A Novel TaqMan Assay for Detection of Rickettsia 364D, the Etiologic Agent of Pacific Coast Tick Fever

ABSTRACT Pacific Coast tick fever is a febrile illness associated with the bite of Dermacentor occidentalis and results from an infection due to the intracellular pathogen Rickettsia 364D (also known by the proposed name “Rickettsia philipii”). Current molecular methods for the detection of this pathogen rely on the amplification of a conserved spotted fever group rickettsial gene (ompA) followed by DNA sequencing of the amplicon to identify the species. This work describes the development of a Rickettsia 364D-specific TaqMan assay to simplify and accelerate the detection and identification processes. The assay demonstrated a sensitivity of 1 genomic copy per 4-μl sample and is highly specific for Rickettsia 364D. The utility of this assay for ecological and diagnostic samples was evaluated using banked specimens collected in a single-blind manner and yielded a clinical sensitivity and specificity of 100%. In conclusion, we describe the development and evaluation of a novel TaqMan real-time PCR assay for the detection and identification of Rickettsia 364D suitable for ecological and diagnostic applications.

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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

Journal of Clinical Microbiology ◽

10.1128/jcm.01802-19 ◽

2020 ◽

Vol 58 (9) ◽

Author(s):

Megan E. Reller ◽

J. Stephen Dumler

Keyword(s):

Quantitative Pcr ◽

Scrub Typhus ◽

Spotted Fever ◽

Febrile Illness ◽

Epidemiologic Studies ◽

Qpcr Assay ◽

Spotted Fever Group ◽

Analytical Sensitivity ◽

Content Type ◽

Real Time Quantitative Pcr

ABSTRACT 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.

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Epidemiology of Spotted Fever Group Rickettsioses and Acute Undifferentiated Febrile Illness in Villeta, Colombia

American Journal of Tropical Medicine and Hygiene ◽

10.4269/ajtmh.16-0442 ◽

2017 ◽

Vol 97 (3) ◽

pp. 782-788 ◽

Cited By ~ 4

Author(s):

Álvaro A. Faccini-Martínez ◽

Elkin Valbuena ◽

Christian Barreto ◽

Ana M. Palomar ◽

Luis J. Polo-Terán ◽

...

Keyword(s):

Spotted Fever ◽

Febrile Illness ◽

Spotted Fever Group

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Searching and Finding the Hidden Treasure: A Retrospective Analysis of Rickettsial Disease Among Dutch International Travelers

Clinical Infectious Diseases ◽

10.1093/cid/ciaa091 ◽

2020 ◽

Cited By ~ 1

Author(s):

Sophia G de Vries ◽

Louise E van Eekeren ◽

Hans van der Linden ◽

Benjamin J Visser ◽

Martin P Grobusch ◽

...

Keyword(s):

Retrospective Analysis ◽

Medical Records ◽

Scrub Typhus ◽

Spotted Fever ◽

Febrile Illness ◽

Empiric Antibiotic Therapy ◽

Spotted Fever Group ◽

Orientia Tsutsugamushi ◽

Rickettsial Disease ◽

Empiric Antibiotic

Abstract Background Rickettsial disease (RD) is a prevalent and underestimated cause of febrile illness worldwide, especially in the absence of an inoculation eschar. We attempted to quantify this underestimation at our clinic, by investigating past cases of febrile illness in travelers who had tested negative for leptospirosis, a disease that can initially present similarly to non-eschar RD, and which we routinely consider when other important causes of unspecified febrile illness have tested negative. Methods We performed a retrospective analysis in febrile returned travelers from Asia, Africa, or the Americas between 2010 and 2017, who had tested negative for leptospirosis. Serologic immunofluorescence assays were performed for Orientia tsutsugamushi (scrub typhus), typhus group, and spotted fever group RD. We performed a medical records review of all patients who tested positive. In case of a fitting medical history, cases were deemed either confirmed (based on convalescent serology) or suspected (based on single serology). Results Among 97 patients, convalescent serology was available in 16 (16.5%) patients, and a single serology in 81 (83.5%) patients. RD was the likely diagnosis in 8 of 16 (50.0%) patients with convalescent serology, and in 8 of 81 (9.9%) with single serology. Of the 16 confirmed/suspected cases, 11 (69%) had been missed and 7 (44%) had not received adequate empiric antibiotic therapy. Conclusions This study shows that non-eschar RD is an important and poorly recognized cause of illness in travelers, even in a specialized travel clinic. A lower threshold to test and treat for RD is warranted in returning travelers with febrile illness.

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Spotted Fever GroupRickettsiaInfection and Transmission Dynamics inAmblyomma maculatum

Infection and Immunity ◽

10.1128/iai.00804-18 ◽

2019 ◽

Vol 87 (4) ◽

Cited By ~ 2

Author(s):

Chanakan Suwanbongkot ◽

Ingeborg M. Langohr ◽

Emma K. Harris ◽

Wellesley Dittmar ◽

Rebecca C. Christofferson ◽

...

Keyword(s):

Salivary Glands ◽

Spotted Fever ◽

Attachment Site ◽

Spotted Fever Group ◽

Tick Feeding ◽

Tick Saliva ◽

Content Type ◽

Feeding Site ◽

Vertebrate Hosts ◽

Tick Salivary Glands

ABSTRACTTick vectors are capable of transmitting several rickettsial species to vertebrate hosts, resulting in various levels of disease. Studies have demonstrated the transmissibility of both rickettsial pathogens and novelRickettsiaspecies or strains with unknown pathogenicity to vertebrate hosts during tick blood meal acquisition; however, the quantitative nature of transmission remains unknown. We tested the hypothesis that if infection severity is a function of the rickettsial load delivered during tick transmission, then a more virulent spotted fever group (SFG)Rickettsiaspecies is transmitted at higher levels during tick feeding. UsingAmblyomma maculatumcohorts infected withRickettsia parkerior “CandidatusRickettsia andeanae,” a quantitative PCR (qPCR) assay was employed to quantify rickettsiae in tick salivary glands and saliva, as well as in the vertebrate hosts at the tick attachment site over the duration of tick feeding. Significantly greater numbers ofR. parkerithan of “Ca. Rickettsia andeanae” rickettsiae were present in tick saliva and salivary glands and in the vertebrate hosts at the feeding site during tick feeding. Microscopy demonstrated the presence of both rickettsial species in tick salivary glands, and immunohistochemical analysis of the attachment site identified localizedR. parkeri, but not “Ca. Rickettsia andeanae,” in the vertebrate host. Lesions were also distinct and more severe in vertebrate hosts exposed toR. parkerithan in those exposed to “Ca. Rickettsia andeanae.” The specific factors that contribute to the generation of a sustained rickettsial infection and subsequent disease have yet to be elucidated, but the results of this study suggest that the rickettsial load in ticks and during transmission may be an important element.

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Rickettsialpox — a rare but not extinct disease: review of the literature and new directions

Russian Journal of Infection and Immunity ◽

10.15789/2220-7619-rar-1294 ◽

2020 ◽

Vol 10 (3) ◽

pp. 477-485

Author(s):

M. E. Eremeeva ◽

K. Muniz-Rodriguez

Keyword(s):

Genetic Diversity ◽

New York ◽

Spotted Fever ◽

Research Priorities ◽

Etiologic Agent ◽

Spotted Fever Group ◽

Public Health Importance ◽

Sexually Transmitted ◽

Worldwide Distribution ◽

Clinical Triad

Rickettsialpox is an urban zoonosis caused by Rickettsia akari. To date R. akari is the only well-characterized mite-borne member of the spotted fever group. It is transmitted by the mouse mite, Liponyssoides sanguineus, commonly found on peridomestic rodents. While the disease was first discovered in New York City in 1946, a few years later a similar outbreak occurred in the Ukraine SSR. Numerous serosurveys and diagnosis of sporadic cases of rickettsialpox suggest its global distribution; however, the actual contemporary geography of rickettsialpox and its incidence are unknown. Rickettsialpox is characterized by the classic clinical triad found in rickettsioses of a black eschar, high fever, and rash but the latter is atypical as it is papulovesicular. Dermatological manifestations and the progression of rickettsialpox may mimic other infectious and noninfectious syndromes, including sexually transmitted diseases. The purpose of this review is to increase awareness of this unique disease through reanalysis of classic and contemporary clinical descriptions of rickettsialpox, evaluation of its worldwide distribution, and updates on the public health importance of the disease as well as the ecology and vector associations of R. akari. Our review data suggests that only limited genetic diversity exists among the available isolates of R. akari associated with previous outbreaks; additional effort is still required to define specific genetic markers permitting direct surveillance, accurate and reliable diagnosis, tracking and studying of the vector and host associations of contemporary isolates. The potential of R. akari to cross into other vector species emphasizes the necessity for detection of outbreaks of the disease in new regions of the world and in novel ecological settings. We describe existing gaps and limitations in our current understanding of the pathogenesis of rickettsialpox, the epidemiology of this disease and the genetic diversity of R. akari. We propose research priorities for what is needed to improve our understanding of this neglected rickettsial disease and its etiologic agent.

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Study of infection by Rickettsiae of the spotted fever group in humans and ticks in an urban park located in the City of Londrina, State of Paraná, Brazil

Revista da Sociedade Brasileira de Medicina Tropical ◽

10.1590/s0037-86822011005000037 ◽

2011 ◽

Vol 44 (3) ◽

pp. 313-317 ◽

Cited By ~ 2

Author(s):

Roberta Santos Toledo ◽

Katia Tamekuni ◽

Mauro de Freitas Silva Filho ◽

Valeska Bender Haydu ◽

Richard Campos Pacheco ◽

...

Keyword(s):

Citrate Synthase ◽

Spotted Fever ◽

Immunofluorescence Assay ◽

Etiologic Agent ◽

Spotted Fever Group ◽

Rickettsia Species ◽

Rickettsia Rickettsii ◽

Serological Studies ◽

Human Sera ◽

Brazilian Spotted Fever

INTRODUCTION: Spotted fevers are emerging zoonoses caused by Rickettsia species in the spotted fever group (SFG). Rickettsia rickettsii is the main etiologic agent of Brazilian spotted fever (BSF) and it is transmitted by Amblyomma spp. ticks. METHODS: The study aimed to investigate SFG rickettsiae in the Arthur Thomas Municipal Park in Londrina, PR, by collecting free-living ticks and ticks from capybaras and blood samples from personnel working in these areas. Samples from A. dubitatum and A. cajennense were submitted for PCR in pools to analyze the Rickettsia spp. gltA (citrate synthase gene). RESULTS: All the pools analyzed were negative. Human sera were tested by indirect immunofluorescence assay with R. rickettsii and R. parkeri as antigens. Among the 34 sera analyzed, seven (20.6%) were reactive for R. rickettsii: four of these had endpoint titers equal to 64, 2 titers were 128 and 1 titer was 256. None of the samples were reactive for R. parkeri. An epidemiological questionnaire was applied to the park staff, but no statistically significant associations were identified. CONCLUSIONS: The serological studies suggest the presence of Rickettsiae related to SFG that could be infecting the human population studied; however, analysis of the ticks collected was unable to determine which species may be involved in transmission to humans.

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Detection and Identification of Spotted Fever Group Rickettsiae and Ehrlichiae in African Ticks

Emerging Infectious Diseases ◽

10.3201/eid0706.010616 ◽

2001 ◽

Vol 7 (6) ◽

pp. 1014-1017 ◽

Cited By ~ 115

Author(s):

Philippe Parola

Keyword(s):

Spotted Fever ◽

Spotted Fever Group ◽

Spotted Fever Group Rickettsiae ◽

Detection And Identification

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Development of a Multiplex Loop-Mediated Isothermal Amplification (LAMP) Method for Simultaneous Detection of Spotted Fever Group Rickettsiae and Malaria Parasites by Dipstick DNA Chromatography

Diagnostics ◽

10.3390/diagnostics10110897 ◽

2020 ◽

Vol 10 (11) ◽

pp. 897

Author(s):

Lavel Chinyama Moonga ◽

Kyoko Hayashida ◽

Naoko Kawai ◽

Ryo Nakao ◽

Chihiro Sugimoto ◽

...

Keyword(s):

Detection System ◽

Spotted Fever ◽

Simultaneous Detection ◽

Febrile Illness ◽

Isothermal Amplification ◽

Lamp Method ◽

Spotted Fever Group ◽

Loop Mediated Isothermal Amplification ◽

Rickettsia Monacensis ◽

Sfg Rickettsia

Spotted fever group (SFG) rickettsiae causes febrile illness in humans worldwide. Since SFG rickettsiosis’s clinical presentation is nonspecific, it is frequently misdiagnosed as other febrile diseases, especially malaria, and complicates proper treatment. Aiming at rapid, simple, and simultaneous detection of SFG Rickettsia spp. and Plasmodium spp., we developed a novel multiple pathogen detection system by combining a loop-mediated isothermal amplification (LAMP) method and dipstick DNA chromatography technology. Two primer sets detecting SFG Rickettsia spp. and Plasmodium spp. were mixed, and amplified products were visualized by hybridizing to dipstick DNA chromatography. The multiplex LAMP with dipstick DNA chromatography distinguished amplified Rickettsia and Plasmodium targeted genes simultaneously. The determined sensitivity using synthetic nucleotides was 1000 copies per reaction for mixed Rickettsia and Plasmodium genes. When genomic DNA from in vitro cultured organisms was used, the sensitivity was 100 and 10 genome equivalents per reaction for Rickettsia monacensis and Plasmodium falciparum, respectively. Although further improvement will be required for more sensitive detection, our developed simultaneous diagnosis technique will contribute to the differential diagnosis of undifferentiated febrile illness caused by either SFG Rickettsia spp. or Plasmodium spp. in resource-limited endemic areas. Importantly, this scheme is potentially versatile for the simultaneous detection of diverse infectious diseases.

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Susceptibility of Inbred Mice to Rickettsia parkeri

Infection and Immunity ◽

10.1128/iai.00109-12 ◽

2012 ◽

Vol 80 (5) ◽

pp. 1846-1852 ◽

Cited By ~ 22

Author(s):

Britton J. Grasperge ◽

Kathryn E. Reif ◽

Timothy D. Morgan ◽

Piyanate Sunyakumthorn ◽

Joseph Bynog ◽

...

Keyword(s):

Inbred Mice ◽

Spotted Fever ◽

Inbred Mouse ◽

Transmission Model ◽

Mouse Strains ◽

Spotted Fever Group ◽

Limited Information ◽

Rickettsia Parkeri ◽

Content Type ◽

Boutonneuse Fever

ABSTRACTRickettsia parkeri, a member of the spotted fever groupRickettsia, is the causative agent of American boutonneuse fever in humans. Despite the increased recognition of human cases, limited information is available regarding the infection of invertebrate and vertebrate hosts for this emerging tick-borne disease. Toward the development of a viable transmission model and to further characterize the pathology associated withR. parkeriinfection, inbred mouse strains (A/J, BALB/c, C3H/HeJ, and C3H/HeN) were intravenously and intradermally inoculated with 105low-passage-numberR. parkeri(Portsmouth strain), and infection, gross pathology, and histopathology were scored. Additionally, a quantitative real-time PCR (qPCR) was performed to estimate rickettsial load in heart, lung, spleen, and liver tissues of infected mice at 19 days postinoculation. Of the A/J, BALB/c, and C3H/HeN mice, none displayed universal pathology consistent with sustained infection. Compared to age-matched control mice, the intravenously inoculated C3H/HeJ mice exhibited marked facial edema and marked splenomegaly upon gross examination, while the intradermally inoculated mice developed characteristic eschar-like lesions. The C3H/HeJ mice also exhibited the greatest concentrations of rickettsial DNA from heart, lung, liver, and spleen samples when examined by qPCR. The similarity of the pathology of human disease and sustained infection suggests that the C3H/HeJ strain of mice is a promising candidate for subsequent experiments to examine the tick transmission, dissemination, and pathology ofR. parkeririckettsiosis.

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