Emerging Tick-borne Infections in the Upper Midwest and Northeast United States Among Patients With Suspected Anaplasmosis.

Background: Emerging tick-transmitted illnesses are increasingly recognized in the United States (US). To identify multiple potential tick-borne pathogens in patients from the Upper Midwest and Northeast US with suspected anaplasmosis, we used state-of-the-art methods (polymerase chain reaction [PCR] and paired serology) to test samples from patients in whom anaplasmosis had been excluded. Methods: Five hundred sixty-eight patients without anaplasmosis had optimal samples available for confirmation of alternative tick-borne pathogens, including PCR and/or paired serology (acute-convalescent interval ≤42 days). Results: Among 266 paired serology evaluations, for which the median acute-convalescent sampling interval was 28 (interquartile range, 21-33) days, we identified 35 acute/recent infections (24 [9%] Borrelia burgdorferi; 6 [2%] Ehrlichia chaffeensis/Ehrlichia muris subsp eauclairensis [EC/EME]; 3 [1%] spotted fever group rickettsioses [SFGR], and 2 [<1%] Babesia microti) in 33 (12%) patients. Two had concurrent or closely sequential infections (1 B burgdorferi and EC/EME, and 1 B burgdorferi and SFGR). Using multiplex PCR and reverse-transcription PCR, we identified 7 acute infections (5/334 [1%] Borrelia miyamotoi and 2/334 [1%] B microti) in 5 (1%) patients, including 2 with B microti-B miyamotoi coinfection, but no Borrelia mayonii, SFGR, Candidatus Anaplasma capra, Heartland virus, or Powassan virus infections. Thus, among 568 patients with ruled-out anaplasmosis, 38 (6.7%) had ≥1 agent of tick-borne illness identified, with 33 patients (35 infections) diagnosed by paired serology and 5 additional patients (7 infections) by PCR. Conclusions: By identifying other tick-borne agents in patients in whom anaplasmosis had been excluded, we demonstrate that emerging tick-borne infections will be identified if specifically sought.

Benidipine impairs innate immunity converting sublethal to lethal infections in a murine model of spotted fever rickettsiosis.

Spotted fever group rickettsiae are tick-borne obligate intracellular bacteria that infect microvascular endothelial cells. Humans and mammalian infection results in endothelial cell barrier dysfunction and increased vascular permeability. We previously demonstrated that treatment of Rickettsia parkeri-infected cells with the calcium channel blocker benidipine significantly delayed vascular barrier permeability. Thus, we hypothesized that benidipine, known to be safe and effective for other clinical processes, could reduce rickettsia-induced vascular permeability in vivo in an animal model of spotted fever rickettsiosis. Based on liver, lung and brain vascular FITC-dextran extravasation studies, benidipine did not reliably impact vascular permeability. However, it precipitated a deleterious effect on responses to control sublethal R. parkeri infection. Animals treated with benidipine alone had no clinical signs or changes in histopathology and splenic immune cell distributions. Benidipine-treated infected animals had marked increases in tissue and blood bacterial loads, more extensive inflammatory histopathologic injury, and changes in splenic architecture and immune cell distributions potentially reflecting diminished Ca2+ signaling, reduced innate immune cell activation, and loss of rickettsial propagation control. Impaired T cell activation by R. parkeri antigen in the presence of benidipine was confirmed in vitro with the use of NKT cell hybridomas. The unexpected findings stand in stark contrast to recent discussions of the benefits of calcium channel blockers for viral infections and chronic infectious or inflammatory diseases. A role for calcium channel blockers in exacerbation of human rickettsiosis and acute inflammatory infections should be evaluated by a retrospective review of patient's outcomes and medications.

Host-response transcriptional biomarkers accurately discriminate bacterial and viral infections of global relevance.

Diagnostic limitations challenge management of clinically indistinguishable acute infectious illness globally. Gene expression classification models show great promise distinguishing causes of fever. We generated transcriptional data for a 294-participant (USA, Sri Lanka) discovery cohort with adjudicated viral or bacterial infections of diverse etiology or non-infectious disease mimics. We then derived and cross-validated gene expression classifiers including: 1) a single model to distinguish bacterial vs. viral (Global Fever-Bacterial/Viral [GF-B/V]) and 2) a two-model system to discriminate bacterial and viral in the context of noninfection (Global Fever-Bacterial/Viral/Non-infectious [GF-B/V/N]). We then translated to a multiplex RT-PCR assay and independent validation involved 101 participants (USA, Sri Lanka, Australia, Cambodia, Tanzania). The GF-B/V model discriminated bacterial from viral infection in the discovery cohort an area under the receiver operator curve (AUROC) of 0.93. Validation in an independent cohort demonstrated the GF-B/V model had an AUROC of 0.84 (95% CI 0.76-0.90) with overall accuracy of 81.6% (95% CI 72.7-88.5). Performance did not vary with age, demographics, or site. Host transcriptional response diagnostics distinguish bacterial and viral illness across global sites with diverse endemic pathogens.

Characterization of the bacterial microbiome of non-hematophagous bats and associated ectoparasites from Brazil.

Introduction: Bats, along with their ectoparasites, harbor a wide diversity of symbiotic and potential pathogenic bacteria. Despite the enormous diversity of bats (181 species), few studies aimed to investigate the bacterial microbiome of Brazilian chiropterans and associated ectoparasites. This study aimed to characterize the bacterial microbiome of non-hematophagous bats and associated Streblidae flies and Macronyssidae and Spinturnicidae mites in the state of Mato Grosso do Sul, midwestern Brazil. Methods: Oral and rectal swabs were collected from 30 bats (Artibeus lituratus [n = 13], Artibeus planirostris [n = 9], Eptesicus furinalis [n = 5], Carollia perspicillata [n = 2], and Platyrrhinus lineatus [n = 1]). In addition, a total of 58 mites (15 Macronyssidae and 43 Spinturnicidae) and 48 Streblidae bat flies were collected from the captured bats. After DNA extraction and purification, each sample's bacterial composition was analyzed with metagenomic sequencing. Results: The microbiome composition of both oral and rectal bat swab samples showed that Gammaproteobacteria was the most abundant bacterial class. Spiroplasma, Wolbachia and Bartonella represented the most abundant genera in Streblidae flies. While Wolbachia (Alphaproteobacteria) was the most abundant genus found in Spinturnicidae, Arsenophonus (Gammaproteobacteria) was found in high abundance in Macronyssidae mites. In addition to characterizing the microbiome of each sample at the class and genus taxonomic levels, we identified medically significant bacteria able to infect both animals and humans in oral (Streptococcus and Anaplasma) and rectal swabs (Enterobacter, Klebsiella, Escherichia, Enterococcus, Streptococcus), Macronyssidae (Anaplasma, Bartonella, Ehrlichia) and Spinturnicidae (Anaplasma, Bartonella) mites as well as Streblidae flies (Spiroplasma, Bartonella). Discussion and conclusion: Besides expanding the knowledge on the bacterial microbiome of non-hematophagous bats and Streblidae flies from Brazil, the present work showed, for the first time, the bacterial community of bat-associated Macronyssidae and Spinturnicidae mites.

Innate immunity in rickettsial infections.

Rickettsial agents are a diverse group of alpha-proteobacteria within the order Rickettsiales, which possesses two families with human pathogens, Rickettsiaceae and Anaplasmataceae. These obligate intracellular bacteria are most frequently transmitted by arthropod vectors, a first step in the pathogens' avoidance of host cell defenses. Considerable study of the immune responses to infection and those that result in protective immunity have been conducted. Less study has focused on the initial events and mechanism by which these bacteria avoid the innate immune responses of the hosts to survive within and propagate from host cells. By evaluating the major mechanisms of evading innate immunity, a range of similarities among these bacteria become apparent, including mechanisms to escape initial destruction in phagolysosomes of professional phagocytes, those that dampen the responses of innate immune cells or subvert signaling and recognition pathways related to apoptosis, autophagy, proinflammatory responses, and mechanisms by which these microbes attach to and enter cells or those molecules that trigger the host responses. To illustrate these principles, this review will focus on two common rickettsial agents that occur globally, Rickettsia species and Anaplasma phagocytophilum.

Spotted fever rickettsia-induced microvascular endothelial barrier dysfunction is delayed by the calcium channel blocker benidipine.

The tick-borne bacterium Rickettsia parkeri is an obligate intracellular pathogen that belongs to spotted fever group rickettsia (SFGR). The SFG pathogens are characterized by their ability to infect and rapidly proliferate inside host vascular endothelial cells that eventually result in impairment of vascular endothelium barrier functions. Benidipine, a wide range dihydropyridine calcium channel blocker, is used to prevent and treat cardiovascular diseases. In this study, we tested whether benidipine has protective effects against rickettsia-induced microvascular endothelial cell barrier dysfunction in vitro. We utilized an in vitro vascular model consisting of transformed human brain microvascular endothelial cells (tHBMECs) and continuously monitored transendothelial electric resistance (TEER) across the cell monolayer. We found that during the late stages of infection when we observed TEER decrease and when there was a gradual increase of the cytoplasmic [Ca2+], benidipine prevented these rickettsia-induced effects. In contrast, nifedipine, another cardiovascular dihydropyridine channel blocker specific for L-type Ca2+ channels, did not prevent R. parkeri-induced drop of TEER. Additionally, neither drug was bactericidal. These data suggest that growth of R. parkeri inside endothelial cells is associated with impairment of endothelial cell monolayer integrity due to Ca2+ flooding through specific, benidipine-sensitive T- or N/Q-type Ca2+ channels but not through nifedipine-sensitive L-type Ca2+ channels. Further study will be required to discern the exact nature of the Ca2+ channels and Ca2+ transporting system(s) involved, any contributions of the pathogen toward this process, as well as the suitability of benidipine and new dihydropyridine derivatives as complimentary therapeutic drugs against Rickettsia-induced vascular failure.

Immune evasion strategies of major tick-transmitted bacterial pathogens.

Tick-transmitted bacterial pathogens thrive in enzootic infection cycles, colonizing disparate vertebrate and arthropod tissues, often establishing persistent infections. Therefore, the evolution of robust immune evasion strategies is central to their successful persistence or transmission between hosts. To survive in nature, these pathogens must counteract a broad range of microbicidal host responses that can be localized, tissue-specific, or systemic, including a mix of these responses at the host-vector interface. Herein, we review microbial immune evasion strategies focusing on Lyme disease spirochetes and rickettsial or tularemia agents as models for extracellular and intracellular tick-borne pathogens, respectively. A better understanding of these adaptive strategies could enrich our knowledge of the infection biology of relevant tick-borne diseases, contributing to the development of future preventions.

Visualization of the Dynamics of Invasion and Intravasation of the Bacterium That Causes Lyme Disease in a Tissue Engineered Dermal Microvessel Model.

Lyme disease is a tick-borne disease prevalent in North America, Europe, and Asia. Despite the accumulated knowledge from epidemiological, in vitro, and in animal studies, the understanding of dissemination of vector-borne pathogens, such as Borrelia burgdorferi (Bb), remains incomplete with several important knowledge gaps, especially related to invasion and intravasation into circulation. To elucidate the mechanistic details of these processes a tissue-engineered human dermal microvessel model is developed. Fluorescently labeled Bb are injected into the extracellular matrix (ECM) to mimic tick inoculation. High resolution, confocal imaging is performed to visualize the sub-acute phase of infection. From analysis of migration paths no evidence to support adhesin-mediated interactions between Bb and ECM components is found, suggesting that collagen fibers serve as inert obstacles to migration. Intravasation occurs at cell-cell junctions and is relatively fast, consistent with Bb swimming in ECM. In addition, it is found that Bb alone can induce endothelium activation, resulting in increased immune cell adhesion but no changes in global or local permeability. Together these results provide new insight into the minimum requirements for Bb dissemination and highlight how tissue-engineered models are complementary to animal models in visualizing dynamic processes associated with vector-borne pathogens.

Assessing the use of a micro-sampling device for measuring blood protein levels in healthy subjects and COVID-19 patients.

BACKGROUND: Venous phlebotomy performed by trained personnel is critical for patient diagnosis and monitoring of chronic disease, but has limitations in resource-constrained settings, and represents an infection control challenge during outbreaks. Self-collection devices have the potential to shift phlebotomy closer to the point of care, supporting telemedicine strategies and virtual clinical trials. Here we assess a capillary blood micro-sampling device, the Tasso Serum Separator Tube (SST), for measuring blood protein levels in healthy subjects and non-hospitalized COVID-19 patients. METHODS: 57 healthy controls and 56 participants with mild/moderate COVID-19 were recruited at two U.S. military healthcare facilities. Healthy controls donated Tasso SST capillary serum, venous plasma and venous serum samples at multiple time points, while COVID-19 patients donated a single Tasso SST serum sample at enrolment. Concentrations of 17 protein inflammatory biomarkers were measured in all biospecimens by Ella multi-analyte immune-assay. RESULTS: Tasso SST serum protein measurements in healthy control subjects were highly reproducible, but their agreements with matched venous samples varied. Most of the selected proteins, including CRP, Ferritin, IL-6 and PCT, were well-correlated between Tasso SST and venous serum with little sample type bias, but concentrations of D-dimer, IL-1B and IL-1Ra were not. Self-collection at home with delayed sample processing was associated with significant concentrations differences for several analytes compared to supervised, in-clinic collection with rapid processing. Finally, Tasso SST serum protein concentrations were significantly elevated in in non-hospitalized COVID-19 patients compared with healthy controls. CONCLUSIONS: Self-collection of capillary blood with micro-sampling devices provides an attractive alternative to routine phlebotomy. However, concentrations of certain analytes may differ significantly from those in venous samples, and factors including user proficiency, temperature control and time lags between specimen collection and processing need to be considered for their effect on sample quality and reproducibility.

Detection of antibodies to Anaplasma phagocytophilum and Babesia microti using linear peptides.

Anaplasma phagocytophilum and Babesia microti are emerging tick-borne pathogens in the United States. Although active infection is typically diagnosed by direct diagnostic tests, such as blood smear or polymerase chain reaction assay, serologic assays can be helpful to identify past infections, and the use of acute plus convalescent testing can potentially identify recent infections. We employed a peptide array to select sets of linear peptides for serologic diagnosis of infections with A. phagocytophilum and B. microti. Three optimal peptides were selected for each agent based on their performance with clinical specimens. All three A. phagocytophilum peptides were located within the conserved fragments of the MSP2 antigen. Two B. microti peptides were located in the N terminus of the SA-1 antigen; the third was in the BMN 1-17 antigen. We found that these peptides can be a useful tool for detection of antibody reactivity to both of these pathogens.

Anaplasma phagocytophilum Ankyrin A Protein (AnkA) Enters the Nucleus Using an Importin-ß-, RanGTP-Dependent Mechanism.

Anaplasma phagocytophilum, a tick-borne obligately intracellular bacterium of neutrophils, causes human granulocytic anaplasmosis. Ankyrin A (AnkA), an effector protein with multiple ankyrin repeats (AR) is injected via type IV-secretion into the host neutrophil to gain access to the nucleus where it modifies the epigenome to promote microbial fitness and propagation. AR proteins transported into the host cell nucleus must use at least one of two known eukaryotic pathways, the classical importin ß-dependent pathway, and/or the RanGDP- and AR (ankyrin-repeat)-dependent importin ß-independent (RaDAR) pathway. Truncation of the first four AnkA N-terminal ARs (AR1-4), but not other regions, prevents AnkA nuclear accumulation. To investigate the mechanism of nuclear import, we created point mutations of AnkA N-terminal ARs, predicted to interfere with RaDAR protein import, and used importazole, a specific inhibitor of the importin α/ß, RanGTP-dependent pathway. Nuclear colocalization analysis shows that nuclear localization of AnkA is unaffected by single AR1-4 mutations but is significantly reduced by single mutations in consecutive ARs suggesting RaDAR protein nuclear import. However, AnkA nuclear localization was also decreased with importazole, and with GTPγS. Furthermore, A. phagocytophilum growth in HL-60 cells was completely suppressed with importazole, indicating that A. phagocytophilum propagation requires a ß-importin-dependent pathway. A typical classical NLS overlapping AR4 was subsequently identified suggesting the primacy of the importin-α/ß system in AnkA nuclear localization. Whether the mutational studies of putative key residues support RaDAR NLS function or simply reflect structural changes that diminish engagement of an AR-NLS-importin pathway needs to be resolved through careful structure-function studies.

Ehrlichiosis and anaplasmosis subcommittee report to the Tick-borne Disease Working Group.

Ehrlichioses and anaplasmosis have undergone dramatic increases in incidence, and the geographic ranges of their occurrence and vectors have also expanded. There is marked underreporting of these diseases owing to deficient physician awareness and knowledge of the illnesses as well as limited access to appropriate diagnostic tests. Human monocytic ehrlichiosis and anaplasmosis are life threatening diseases with estimated case fatality rates of 2.7 and 0.3%, respectively. However, knowledge of their full range of signs and symptoms is incomplete, and the incidence of subclinical infections is unknown. Currently available laboratory diagnostic methods are poorly utilized, and with the exception of nucleic acid amplification tests are not useful for diagnosis during the acute stage of illness when timely treatment is needed. The Ehrlichiosis and Anaplasmosis Subcommittee of the Tick-Borne Disease Working Group recommended active clinical surveillance to determine the true incidence, full clinical spectrum, and risk factors for severe illness, as well as standardized surveillance of ticks for these pathogens, and enhanced education of primary medical caregivers and the public regarding these diseases. The subcommittee identified the needs to develop sensitive, specific acute stage diagnostic tests for local clinical laboratories and point-of-care testing, to develop approaches for utilizing electronic medical records, data mining, and artificial intelligence for assisting early diagnosis and treatment, and to develop adjunctive therapies for severe disease.

Anaplasma phagocytophilum Activates NF-κB Signaling via Redundant Pathways.

Anaplasma phagocytophilum subverts neutrophil function permitting intracellular survival, propagation and transmission. Sustained pro-inflammatory response, recruitment of new host cells for population expansion, and delayed apoptosis are associated with prolonged nuclear presence of NF-κB. We investigated NF-κB signaling and transcriptional activity with A. phagocytophilum infection using inhibitors of NF-κB signaling pathways, and through silencing of signaling pathway genes. How inhibitors or silencing affected A. phagocytophilum growth, inflammatory response (transcription of the κB-enhanced genes CXCL8 and MMP9), and NF-κB signaling pathway gene expression were tested. Among A. phagocytophilum-infected HL-60 cells, nuclear NF-κB p50, p65, and p52 were detected by immunoblots or iTRAQ proteomics. A. phagocytophilum growth was affected most by the IKKαß inhibitor wedelolactone (reductions of 96 to 99%) as compared with SC-514 that selectively inhibits IKKß, illustrating a role for the non-canonical pathway. Wedelolactone inhibited transcription of both CXCL8 (p = 0.001) and MMP9 (p = 0.002) in infected cells. Compared to uninfected THP-1 cells, A. phagocytophilum infection led to >2-fold down regulation of 64 of 92 NF-κB signaling pathway genes, and >2-fold increased expression in only 4. Wedelolactone and SC-514 reversed downregulation in all 64 and 45, respectively, of the genes down-regulated by infection, but decreased expression in 1 gene with SC-514 only. Silencing of 20 NF-κB signal pathway genes increased bacterial growth in 12 (IRAK1, MAP3K1, NFKB1B, MAP3K7, TICAM2, TLR3, TRADD, TRAF3, CHUK, IRAK2, LTBR, and MALT1). Most findings support canonical pathway activation; however, the presence of NFKB2 in infected cell nuclei, selective non-canonical pathway inhibitors that dampen CXCL8 and MMP9 transcription with infection, upregulation of non-canonical pathway target genes CCL13 and CCL19, enhanced bacterial growth with TRAF3 and LTBR silencing provide evidence for non-canonical pathway signaling. Whether this impacts distinct inflammatory processes that underlie disease, and whether and how A. phagocytophilum subverts NF-κB signaling via these pathways, need to be investigated.

Optimization and Evaluation of a Multiplex Quantitative PCR Assay for Detection of Nucleic Acids in Human Blood Samples from Patients with Spotted Fever Rickettsiosis, Typhus Rickettsiosis, Scrub Typhus, Monocytic Ehrlichiosis, and Granulocytic Anaplasmosis.

Spotted fever group rickettsioses (SFGR), typhus group rickettsioses (TGR), scrub typhus (caused by Orientia tsutsugamushi), ehrlichiosis, and anaplasmosis often present as undifferentiated fever but are not treated by agents (penicillins and cephalosporins) typically used for acute febrile illness. Inability to diagnose these infections when the patient is acutely ill leads to excess morbidity and mortality. Failure to confirm these infections retrospectively if a convalescent blood sample is not obtained also impairs epidemiologic and clinical research. We designed a multiplex real-time quantitative PCR (qPCR) assay to detect SFGR, TGR, O. tsutsugamushi, and infections caused by Anaplasma phagocytophilum and Ehrlichia chaffeensis with the ompA, 17-kDa surface antigen gene, tsa56, msp2 (p44), and vlpt gene targets, respectively. Analytical sensitivity was ≥2 copies/µl (linear range, 2 to 2 × 105) and specificity was 100%. Clinical sensitivities for SFGR, TGR, and O. tsutsugamushi were 25%, 20%, and 27%, respectively, and specificities were 98%, 99%, and 100%, respectively. Clinical sensitivities for A. phagocytophilum and E. chaffeensis were 93% and 84%, respectively, and specificities were 99% and 98%, respectively. This multiplex qPCR assay could support early clinical diagnosis and treatment, confirm acute infections in the absence of a convalescent-phase serum sample, and provide the high-throughput testing required to support large clinical and epidemiologic studies. Because replication of SFGR and TGR in endothelial cells results in very low bacteremia, optimal sensitivity of qPCR for these rickettsioses will require use of larger volumes of input DNA, which could be achieved by improved extraction of DNA from blood and/or extraction of DNA from a larger initial volume of blood.

Rickettsioses as Major Etiologies of Unrecognized Acute Febrile Illness, Sabah, East Malaysia.

Orientia tsutsugamushi, spotted fever group rickettsioses, and typhus group rickettsioses (TGR) are reemerging causes of acute febrile illness (AFI) in Southeast Asia. To further delineate extent, we enrolled patients >4 weeks of age with nonmalarial AFI in Sabah, Malaysia, during 2013-2015. We confirmed rickettsioses (past or acute, IgG titer >160) in 126/354 (36%) patients. We confirmed acute rickettsioses (paired 4-fold IgG titer rise to >160) in 38/145 (26%) patients: 23 O. tsutsugamushi, 9 spotted fever group, 4 TGR, 1 O. tsutsugamushi/spotted fever group, and 1 O. tsutsugamushi/TGR. PCR results were positive in 11/319 (3%) patients. Confirmed rickettsioses were more common in male adults; agricultural/plantation work and recent forest exposure were risk factors. Dizziness and acute hearing loss but not eschars were reported more often with acute rickettsioses. Only 2 patients were treated with doxycycline. Acute rickettsioses are common (>26%), underrecognized, and untreated etiologies of AFI in East Malaysia; empirical doxycycline treatment should be considered.

Development of a Sensitive and Rapid Recombinase Polymerase Amplification Assay for Detection of Anaplasma phagocytophilum.

Human granulocytic anaplasmosis (HGA) is a tick-borne disease caused by the obligate intracellular Gram-negative bacterium Anaplasma phagocytophilum The disease often presents with nonspecific symptoms with negative serology during the acute phase. Direct pathogen detection is the best approach for early confirmatory diagnosis. Over the years, PCR-based molecular detection methods have been developed, but optimal sensitivity is not achieved by conventional PCR while real-time PCR requires expensive and sophisticated instruments. To improve the sensitivity and also develop an assay that can be used in resource-limited areas, an isothermal DNA amplification assay based on recombinase polymerase amplification (RPA) was developed. To do this, we identified a 171-bp DNA sequence within multiple paralogous copies of msp2 within the genome of A. phagocytophilum Our novel RPA assay targeting this sequence has an analytical limit of detection of one genome equivalent copy of A. phagocytophilum and can reliably detect 125 bacteria/ml in human blood. A high level of specificity was demonstrated by the absence of nonspecific amplification using genomic DNA from human or DNA from other closely-related pathogenic bacteria, such as Anaplasma platys, Ehrlichia chaffeensis, Orientia tsutsugamushi, and Rickettsia rickettsii, etc. When applied to patient DNA extracted from whole blood, this new RPA assay was able to detect 100% of previously diagnosed A. phagocytophilum cases. The sensitivity and rapidness of this assay represents a major improvement for early diagnosis of A. phagocytophilum in human patients and suggest a role for better surveillance in its reservoirs or vectors, especially in remote regions where resources are limited.

A Novel Laminin-Binding Protein Mediates Microbial-Endothelial Cell Interactions and Facilitates Dissemination of Lyme Disease Pathogens.

Borrelia burgdorferi conserved gene products BB0406 and BB0405, members of a common B. burgdorferi paralogous gene family, share 59% similarity. Although both gene products can function as potential porins, only BB0405 is essential for infection. Here we show that, despite sequence homology and coexpression from the same operon, both proteins differ in their membrane localization attributes, antibody accessibility, and immunogenicity in mice. BB0406 is required for spirochete survival in mammalian hosts, particularly for the disseminated infection in distant organs. We identified that BB0406 interacts with laminin, one of the major constituents of the vascular basement membrane, and facilitates spirochete transmigration across host endothelial cell barriers. A better understanding of how B. burgdorferi transmigrates through dermal and tissue vascular barriers and establishes disseminated infections will contribute to the development of novel therapeutics to combat early infection.

Using detergent-enhanced LAMP for African trypanosome detection in human cerebrospinal fluid and implications for disease staging.

OBJECTIVE: Where human African trypanosomiasis (HAT) patients are seen, failure to microscopically diagnose infections by Trypanosoma brucei gambiense in blood smears and/or cerebrospinal fluid (CSF) in the critical early stages of the disease is the single most important factor in treatment failure, a result of delayed treatment onset or its absence. We hypothesized that the enhanced sensitivity of detergent-enhanced loop-mediated isothermal amplification (LAMP) will allow for point of care (POC) detection of African trypanosomes in the CSF of HAT patients where the probability for detecting a single parasite or parasite DNA molecule in 1 µL of CSF sample is negligible by current methods. METHODOLOGY: We used LAMP targeting the multicopy pan-T. brucei repetitive insertion mobile element (RIME LAMP) and the Trypanosoma brucei gambiense 5.8S rRNA-internal transcribed spacer 2 gene (TBG1 LAMP). We tested 1 µL out of 20 µL sham or Triton X-100 treated CSFs from 73 stage-1 and 77 stage-2 HAT patients from the Central African Republic and 100 CSF negative controls. RESULTS: Under sham conditions, parasite DNA was detected by RIME and TBG1 LAMP in 1.4% of the stage-1 and stage-2 gambiense HAT CSF samples tested. After sample incubation with detergent, the number of LAMP parasite positive stage-2 CSF's increased to 26%, a value which included the 2 of the 4 CSF samples where trypanosomes were identified microscopically. Unexpected was the 41% increase in parasite positive stage-1 CSF's detected by LAMP. Cohen's kappa coefficients for RIME versus TBG1 LAMP of 0.92 (95%CI: 0.82-1.00) for stage-1 and 0.90 (95%CI: 0.80-1.00) for stage-2 reflected a high level of agreement between the data sets indicating that the results were not due to amplicon contamination, data confirmed in χ2 tests (p<0.001) and Fisher's exact probability test (p = 4.7e-13). CONCLUSION: This study detected genomic trypanosome DNA in the CSF independent of the HAT stage and may be consistent with early CNS entry and other scenarios that identify critical knowledge gaps for future studies. Detergent-enhanced LAMP could be applicable for non-invasive African trypanosome detection in human skin and saliva or as an epidemiologic tool for the determination of human (or animal) African trypanosome prevalence in areas where chronically low parasitemias are present.

Development and Clinical Validation of a Multiplex Real-Time Quantitative PCR Assay for Human Infection by Anaplasma phagocytophilum and Ehrlichia chaffeensis.

BACKGROUND: Human granulocytic anaplasmosis (HGA), caused by Anaplasma phagocytophilum, and human monocytic ehrlichiosis (HME), caused by Ehrlichia chaffeensis, often present as undifferentiated fever but are not treated by typical empiric regimens for acute febrile illness. Their role as agents of vector-borne febrile disease in tropical regions is more poorly studied than for other rickettsial infections. Limitations in diagnosis have impaired epidemiologic and clinical research and needless morbidity and mortality occur due to untreated illness. METHODS: We designed and clinically validated a multiplex real-time quantitative PCR assay for Anaplasma phagocytophilum and Ehrlichia chaffeensis using samples confirmed by multiple gold-standard methods. RESULTS: Clinical sensitivity and specificity for A. phagocytophilum were 100% (39/39) and 100% (143/143), respectively, and for E. chaffeensis 95% (20/21) and 99% (159/161), respectively. CONCLUSIONS: These assays could support early diagnosis and treatment as well as the high-throughput testing required for large epidemiologic studies.