Global Health Initiatives: International Physician-to-Physician Consultation Programs.

PURPOSE: The goal of this article is to provide technical and operational blueprints for two successful global telehealth programs. METHODS: The authors designed a physician-to-physician consultation program to provide subspecialty expertise to physicians in war-torn Ukraine. Leveraging secure web applications, telehealth platforms, and image-sharing platforms, the authors repeatedly iterated upon infrastructure and workflows, which in turn facilitated the development of a parallel international program for US Department of State (DOS) employees and families. The authors provide descriptive statistics and metrics of both programs' successes and failures and detail iterative improvements with workflow visuals. To measure the added value of subspecialty imaging consultation, two radiologists performed a retrospective comparative review of the DOS program imaging reports, comparing the initial report to the consult report in consensus, measuring diagnostic report agreement, and rating the clinical impact of identified discrepancies on a three-point scale (mild, moderate, or major). Bivariate analyses using χ2 tests were conducted to assess associations between diagnostic discrepancies and patient or imaging factors. P values <.05 were considered to indicate statistical significance. RESULTS: The Ukraine program (May 2022 to August 2023) provided 114 patient consultations with 77 subspecialty radiology consults, >50 WhatsApp chats, and >1,000 messages exchanged, with a 92% overall consult request response rate. The DOS program (November 2022 to July 2023) provided 275 consultations with 70 subspecialty radiology consults and a 36% to 38% rate of alternative diagnoses, with 20% rated as incurring moderate or major clinical impact. Bivariate analyses demonstrated no significant patient or imaging association with diagnostic disagreements (P > .05 for all). CONCLUSIONS: Global telehealth infrastructure and multiple applications and platforms can be optimized in a workflow to provide efficient, high-level clinical and imaging consultation services across the globe.

Correction to "Gravimetric and Thermal-Imaging Characterization of Water-Free Reflux Condensers for Laboratory Applications".

[This corrects the article DOI: 10.1021/acsomega.4c00411.].

Gravimetric and Thermal-Imaging Characterization of Water-Free Reflux Condensers for Laboratory Applications.

Water-free reflux condensers, which use convective cooling from the surrounding air to condense vapors, avoid the need for cooling water, which is more sustainable than water-cooled condensers, and eliminates the risk of flooding, but these devices are newer and less familiar to many chemists, who may never have used them before. To facilitate the shift to water-free condensers, several types of water-free condensers (simple glass tube, Vigreux column, Condensyn, Findenser, and air-cooled Dimroth) were characterized using three different solvents (ethyl acetate, acetone, and tetrahydrofuran) under both gentle and vigorous refluxing conditions to compare their relative performance and determine the condensing capacity/failure point. In addition to experimentally quantifying the performance of each condenser both gravimetrically and via infrared thermal imaging, energy-balance models were developed to gain insight into which factors were most important in driving their performance. Several of the water-free condensers, including the Findenser, Condensyn, and air-cooled Dimroth condenser, were shown to provide suitable performance for most refluxing operations.

Borrelia miyamotoi BipA-like protein, BipM, is a candidate serodiagnostic antigen distinguishing between Lyme disease and relapsing fever Borrelia infections.

A Borrelia miyamotoi gene with partial homology to bipA of relapsing fever spirochetes Borrelia hermsii and Borrelia turicatae was identified by a GenBank basic alignment search analysis. We hypothesized that this gene product may be an immunogenic antigen as described for other relapsing fever Borrelia (RFB) and could serve as a serological marker for B. miyamotoi infections. The B. miyamotoi gene was a truncated version about half the size of the B. hermsii and B. turicatae bipA with a coding sequence of 894 base pairs. The gene product had a calculated molecular size of 32.7 kDa (including the signal peptide). Amino acid alignments with B. hermsii and B. turicatae BipA proteins and with other B. miyamotoi isolates showed conservation at the carboxyl end. We cloned the B. miyamotoi bipA-like gene (herein named bipM) and generated recombinant protein for serological characterization and for antiserum production. Protease protection analysis demonstrated that BipM was surface exposed. Serologic analyses using anti-B. miyamotoi serum samples from tick bite-infected and needle inoculated mice showed 94 % positivity against BipM. The 4 BipM negative serum samples were blotted against another B. miyamotoi antigen, BmaA, and two of them were seropositive resulting in 97 % positivity with both antigens. Serum samples from B. burgdorferi sensu stricto (s.s.)-infected mice were non-reactive against rBipM by immunoblot. Serum samples from Lyme disease patients were also serologically negative against BipM except for 1 sample which may have indicated a possible co-infection. A recently published study demonstrated that B. miyamotoi BipM was non-reactive against serum samples from B. hermsii, Borrelia parkeri, and B. turicatae infected animals. These results show that BipM has potential for a B. miyamotoi-infection specific and sensitive serodiagnostic to differentiate between Lyme disease and various RFB infections.

Analysis of variable major protein antigenic variation in the relapsing fever spirochete, Borrelia miyamotoi, in response to polyclonal antibody selection pressure.

Borrelia miyamotoi is a tick-transmitted spirochete that is genetically grouped with relapsing fever Borrelia and possesses multiple archived pseudogenes that encode variable major proteins (Vmps). Vmps are divided into two groups based on molecular size; variable large proteins (Vlps) and variable small proteins (Vsps). Relapsing fever Borrelia undergo Vmp gene conversion at a single expression locus to generate new serotypes by antigenic switching which is the basis for immune evasion that causes relapsing fever in patients. This study focused on B. miyamotoi vmp expression when spirochetes were subjected to antibody killing selection pressure. We incubated a low passage parent strain with mouse anti-B. miyamotoi polyclonal antiserum which killed the majority population, however, antibody-resistant reisolates were recovered. PCR analysis of the gene expression locus in the reisolates showed vsp1 was replaced by Vlp-encoded genes. Gel electrophoresis protein profiles and immunoblots of the reisolates revealed additional Vlps indicating that new serotype populations were selected by antibody pressure. Sequencing of amplicons from the expression locus of the reisolates confirmed the presence of a predominant majority serotype population with minority variants. These findings confirm previous work demonstrating gene conversion in B. miyamotoi and that multiple serotype populations expressing different vmps arise when subjected to antibody selection. The findings also provide evidence for spontaneous serotype variation emerging from culture growth in the absence of antibody pressure. Validation and determination of the type, number, and frequency of serotype variants that arise during animal infections await further investigations.

Evaluation of Immunocompetent Mouse Models for Borrelia miyamotoi Infection.

Borrelia miyamotoi is a relapsing fever spirochete that is harbored by Ixodes spp. ticks and is virtually uncharacterized, compared to other relapsing fever Borrelia vectored by Ornithodoros spp. ticks. There is not an immunocompetent mouse model for studying B. miyamotoi infection in vivo or for transmission in the vector-host cycle. Our goal was to evaluate B. miyamotoi infections in multiple mouse breeds/strains as a prelude to the ascertainment of the best experimental infection model. Two B. miyamotoi strains, namely, LB-2001 and CT13-2396, as well as three mouse models, namely, CD-1, C3H/HeJ, and BALB/c, were evaluated. We were unable to observe B. miyamotoi LB-2001 spirochetes in the blood via darkfield microscopy or to detect DNA via real-time PCR post needle inoculation in the CD-1 and C3H/HeJ mice. However, LB-2001 DNA was detected via real-time PCR in the blood of the BALB/c mice after needle inoculation, although spirochetes were not observed via microscopy. CD-1, C3H/HeJ, and BALB/c mice generated an antibody response to B. miyamotoi LB-2001 following needle inoculation, but established infections were not detected, and the I. scapularis larvae failed to acquire spirochetes from the exposed CD-1 mice. In contrast, B. miyamotoi CT13-2396 was visualized in the blood of the CD-1 and C3H/HeJ mice via darkfield microscopy and detected by real-time PCR post needle inoculation. Both mouse strains seroconverted. However, no established infection was detected in the mouse organs, and the I. scapularis larvae failed to acquire Borrelia after feeding on CT13-2396 exposed CD-1 or C3H/HeJ mice. These findings underscore the challenges in establishing an experimental B. miyamotoi infection model in immunocompetent laboratory mice. IMPORTANCE Borrelia miyamotoi is a causative agent of hard tick relapsing fever, was first identified in the early 1990s, and was characterized as a human pathogen in 2011. Unlike other relapsing fever Borrelia species, B. miyamotoi spread by means of Ixodes ticks. The relatively recent recognition of this human pathogen means that B. miyamotoi is virtually uncharacterized, compared to other Borrelia species. Currently there is no standard mouse-tick model with which to study the interactions of the pathogen within its vector and hosts. We evaluated two B. miyamotoi isolates and three immunocompetent mouse models to identify an appropriate model with which to study tick-host-pathogen interactions. With the increased prevalence of human exposure to Ixodes ticks, having an appropriate model with which to study B. miyamotoi will be critical for the future development of diagnostics and intervention strategies.

Characterization of the arthropod associated lipoprotein (Alp) in the tick-mammalian transmission cycle of Borrelia turicatae.

Pathogenic species of Borrelia are etiological agents of tick-borne relapsing fever (TBRF). Most species of TBRF Borrelia are transmitted by argasid ticks, and persistent colonization of the salivary glands is vital for spirochete transmission. This is due to the fast-feeding dynamics of the vector. However, the molecular mechanisms leading to vector colonization by the spirochete and their transmission to the vertebrate host remain vague. Previous work in Borrelia hermsii identified the arthropod associated lipoprotein (Alp) as being produced by spirochetes colonizing tick salivary glands. Upon transmission to mice, alp expression was down-regulated and the protein was undetectable in B. hermsii infecting mouse blood. Furthermore, Alp has homologs in multiple TBRF Borrelia species including Borrelia turicatae, Borrelia duttonii, and Borrelia recurrentis. To further evaluate the role of Alp in tick colonization and transmission, the gene was deleted in B. turicatae and the mutant's phenotype was evaluated. Our findings indicate that Alp is dispensable for colonization of the tick salivary glands and for the establishment of infection in laboratory mice.

Modification of the multiplex plasmid PCR assay for Borrelia miyamotoi strain LB-2001 based on the complete genome sequence reflecting genomic rearrangements differing from strain CT13-2396.

The genome of Borrelia spp. consists of an approximate 1 megabase chromosome and multiple linear and circular plasmids. We previously described a multiplex PCR assay to detect plasmids in the North American Borrelia miyamotoi strains LB-2001 and CT13-2396. The primer pair sets specific for each plasmid were derived from the genome sequence for B. miyamotoi strain CT13-2396, because the LB-2001 complete sequence had not been generated. The recent completion of the LB-2001 genome sequence revealed a distinct number of plasmids (n = 12) that differed from CT13-2396 (n = 14). Notable was a 97-kilobase plasmid in LB-2001, not present in CT13-2396, that appeared to be a rearrangement of the circular plasmids of strain CT13-2396. Strain LB-2001 contained two plasmids, cp30-2 and cp24, that were not annotated for strain CT13-2396. Therefore, we re-evaluated the original CT13-2396-derived multiplex PCR primer pairs and determined their location in the LB-2001 plasmids. We modified the original multiplex plasmid PCR assay for strain LB-2001 to include cp30-2 and cp24. We also determined which LB-2001 plasmids corresponded to the amplicons generated from the original CT13-2396 primer sets. These observations provide a more precise plasmid profile based on the multiplex PCR assay and reflect the complexity of gene rearrangements that occur in B. miyamotoi strains isolated from the same geographic region.

Diversity and distribution of the tick-borne relapsing fever spirochete Borrelia turicatae.

Borrelia turicatae is a causative agent of tick-borne relapsing fever (TBRF) in the subtropics and tropics of the United States and Latin America. Historically, B. turicatae was thought to be maintained in enzootic cycles in rural areas. However, there is growing evidence that suggests the pathogen has established endemic foci in densely populated regions of Texas. With the growth of homelessness in the state and human activity in city parks, it was important to implement field collection efforts to identify areas where B. turicatae and its vector circulate. Between 2017 and 2020 we collected Ornithodoros turicata ticks in suburban and urban areas including public and private parks and recreational spaces. Ticks were fed on naïve mice and spirochetes were isolated from the blood. Multilocus sequence typing (MLST) was performed on eight newly obtained isolates and included previously reported sequences. The four chromosomal loci targeted for MLST were 16S ribosomal RNA (rrs), flagellin B (flaB), DNA gyrase B (gyrB), and the intergenic spacer (IGS). Given the complexity of Borrelia genomes, plasmid diversity was also evaluated. These studies indicate that the IGS locus segregates B. turicatae into four genomic types and plasmid diversity is extensive between isolates. Furthermore, B. turicatae and its vector have established endemic foci in parks and recreational areas in densely populated settings of Texas.

Isolation and genetic characterization of a relapsing fever spirochete isolated from Ornithodoros puertoricensis collected in central Panama.

Tick-borne relapsing fever (TBRF) spirochetes are likely an overlooked cause of disease in Latin America. In Panama, the pathogens were first reported to cause human disease in the early 1900s. Recent collections of Ornithodoros puertoricensis from human dwellings in Panama prompted our interest to determine whether spirochetes still circulate in the country. Ornithodoros puertoricensis ticks were collected at field sites around the City of Panama. In the laboratory, the ticks were determined to be infected with TBRF spirochetes by transmission to mice, and we report the laboratory isolation and genetic characterization of a species of TBRF spirochete from Panama. Since this was the first isolation of a species of TBRF spirochete from Central America, we propose to designate the bacteria as Borrelia puertoricensis sp. nov. This is consistent with TBRF spirochete species nomenclature from North America that are designated after their tick vector. These findings warrant further investigations to assess the threat B. puertoricensis sp. nov. may impose on human health.

The impact of in vitro cultivation on the natural life cycle of the tick-borne relapsing fever spirochete Borrelia turicatae.

Tick-borne relapsing fever is an infectious disease caused by Borrelia species and are primarily transmitted by Ornithodoros ticks. Prior work indicated that in vitro cultivated spirochetes remain infectious to mice by needle inoculation; however, the impact of laboratory propagation on the pathogens natural life cycle has not been determined. Our current study assessed the effect of serial cultivation on the natural tick-mammalian transmission cycle. First, we evaluated genomic DNA profiles from B. turicatae grown to 30, 60, 120, and 300 generations, and these spirochetes were used to needle inoculate mice. Uninfected nymphal ticks were fed on these mice and acquisition, transstadial maintenance, and subsequent transmission after tick bite was determined. Infection frequencies in mice that were fed upon by ticks colonized with B. turicatae grown to 30, 60, and 120 generations were 100%, 100%, and 30%, respectively. Successful infection of mice by tick feeding was not detected after 120 generations. Quantifying B. turicatae in tick tissues indicated that by 300 generations they no longer colonized the vector. The results indicate that in vitro cultivation significantly affects the establishment of tick colonization and murine infection. This work provides a foundation for the identification of essential genetic elements in the tick-mammalian infectious cycle.

Characterization of a Borrelia miyamotoi membrane antigen (BmaA) for serodiagnosis of Borrelia miyamotoi disease.

Borrelia miyamotoi is a tick-borne pathogen that causes Borrelia miyamotoi disease (BMD), an emerging infectious disease of increasing public health significance. B. miyamotoi is transmitted by the same tick vector (Ixodes spp.) as B. burgdorferi sensu lato (s.l.), the causative agent of Lyme disease, therefore laboratory assays to differentiate BMD from Lyme disease are needed to avoid misdiagnoses and for disease confirmation. We previously performed a global immunoproteomic analysis of the murine host antibody response against B. miyamotoi infection to discover antigens that could serologically distinguish the two infections. An initial assessment identified a putative lipoprotein antigen, here termed BmaA, as a promising candidate to augment current research-based serological assays. In this study, we show that BmaA is an outer surface-associated protein by its susceptibility to protease digestion. Synthesis of BmaA in culture was independent of temperature at either 23 °C or 34 °C. The BmaA gene is present in two identical loci harbored on separate plasmids in North American strains LB-2001 and CT13-2396. bmaA-like sequences are present in other B. miyamotoi strains and relapsing fever borrelia as multicopy genes and as paralogous or orthologous gene families. IgM and IgG antibodies in pooled serum from BMD patients reacted with native BmaA fractionated by 2-dimensional gel electrophoresis and identified by mass spectrometry. IgG against recombinant BmaA was detected in 4 of 5 BMD patient serum samples as compared with 1 of 23 serum samples collected from patients with various stages of Lyme disease. Human anti-B. turicatae serum did not seroreact with recombinant BmaA suggesting a role as a species-specific diagnostic antigen. These results demonstrated that BmaA elicits a human host antibody response during B. miyamotoi infection but not in a tested group of B. burgdorferi-infected Lyme disease patients, thereby providing a potentially useful addition for developing BMD serodiagnostic tests.

Corrigendum: Differential Expression of Putative Ornithodoros turicata Defensins Mediated by Tick Feeding.

[This corrects the article DOI: 10.3389/fcimb.2020.00152.].

Ornithodoros capensis sensu stricto (Ixodida: Argasidae) in Coiba National Park: first report for Panama, with notes on the O. capensis group in Panamanian shores and Costa Rica.

Ornithodoros capensis sensu lato (s.l.) is a morphologically similar group of soft ticks that parasitizes mostly seabirds in continental and offshore territories worldwide. Ornithodoros capensis sensu stricto (s.s.) has been previously recorded in many islands and coastal localities along the American continent; however, some records from Central America remain obscure. In this work we performed morphological and molecular analyses on soft ticks collected in Coiba National Park, an archipelago located in the Pacific Ocean off the coast of Panama, confirming the occurrence of O. capensis s.s. in this country for the first time. In addition, a morphological examination of museum specimens collected in Costa Rica, and a further locality in Panama, confirmed that O. capensis s.l. is established in the former country, and that its distribution along Panamanian shores is likely larger.

Differential Expression of Putative Ornithodoros turicata Defensins Mediated by Tick Feeding.

Additional research on soft ticks in the family Argasidae is needed to bridge the knowledge gap relative to hard ticks of the family Ixodidae; especially, the molecular mechanisms of Ornithodoros biology. Ornithodoros species are vectors of human and animal pathogens that include tick-borne relapsing fever spirochetes and African swine fever virus. Soft tick vector-pathogen interactions involving components of the tick immune response are not understood. Ticks utilize a basic innate immune system consisting of recognition factors and cellular and humoral responses to produce antimicrobial peptides, like defensins. In the present study, we identified and characterized the first putative defensins of Ornithodoros turicata, an argasid tick found primarily in the southwestern United States and regions of Latin America. Four genes (otdA, otdB, otdC, and otdD) were identified through sequencing and their predicted amino acid sequences contained motifs characteristic of arthropod defensins. A phylogenetic analysis grouped these four genes with arthropod defensins, and computational structural analyses further supported the identification. Since pathogens transmitted by O. turicata colonize both the midgut and salivary glands, expression patterns of the putative defensins were determined in these tissues 1 week post engorgement and after molting. Defensin genes up-regulated in the tick midgut 1 week post blood feeding were otdA and otdC, while otdD was up-regulated in the midgut of post-molt ticks. Moreover, otdB and otdD were also up-regulated in the salivary glands of flat post-molt ticks, while otdC was up-regulated within 1 week post blood-feeding. This work is foundational toward additional studies to determine mechanisms of vector competence and pathogen transmission from O. turicata.

Humoral immune response of pigs, Sus scrofa domesticus, upon repeated exposure to blood-feeding by Ornithodoros turicata Duges (Ixodida: Argasidae).

BACKGROUND: Ornithodoros turicata is an important vector of both human and veterinary pathogens. One primary concern is the global spread of African swine fever virus and the risk of its re-emergence in the Americas through potential transmission by O. turicata to domestic pigs and feral swine. Moreover, in Texas, African warthogs were introduced into the state for hunting purposes and evidence exists that they are reproducing and have spread to three counties in the state. Consequently, it is imperative to develop strategies to evaluate exposure of feral pigs and African warthogs to O. turicata. RESULTS: We report the development of an animal model to evaluate serological responses of pigs to O. turicata salivary proteins after three exposures to tick feeding. Serological responses were assessed for ~ 120 days by enzyme-linked immunosorbent assay and immunoblotting using salivary gland extracts from O. turicata. CONCLUSIONS: Our findings indicate that domestic pigs seroconverted to O. turicata salivary antigens that is foundational toward the development of a diagnostic assay to improve soft tick surveillance efforts.

Isolation and Molecular Characterization of Tick-Borne Relapsing Fever Borrelia Infecting Ornithodoros (Pavlovskyella) verrucosus Ticks Collected in Ukraine.

BACKGROUND: Tick-borne relapsing fever (TBRF) is a neglected zoonotic bacterial disease known to occur on 5 continents. We report a laboratory-acquired case of TBRF caused by Borrelia caucasica, which is endemic in Ukraine and transmitted by Ornithodoros verrucosus ticks. METHODS: We isolated spirochetes and characterized them by partially sequencing the 16s ribosomal ribonucleic acid (rrs), flagellin (flaB), and deoxyribonucleic acid gyrase (gyrB) genes and conducting a phylogenetic analysis. RESULTS: These analyses revealed a close relationship of Ukrainian spirochetes with the Asian TBRF species, Borrelia persica. The taxonomic and nomenclature problems related to insufficient knowledge on the spirochetes and their vectors in the region are discussed. CONCLUSIONS: Although these findings enhance our understanding of species identities for TBRF Borrelia in Eurasia, further work is required to address the neglected status of TBRF in this part of the world. Public health practitioners should consider TBRF and include the disease into differential diagnosis of febrile illnesses with unknown etiology.

NMR Quantification of the Effects of Ligands and Counterions on Lewis Acid Catalysis.

The relative Lewis acidity of a variety of metal-ligand catalyst complexes is quantified using 31P NMR spectroscopy. Three 31P NMR probes, including two new bidentate binding probes, are compared on the basis of different binding modes (i.e., monodentate vs bidentate) and the relative scale of their downfield shift upon binding to Lewis acid complexes. Bidentate coordination of catalyst complexes including metal catalysts, ligands, and counterions were assessed due to their importance to asymmetric catalysis. The effect of ligands, counterions, and additives on Lewis acidity is quantified and correlated to reaction yield at an early time point as an approximation for catalytic activity/efficiency and chelation mode in two organic transformations. Binding studies were performed under catalytically relevant conditions, giving further applicability to synthesis. Insight into activation modes are revealed through this analysis.

Catalytic Asymmetric Synthesis of Cyclopentene-spirooxindoles Bearing Vinylsilanes Capable of Further Transformations.

We report a scandium-catalyzed [3 + 2] annulation of alkylideneoxindoles with allenylsilanes for the enantioselective formation of cyclopentene-spirooxindoles containing vinylsilanes. Using a Sc(OTf)2/PyBOX/BArF complex, the spiroannulation of allenylsilanes affords products with >94:6 dr and >90:10 er. The effect of the counterion and ligand to control selectivity is discussed. The transformation of the vinylsilane is demonstrated using cross-coupling, epoxidation, and Tamao-Fleming oxidation reactions. A series of competition experiments provide a comparison of nucleophilicity between allyl- and allenylsilanes.