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We conducted surveillance studies in Sinaloa, Mexico, to determine the circulation of tick-borne relapsing fever spirochetes. We collected argasid ticks from a home in the village of Camayeca and isolated spirochetes. Genomic analysis indicated that Borrelia turicatae infection is a threat to those living in resource-limited settings. [ABSTRACT FROM AUTHOR]

Territories in southern parts of Eastern Europe and in the Caucasus are endemic for tick-borne relapsing fever (TBRF), caused by Borrelia caucasica. This spirochete is transmitted exclusively by the bites of Ornithodoros verrucosus ; however, the distribution and genetic diversity of the tick vector have not been explored. To address this, we performed a phylogeographic study of O. verrucosus specimens collected across a large geographic distribution. We sequenced and analyzed complete mitochondrial genomes of 54 individual O. verrucosus ticks representing 23 geographically diverse populations from Ukraine, Georgia, and Azerbaijan. We detected 47 unique haplotypes, with every collection site exhibiting distinct polymorphisms. This, along with other population genetic indices, suggests little evidence of gene flow between populations. The Bayesian coalescent analysis revealed the presence of four lineages that diverged in the Middle Pleistocene (770–126 kya). Two lineages were widespread and present in all study regions, while the other two were restricted to the southern foothills of the Lesser Caucasus mountain range. The sympatry of these ancient lineages suggests that isolation by environment, in addition to geographic distance, may play a role in the intraspecific divergence of tick populations. Using a phylogeographic approach, we provide a snapshot of genetic diversity in O. verrucosus and discuss the evolutionary history of the tick vector. • Mitogenomes of 54 Ornithodoros verrucosus from Ukraine, Georgia, and Azerbaijan; • High level of identity (average 99.29 %) at the mitogenome level revealed; • 47 unique haplotypes highlighted the lack of gene flow between populations; • Four lineages diverged in the Middle Pleistocene (770–126 kya); • Low prevalence of Borrelia caucasica. [ABSTRACT FROM AUTHOR]

The mitochondrial genome (mitogenome) has proven to be important for the taxonomy, systematics, and population genetics of ticks. However, current methods to generate mitogenomes can be cost-prohibitive at scale. To address this issue, we developed a cost-effective approach to amplify and sequence the whole mitogenome of individual tick specimens. Using two different primer sites, this approach generated two full-length mitogenome amplicons that were sequenced using the Oxford Nanopore Technologies' Mk1B sequencer. We used this approach to generate 85 individual tick mitogenomes from samples comprised of the three tick families, 11 genera, and 57 species. Twenty-six of these species did not have a complete mitogenome available on GenBank prior to this work. We benchmarked the accuracy of this approach using a subset of samples that had been previously sequenced by low-coverage Illumina genome skimming. We found our assemblies were comparable or exceeded the Illumina method, achieving a median sequence concordance of 99.98%. We further analyzed our mitogenome dataset in a mitophylogenomic analysis in the context of all three tick families. We were able to sequence 72 samples in one run and achieved a cost/sample of ~ $10 USD. This cost-effective strategy is applicable for sample identification, taxonomy, systematics, and population genetics for not only ticks but likely other metazoans; thus, making mitogenome sequencing equitable for the wider scientific community. [ABSTRACT FROM AUTHOR]

Background: Tick-borne relapsing fever (TBRF) is a globally prevalent, yet under-studied vector-borne disease transmitted by soft and hard bodied ticks. While soft TBRF (sTBRF) spirochetes have been described for over a century, our understanding of the molecular mechanisms facilitating vector and host adaptation is poorly understood. This is due to the complexity of their small (~ 1.5 Mb) but fragmented genomes that typically consist of a linear chromosome and both linear and circular plasmids. A majority of sTBRF spirochete genomes' plasmid sequences are either missing or are deposited as unassembled sequences. Consequently, our goal was to generate complete, plasmid-resolved genomes for a comparative analysis of sTBRF species of the Western Hemisphere. Results: Utilizing a Borrelia specific pipeline, genomes of sTBRF spirochetes from the Western Hemisphere were sequenced and assembled using a combination of short- and long-read sequencing technologies. Included in the analysis were the two recently isolated species from Central and South America, Borrelia puertoricensis n. sp. and Borrelia venezuelensis, respectively. Plasmid analyses identified diverse sequences that clustered plasmids into 30 families; however, only three families were conserved and syntenic across all species. We also compared two species, B. venezuelensis and Borrelia turicatae, which were isolated ~ 6,800 km apart and from different tick vector species but were previously reported to be genetically similar. Conclusions: To truly understand the biological differences observed between species of TBRF spirochetes, complete chromosome and plasmid sequences are needed. This comparative genomic analysis highlights high chromosomal synteny across the species yet diverse plasmid composition. This was particularly true for B. turicatae and B. venezuelensis, which had high average nucleotide identity yet extensive plasmid diversity. These findings are foundational for future endeavors to evaluate the role of plasmids in vector and host adaptation. [ABSTRACT FROM AUTHOR]

Chagas disease is a leading cause of non-ischemic cardiomyopathy in endemic regions of Central and South America. In Belize, Triatoma dimidiata sensu lato has been identified as the predominate taxon but vectorial transmission of Chagas disease is considered to be rare in the country. We recently identified an acute case of vector-borne Chagas disease in the northern region of Belize. Here we present a subsequent investigation of triatomines collected around the case-patient's home. We identified yet undescribed species, closely related to Triatoma huehuetenanguensis vector by molecular systematics methods occurring in the peridomestic environment. The identification of a T. cruzi-positive, novel species of Triatoma in Belize indicates an increased risk of transmission to humans in the region and warrants expanded surveillance and further investigation. [ABSTRACT FROM AUTHOR]

Mosquito saliva is a mix of numerous proteins that are injected into the skin while the mosquito searches for a blood meal. While mosquito saliva is known to be immunogenic, the salivary components driving these immune responses, as well as the types of immune responses that occur, are not well characterized. We investigated the effects of one potential immunomodulatory mosquito saliva protein, sialokinin, on the human immune response. We used flow cytometry to compare human immune cell populations between humanized mice bitten by sialokinin knockout mosquitoes or injected with sialokinin, and compared them to those bitten by wild-type mosquitoes, unbitten, or saline-injected control mice. Humanized mice received 4 mosquito bites or a single injection, were euthanized after 7 days, and skin, spleen, bone marrow, and blood were harvested for immune cell profiling. Our results show that bites from sialokinin knockout mosquitoes induced monocyte and macrophage populations in the skin, blood, bone marrow, and spleens, and primarily affected CD11c- cell populations. Other increased immune cells included plasmacytoid dendritic cells in the blood, natural killer cells in the skin and blood, and CD4+ T cells in all samples analyzed. Conversely, we observed that mice bitten with sialokinin knockout mosquitoes had decreased NKT cell populations in the skin, and fewer B cells in the blood, spleen, and bone marrow. Taken together, we demonstrated that sialokinin knockout saliva induces elements of a TH1 cellular immune response, suggesting that the sialokinin peptide is inducing a TH2 cellular immune response during wild-type mosquito biting. These findings are an important step towards understanding how mosquito saliva modulates the human immune system and which components of saliva may be critical for arboviral infection. By identifying immunomodulatory salivary proteins, such as sialokinin, we can develop vaccines against mosquito saliva components and direct efforts towards blocking arboviral infections. Author summary: Numerous studies have shown the effects of mosquito saliva proteins on the immune system of animals and humans with disease caused by mosquito-borne pathogens. We have previously described some of these effects in humanized mice (which contain specific human immune system cells and develop arboviral diseases similar to humans) infected by mosquito bite with dengue and chikungunya viruses. In this study, we show that humanized mice have altered cellular immune responses after they are bitten by uninfected mosquitoes lacking the sialokinin salivary protein. Our results suggest that sialokinin alone shifts mammalian immunity towards a TH2 response, away from the anti-viral, cell-mediated, and humoral responses that would protect against viruses included in the saliva. This is the first study of its kind, and it highlights how the effects of specific saliva components can be evaluated for human therapeutic intervention. [ABSTRACT FROM AUTHOR]

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. Author summary: In the Americas, the range and genetic diversity of tick-borne relapsing fever spirochetes remains poorly defined. Borrelia turicatae is the predominant species in the southwestern and eastern portions of the United States and to Latin America, and recent studies suggests the pathogens and their vectors have established endemic foci in highly populated areas of the United States. Given the rate of urban growth and the incorporation of green spaces into large cities, this study supports previous work suggesting the expansion of B. turicatae. [ABSTRACT FROM AUTHOR]

Chikungunya virus (CHIKV) is an emerging, mosquito-borne alphavirus responsible for acute to chronic arthralgias and neuropathies. Although it originated in central Africa, recent reports of disease have come from many parts of the world, including the Americas. While limiting human CHIKV cases through mosquito control has been used, it has not been entirely successful. There are currently no licensed vaccines or treatments specific for CHIKV disease, thus more work is needed to develop effective countermeasures. Current animal research on CHIKV is often not representative of human disease. Most models use CHIKV needle inoculation via unnatural routes to create immediate viremia and localized clinical signs; these methods neglect the natural route of transmission (the mosquito vector bite) and the associated human immune response. Since mosquito saliva has been shown to have a profound effect on viral pathogenesis, we evaluated a novel model of infection that included the natural vector, Aedes species mosquitoes, transmitting CHIKV to mice containing components of the human immune system. Humanized mice infected by 3–6 mosquito bites showed signs of systemic infection, with demonstrable viremia (by qRT-PCR and immunofluorescent antibody assay), mild to moderate clinical signs (by observation, histology, and immunohistochemistry), and immune responses consistent with human infection (by flow cytometry and IgM ELISA). This model should give a better understanding of human CHIKV disease and allow for more realistic evaluations of mechanisms of pathogenesis, prophylaxis, and treatments. Author summary: In humans, CHIKV infections are mostly symptomatic, resulting in fever, viremia, and debilitating joint disease; viremia is present up to 13 days after the onset of fever and peaks at the onset of clinical signs. Previous studies in mice and nonhuman primates (NHPs) have often neglected the mosquito vector, leading to variable viremias and infection of tissues primarily at the sites of CHIKV injection (legs in mice, ankles or spleen/liver in NHPs). This is the first study to demonstrate systemic CHIKV infection caused by infected mosquito bites in humanized laboratory mice. Disease seen with this humanized mouse model mimics human clinical signs and symptoms, with signs of inflammation in muscle and joints due to viral replication. This humanized mouse model provides a basis for assessing human CHIKV pathogenesis, prophylaxis, and treatment, under natural conditions of infection. [ABSTRACT FROM AUTHOR]

(B) Infected, degenerate muscle fibrils from minimal (score 1) myositis at day 7 post mosquito bite, 40X magnification. (C) Infected, degenerate muscle fibrils from mild (score 2) myositis at day 14 post mosquito bite, 40X magnification. [Extracted from the article]

To assess changes in SARS-CoV-2 spike binding antibody prevalence in the Dominican Republic and implications for immunologic protection against variants of concern, we prospectively enrolled 2,300 patients with undifferentiated febrile illnesses in a study during March 2021-August 2022. We tested serum samples for spike antibodies and tested nasopharyngeal samples for acute SARS-CoV-2 infection using a reverse transcription PCR nucleic acid amplification test. Geometric mean spike antibody titers increased from 6.6 (95% CI 5.1-8.7) binding antibody units (BAU)/mL during March-June 2021 to 1,332 (95% CI 1,055-1,682) BAU/mL during May-August 2022. Multivariable binomial odds ratios for acute infection were 0.55 (95% CI 0.40-0.74), 0.38 (95% CI 0.27-0.55), and 0.27 (95% CI 0.18-0.40) for the second, third, and fourth versus the first anti-spike quartile; findings were similar by viral strain. Combining serologic and virologic screening might enable monitoring of discrete population immunologic markers and their implications for emergent variant transmission. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Zika virus (Flavivirus genus) is the first mosquito-borne virus known to cause high rates of microcephaly and abortion in humans. Typically, Zika virus causes a self-limiting, systemic illness; however, the current outbreak of Zika virus in the Americas has been associated with increased rates of fetal malformations and Guillain-Barré syndrome. Very few Zika virus isolates have been described in the literature, and live viruses are needed to perform studies of pathogenesis and to develop vaccines and treatments. Methodology/Clinical findings: We isolated Zika virus, strain FLR, directly from the serum of an individual infected in Barranquilla, Colombia (December, 2015). Here, we describe the patient’s clinical course and characterize strain FLR by its growth characteristics in mosquito and mammalian cells and its partial resistance to UV-inactivation. The full genome sequence of FLR was also analyzed (including the 3’ un-translated region), to determine its probable geographic origin, and to pinpoint structural differences from other Zika virus strains. Conclusions/Significance: We anticipate that the study of this low passage, clinical isolate of Zika virus, which is available for worldwide distribution, will help uncover the mechanisms of viral replication and host immune responses contributing to the varied and sometimes severe clinical presentations seen during the current epidemic in the Americas. [ABSTRACT FROM AUTHOR]

Ethnopharmacological relevance Many different tribes of American Indians used jewelweed, Impatiens capensis Meerb, as a plant mash to reduce development of poison ivy dermatitis. Saponins are a natural soapy constituent found within plants. A 2012 study suggested that saponins may be present in jewelweed which could be responsible for its efficacy in preventing rash development following contact with Toxicodendron radicans (L.) Kuntze (poison ivy). This study validated this hypothesis and demonstrated additional biological activity of the jewelweed saponin containing extract. Materials and methods Fresh I. capensis leaves were extracted with methanol and further partitioned between ethyl acetate and water, with a final separation between water and n-butanol, to obtain a saponin containing extract. The presence of saponins in the extract was demonstrated by the observation of foaming and using a vanillin colorimetric assay for total saponins. Efficacy of the saponin containing extracts in rash reduction was tested by brushing poison ivy (PI) onto the forearms of volunteers ( N =23) in six locations and treating these PI exposed areas with distilled water (control), saponin containing extracts, fresh plant mashes, and soaps made with and without plant extracts. Saponin containing extracts were further tested for biological activity against both gram negative and gram positive bacteria and against cancer cell lines A-375, HT-29, and MCF-7. Additionally, because saponins have been shown to have a stimulatory effect in cardiac muscle 2 µl saponin extract was applied superficially to black worms, Lumbriculus variegatus ( N =5). Results, and conclusions Both saponin containing extracts and all soaps tested were effective in reducing poison ivy dermatitis; thus, saponin content correlates with PI rash prevention. No apparent antibiosis was observed against any bacteria tested; however, dose response cytotoxicity was documented against MCF-7 breast cancer cells and cytostatic activity was seen against the HT-29 colon cancer cell lines. Lumbriculus variegatus exhibited a 138% increase in heart rate over baseline rate five minutes post treatment implying a possible positive chronotropic effect. [ABSTRACT FROM AUTHOR]

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. Author summary: Tick-borne relapsing fever (TBRF) is an often misdiagnosed neglected tropical disease primarily impacting those in resource limited settings. Most species are transmitted by argasid ticks, but ixodid ticks and the human body louse are also competent vectors. Infection of TBRF spirochetes is challenging to diagnose because argasid ticks are rapid feeders and rarely spotted on the patient. Moreover, given the nonspecific manifestation of disease and poor clinical awareness, TBRF is overlooked. In Central America, TBRF was studied in the Canal Zone of Panama until the 1930s. Over the last 10 years argasid ticks have been reported to colonize human dwellings in the country but it has remained vague if the disease persists. In this study, we demonstrate the transmission of TBRF spirochetes from field collected ticks and the laboratory isolation and genetic characterization of the species. These findings suggest that TBRF spirochetes continue to circulate in Panama and should prompt health care providers to consider this disease when patients present with a fever of unknown origin. Furthermore, with the laboratory isolation of this bacteria, molecular diagnostic tools can be developed to improve epidemiological studies of TBRF spirochetes in Central America. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Mosquito saliva is a very complex concoction of >100 proteins, many of which have unknown functions. The effects of mosquito saliva proteins injected into our skin during blood feeding have been studied mainly in mouse models of injection or biting, with many of these systems producing results that may not be relevant to human disease. Here, we describe the numerous effects that mosquito bites have on human immune cells in mice engrafted with human hematopoietic stem cells. We used flow cytometry and multiplex cytokine bead array assays, with detailed statistical analyses, to detect small but significant variations in immune cell functions after 4 mosquitoes fed on humanized mice footpads. After preliminary analyses, at different early times after biting, we focused on assessing innate immune and subsequent cellular responses at 6 hours, 24 hours and 7 days after mosquito bites. We detected both Th1 and Th2 human immune responses, and delayed effects on cytokine levels in the blood, and immune cell compositions in the skin and bone marrow, up to 7 days post-bites. These are the first measurements of this kind, with human immune responses in whole animals, bitten by living mosquitoes, versus previous studies using incomplete mouse models and salivary gland extracts or needle injected saliva. The results have major implications for the study of hematophagous insect saliva, its effects on the human immune system, with or without pathogen transmission, and the possibility of determining which of these proteins to target for vaccination, in attempts to block transmission of numerous tropical diseases. [ABSTRACT FROM AUTHOR]

Background: While Zika virus (ZIKV) is mainly transmitted by mosquitoes, numerous cases of sexual transmission have been reported during recent outbreaks. Little is known about which host cell types or entry factors aid in mediating this sexual transmission.Methods: In this study, we investigated ZIKV cell tropism by infecting 2 types of human prostate cells with 3 contemporary ZIKV isolates from persons infected in the Americas. We used real-time quantitative polymerase chain reaction and immunofluorescence analyses to measure infection and flow cytometry to detect entry factor expression.Results: Here we show that ZIKV infects, replicates, and produces infectious virus in prostate stromal mesenchymal stem cells, epithelial cells, and organoids made with a combination of these cells. We also show that prostate cells express several well-characterized flavivirus attachment factors. In contrast, dengue virus does not infect or does not replicate in these prostate cells, although it is known to use similar receptors.Conclusions: Our results indicate that ZIKV favors infection of stromal cells more so than epithelial cells in organoids, possibly indicating a preference for stem cells in general. Overall, these results suggest that ZIKV replication occurs in the human prostate and can account for ZIKV secretion in semen, thus leading to sexual transmission. [ABSTRACT FROM AUTHOR]

Zika virus belongs to the Flavivirus family of RNA viruses, which include other important human pathogens such as dengue and West Nile virus. There are no approved antiviral drugs for these viruses. The highly conserved NS2B-NS3 protease of Flavivirus is essential for the replication of these viruses and it is therefore a drug target. Compound screen followed by medicinal chemistry optimization yielded a novel series of 2,6-disubstituted indole compounds that are potent inhibitors of Zika virus protease (ZVpro) with IC 50 values as low as 320 nM. The structure-activity relationships of these and related compounds are discussed. Enzyme kinetics studies show the inhibitor 66 most likely exhibited a non-competitive mode of inhibition. In addition, this series of ZVpro inhibitors also inhibit the NS2B-NS3 protease of dengue and West Nile virus with reduced potencies. The most potent compounds 66 and 67 strongly inhibited Zika virus replication in cells with EC 68 values of 1–3 μM. These compounds are novel pharmacological leads for further drug development targeting Zika virus. [Display omitted] • Compound screening identified indole-containing compounds 1 and 2 to be novel inhibitors of Zika virus NS2B-NS3 protease. • Medicinal chemistry optimization gave a significantly more potent inhibitor 66. • Compound 66 exhibited a strong antiviral activity against cellular replication of Zika virus. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]