow immunoassay. This so-called 'Rapid Test' used 5microl of serum and gave unambiguous results within 10 min. When applied to 178 badger sera, the Rapid Test had a sensitivity of 53% and a specificity of 95%. This represented an improvement over the performance of the existing ELISA Test, which had a sensitivity of 47% and a specificity of 89% on the same sera. This is the first report of a diagnostic test for badger tuberculosis that can be performed alongside the captive animal.

Serum antibodies to lipophilin B detected in late stage breast cancer patients.

Lipophilin B mRNA is overexpressed in approximately 70% of breast tumors and shows a high degree of correlation with the mRNA expression profile of mammaglobin. This is further supported by the recent finding that, like other members of the secretoglobulin-uteroglobin family, mammaglobin and lipophilin B form a heteroduplex. The studies described show that there are pre-existing antibodies to lipophilin B peptide in the sera of breast cancer patients with different stages and grade of tumor and that this response is different from that seen to recombinant mammaglobin and native mammaglobin-lipophilin B complex. The highest titers were observed in later stage tumors. In addition, low levels of antibody were also seen in some patients with prostate and ovarian cancers, consistent with lipophilin B mRNA expression in these tumors at lower abundance than in breast tumors. In contrast, lipophilin B antibodies were absent in 20 healthy donor sera and 30 lung cancer sera. A polymorphism identified in Lipophilin B did not appear to influence human sera reactivity. The data indicate that humoral immune responses to lipophilin B may serve as a diagnostic indicator, particularly for breast cancer.

Identification and characterization of putative secreted antigens from Babesia microti.

The need for improved diagnostic reagents to identify human long-term carriers of the zoonotic parasite Babesia microti is evidenced by numerous reported cases of transfusion-acquired infections. This report describes the identification and initial characterization of 27 clones representing seven genes or gene families that were isolated through serological expression cloning by using a technique that we specifically designed to screen for shed antigens. In this screen, sera from B. microti-infected SCID mice, putatively containing secreted or shed antigens from the parasites, were harvested and used to immunize syngeneic immunocompetent mice (BALB/c). After boosting, the sera from the BALB/c mice, containing antibodies against the immunodominant secreted antigens, were used to screen a B. microti genomic expression library. Analyses of the putative peptides encoded by the novel DNA sequences revealed characteristics indicating that these peptides might be secreted. Initial serological data obtained with recombinant proteins and a patient serum panel demonstrated that several of the proteins could be useful in developing diagnostic tests for detection of B. microti antibodies and antigens in serum.

Relationship between tick bites and the seroprevalence of Babesia microti and Anaplasma phagocytophila (previously Ehrlichia sp.) in blood donors.

BACKGROUND: Tick-borne diseases, particularly babesiosis and ehrlichiosis, represent recently emerging infections. Despite an increased recognition of the threat tick-borne agents pose to blood safety, our understanding of the prevalence and transmissibility of these agents in blood donors is limited. STUDY DESIGN AND METHODS: Babesia microti and Anaplasma phagocytophila (previously Ehrlichia sp.) seroprevalence was determined in random Connecticut and Wisconsin donors, and subsequently in Connecticut donors reporting tick bites. In the interim, a postcard survey regarding tick bites during the previous 6 months was sent to 6,000 random donors in six geographically distinct collection regions. RESULTS: In total, 3 of 999 Wisconsin donors (0.3%) and 6 of 1,007 Connecticut donors (0.6%) had antibodies to B. microti. Of 992 donors tested for A. phagocytophila, 5 Wisconsin donors (0.5%) and 35 Connecticut donors (3.5%) were seropositive. A total of 2,482 donors (41.4%) completed the survey; 103 (4.1%) reported a tick bite. Of 848 Connecticut donors (0.4%) reporting tick bites, 3 had B. microti antibodies, while 8 (0.9%) had A. phagocytophila antibodies. These rates were not significantly different from control donors. CONCLUSION: Blood donors seropositive for B. microti and A. phagocytophila are present in Connecticut and Wisconsin. Donors readily recall previous tick bites, but self-reported bites are not reliable indicators of serologic status. The exposure of blood donors to tick-borne pathogens does suggest a need to better understand the transfusion transmission potential of these agents.