Duplex Reverse-Transcription Real-Time Polymerase Chain Reaction Assay Targeting 23S rRNA Single Nucleotide Polymorphisms for the Detection of Flea-Borne Rickettsioses.

Flea-borne spotted fever and flea-borne (murine) typhus are rickettsioses caused by Rickettsia felis and Rickettsia typhi, respectively, and typically present as undifferentiated febrile illnesses. The relative contribution of these agents to flea-borne rickettsioses in California is unclear. We have developed a duplex reverse transcription real-time polymerase chain reaction (RT-rtPCR) assay targeting R. felis- and R. typhi-specific 23S ribosomal RNA single nucleotide polymorphisms to better understand the respective roles of these agents in causing flea-borne rickettsioses in California. This assay was compared with an established duplex R. felis- and R. typhi-ompB rt-PCR assay and was shown to have 1,000-fold and 10-fold greater analytical sensitivity for the detection of R. felis and R. typhi, respectively. Retrospective testing of clinical specimens with both assays established R. typhi as the major etiologic agent of flea-borne rickettsioses in California.

Sunbathing, a possible risk factor of murine typhus infection in Greece.

BACKGROUND: There are few studies about the presence of murine typhus in Greece. Our objective was to conduct a large scale retrospective investigation to determine the clinical and epidemiological features of patients diagnosed with murine typhus in Greece. METHODOLOGY/PRINCIPAL FINDINGS: From 2012 to 2019 serum samples from hospitalized patients and outpatients throughout Greece suspected for murine typhus infection were tested by immunofluorescence assay for Rickettsia typhi. Immunofluorescence positive samples obtained since 2016 were also tested by qPCR targeting R. typhi. Clinical and epidemiological data were retrospectively collected for the patients with confirmed murine typhus. Overall, we tested 5,365 different patients and, in total, 174 patients from all geographic regions of Greece were diagnosed with murine typhus. The most frequently reported sign or symptom was fever (89%), followed by headache (84%) and rash (81%). The classical triad of fever, headache, and rash was present in 72% of patients during their illness. Severe infections with complications including acute renal failure or septic shock were not recorded. The majority of cases (81%) occurred during May-October and peaked in June and September. Most of patients (81%) infected in Athens, recalled that their only activity the last weeks before symptoms onset was swimming on the beach and 59% of them also reported an insect bite while sunbathing. CONCLUSIONS/SIGNIFICANCE: Our results may reflect the reemergence of murine typhus in Greece and we highlight the importance of awareness of this difficult-to-recognize undifferentiated febrile illness.

Urban foci of murine typhus involving cat fleas (Ctenocephalides felis felis) collected from opossums in Mexico City.

Murine typhus, a neglected rickettsiosis caused by Rickettsia typhi, is a common disease in several Latin-American countries. The sylvatic life cycle of R. typhi encompasses the presence of several wild mammals, particularly opossums of the genus Didelphis and their associated fleas. Due to the colonization of wild environments by human populations, the increase in contact with opossum fleas has generated the presence of urban outbreaks of typhus. For this reason, the aim of our study was to identify the presence and diversity of Rickettsia sp. in fleas collected from opossums of an urban reserve in Mexico City. Opossums were captured from February to September 2017. For the detection of Rickettsia DNA, fragments of 800 bp of the citrate synthase (gltA) and the outer membrane protein B (ompB) were amplified. A total of 141 fleas (111 ♀, 30 ♂) of a single species (Ctenocephalides felis felis) were recovered from 31 Didelphis virginiana. Rickettsia DNA was detected in 17.7% (25/141) of the analysed fleas, recovered from seven infested opossums. The Maximum likelihood of sequences exhibited an identity of 99%-100% with sequences of R. typhi from southern United States. This work represents the first record of R. typhi in fleas from opossums in Mexico.

Fatal murine typhus with hemophagocytic lymphohistiocytosis in a child.

Hemophagocytic lymphohistiocytosis is a rare complication in Rickettsia typhi infections. We report the case of a 2-year-old boy with sudden night-onset fever, pallor, neck adenopathy and erythematous macular rash on the thorax, thighs and buttocks. During admission, he developed hyponatremia, hypoalbuminemia, anemia, thrombocytopenia, leukopenia, neutropenia, liver damage, hemorrhages and persistent fever. No hematological improvement was observed after the initial management, neoplastic diseases were discarded by bone marrow aspiration and lymph node biopsy; hemophagocytic lymphohistiocytosis was diagnosed. By immunohistochemistry and indirect immunofluorescence, murine typhus was also diagnosed and doxycycline was started with transitory recovery. Later, the child developed kidney failure and distributive shock that evolved to cardiac arrest and death. This is the first case report in Mexico on a fatal murine typhus associated with hemophagocytic lymphohistiocytosis in which the etiology was evidenced by histopathology.

Murine typhus mistaken for COVID-19 in a young man.

Fever is a widely recognised presenting symptom of COVID-19. Consequently, other febrile illnesses may be difficult to distinguish from COVID-19-leading to delays in diagnosis and treatment. One such illness is murine typhus, a fleaborne illness with worldwide distribution caused by Rickettsia typhi It often presents with fever, headache and myalgia, all of which have been commonly reported with COVID-19. Although the disease is usually mild with a good prognosis, there have been reports of severe illness and death. I present a case of murine typhus in a young male who had 2 weeks of headaches and daily fevers during the COVID-19 pandemic. He was ultimately tested for murine typhus when his occupation as a dog trainer was queried, and he experienced resolution of symptoms after treatment with doxycycline. During this pandemic, clinicians must be vigilant of other febrile illnesses whose symptoms overlap with COVID-19.

Detección de Rickettsia typhi en Rhipicephalus sanguineus s.l. y Amblyomma mixtum en el sur de México / Detection of Rickettsia typhi in Rhipicephalus sanguineus s.l. and Amblyomma mixtum in South Mexico

Resumen: Objetivo: Determinar la presencia de Rickettsia typhi en Rhipicephalus sanguineus s.l. y Amblyomma mixtum, en el sur de México. Material y métodos: Las garrapatas fueron colectadas en humanos y animales domésticos. Se determinó la presencia de Rickettsia por reacción en cadena de la polimerasa (PCR, por sus siglas en inglés) y secuenciación. Resultados: 10/39 viales de trabajo amplificaron fragmentos de los genes gltA, htrA y ompB, en 7/10 proveniente de Rh. sanguineus s.l. colectadas de perros y en 3/10 de A. mixtum colectadas de caballo y humano. La secuenciación indicó R. typhi en Rh. sanguineus y A. mixtum con homología de 100% (LS992663.1), para una región del gen de htrA, y de 99% (LS992663.1), con las regiones de los genes de gltA y OmpB. La tasa mínima de infección (TMI) para R. typhi fue de 3.88. Conclusiones: Las garrapatas Rhipicephalus sanguineus s.l. y Amblyomma mixtum están infectadas naturalmente con R. typhi en el sur de México.

Abstract: Objective: To determine the presence of Rickettsia typhi in Rhipicephalus sanguineuss.l. and Amblyomma mixtum in southern Mexico. Materials and methods: Ticks were collected in humans and domestic animals. The presence of Rickettsia was determined by PCR and sequencing. Results: 10/39 work vials amplified fragments of the gltA,htrA and ompB genes. On 7/10 from Rh. sanguineus s.l collected from dogs and in 3/10 of A. mixtum collected from horse and human. Sequencing indicated R. typhi in Rh. Sanguineus and A. mixtum with 100% homology (LS992663.1) for a region of the htrA gene and 99% (LS992663.1) with the regions of the gltA and OmpB genes. The minimum infection rate (TMI) for R. typhi was 3.88. Conclusions: Rhipicephalus sanguineuss.l. and Amblyomma mixtum are naturally infected with R. typhi in Southern Mexico.

[Detection of Rickettsia typhi in Rhipicephalus sanguineus s.l. and Amblyomma mixtum in South of Mexico]. / Detección de Rickettsia typhi en Rhipicephalus sanguineus s.l. y Amblyomma mixtum en el sur de México.

OBJECTIVE: To determine the presence of Rickettsia typhi in Rhipicephalus sanguineus s.l. and Amblyomma mixtum in southern Mexico. MATERIALS AND METHODS: Ticks were collected in humans and domestic animals. The presence of Rickettsia was determined by PCR and sequencing. RESULTS: 10/39 work vials amplified fragments of the gltA, htrA and ompB genes. On 7/10 from Rh. sanguineus s.l. collected from dogs and in 3/10 of A. mixtum collected from horse and human. Sequencing indicated R. typhi in Rh. sanguineus and A. mixtum with 100% homology (LS992663.1) for a region of the htrA gene and 99% (LS992663.1) with the regions of the gltA and OmpB genes. The minimum infection rate (TMI) for R. typhi was 3.88. CONCLUSIONS: Rhipicephalus sanguineus s.l. and Amblyomma mixtum are naturally infected with R. typhi in Southern Mexico.

OBJETIVO: Determinar la presencia de Rickettsia typhi en Rhipicephalus sanguineus s.l. y Amblyomma mixtum, en el sur de México. MATERIAL Y MÉTODOS: Las garrapatas fueron colectadas en humanos y animales domésticos. Se determinó la presencia de Rickettsia por reacción en cadena de la polimerasa (PCR, por sus siglas en inglés) y secuenciación. RESULTADOS: 10/39 viales de trabajo amplificaron fragmentos de los genes gltA, htrA y ompB, en 7/10 proveniente de Rh. sanguineus s.l. colectadas de perros y en 3/10 de A. mixtum colectadas de caballo y humano. La secuenciación indicó R. typhi en Rh. sanguineus y A. mixtum con homología de 100% (LS992663.1), para una región del gen de htrA, y de 99% (LS992663.1), con las regiones de los genes de gltA y OmpB. La tasa mínima de infección (TMI) para R. typhi fue de 3.88. CONCLUSIONES: Las garrapatas Rhipicephalus sanguineus s.l. y Amblyomma mixtum están infectadas naturalmente con R. typhi en el sur de México.

Selection of Diagnostic Cutoffs for Murine Typhus IgM and IgG Immunofluorescence Assay: A Systematic Review.

Murine typhus is a neglected but widespread infectious disease that results in acute fever. The immunofluorescence assay (IFA) is the "gold standard" to identify IgM or IgG antibodies, although there is a lack of standardization in methodologies. The objective of this review is to summarize 1) the differences in published methodologies, 2) the diagnostic cutoff titers, and 3) the justification of diagnostic cutoffs. Searches were performed by combining the following search terms: "murine typhus," "rickettsia typhi," "immunofluorescence," "IFA," and "serologic" with restrictions (i.e., "rickettsia typhi" or "murine typhus," and "IFA" or "immunofluorescence," or "serologic*"). The search identified 78 studies that used IFA or immunoperoxidase assay (IIP) antibody cutoffs to diagnose murine typhus, 39 of which were case series. Overall, 45 studies (57.7%) provided little to no rationale as to how the cutoff was derived. Variation was seen locally in the cutoff titers used, but a 4-fold or greater increase was often applied. The cutoffs varied depending on the antibody target. No consensus was observed in establishing a cutoff, or for a single-value diagnostic cutoff. In conclusion, there is a lack of consensus in the establishment of a single-value cutoff. Further studies will need to be executed at each distinct geographic location to identify region-specific cutoffs, while also considering background antibody levels to distinguish between healthy and infected patients.

Rickettsial Infections among Cats and Cat Fleas in Riverside County, California.

Presently, few studies have investigated the role of domestic cats (Felis catus) in the recrudescence of flea-borne rickettsioses in California and the southern United States. In this study, we aimed to investigate the presence of Rickettsia typhi or Rickettisa felis in domestic cats (F. catus) and the fleas (primarily Ctenocephalides felis, the cat flea) associated with these cats in Riverside County, California. Thirty cats and 64 pools of fleas collected from these cats were investigated for rickettsial infections. Three cats and 17 flea pools (from 10 cats) tested positive for rickettsial infections. polymerase chain reaction and DNA sequencing indicated that one of the cats was positive for R. felis infections, whereas two were positive for Candidatus Rickettsia senegalensis infection. In addition, 12 of the flea pools were positive for R. felis, whereas five were positive for Ca. R. senegalensis. By contrast, no cats or their associated fleas tested positive for R. typhi. Finally, eight sera from these cats contained spotted fever group Rickettsia (SFGR) antibodies. The detection of R. felis and SFGR antibodies and the lack of R. typhi and TGR antibodies support R. felis as the main rickettsial species infecting cat fleas. The detection of Ca. R. senegalensis in both fleas and cats also provides additional evidence that cats and their associated fleas are infected with other R. felis-like organisms highlighting the potential risk for human infections with R. felis or R. felis-like organisms.

Murine Typhus with Marked Thrombocytopenia in a Child in Northern Greece and Literature Review.

We report a case of murine typhus in a 4-year-old boy living in northern Greece. Although the illness started with mild symptoms, a maculopapular rash appeared by the end of the first week of illness followed by marked thrombocytopenia. The detection of IgM antibodies against Rickettsia typhi in the patient's blood and a positive polymerase chain reaction result combined with sequencing confirmed the diagnosis of infection by Rickettsia typhi. Clinicians in northern Greece should be aware of the disease, even in cases presenting with no specific initial symptoms.

Murine Typhus Mimicking Kawasaki Disease in a Preadolescent Girl.

We report the case of an 11-year-old preadolescent girl presenting with prolonged fever, lymphadenitis, nonpurulent conjunctivitis, a generalized maculopapular rash, erythematous lips and edema of hands/feet. Although major diagnostic criteria for Kawasaki disease were met, local epidemiologic data suggested a possible vector-borne etiology. Treatment with doxycycline was initiated, and defervescence occurred. Laboratory investigation confirmed the diagnosis of Rickettsia typhi infection.

Fatal Flea-Borne Typhus in Texas: A Retrospective Case Series, 1985-2015.

AbstractFlea-borne (murine) typhus is a global rickettsiosis caused by Rickettsia typhi. Although flea-borne typhus is no longer nationally notifiable, cases are reported for surveillance purposes in a few U.S. states. The infection is typically self-limiting, but may be severe or life-threatening in some patients. We performed a retrospective review of confirmed or probable cases of fatal flea-borne typhus reported to the Texas Department of State Health Services during 1985-2015. When available, medical charts were also examined. Eleven cases of fatal flea-borne typhus were identified. The median patient age was 62 years (range, 36-84 years) and 8 (73%) were male. Patients presented most commonly with fever (100%), nausea and vomiting (55%), and rash (55%). Respiratory (55%) and neurologic (45%) manifestations were also identified frequently. Laboratory abnormalities included thrombocytopenia (82%) and elevated hepatic transaminases (63%). Flea or animal contact before illness onset was frequently reported (55%). The median time from hospitalization to administration of a tetracycline-class drug was 4 days (range, 0-5 days). The median time from symptom onset to death was 14 days (range, 1-34 days). Flea-borne typhus can be a life-threatening disease if not treated in a timely manner with appropriate tetracycline-class antibiotics. Flea-borne typhus should be considered in febrile patients with animal or flea exposure and respiratory or neurologic symptoms of unknown etiology.

First Detection of Rickettsia typhi and Rickettsia felis in Fleas Collected From Client-Owned Companion Animals in the Southern Great Plains.

Flea-borne rickettsiosis occurs worldwide and includes a number of pathogens, namely, Rickettsia typhi and Rickettsia felis. Most studies in the United States have occurred in southern Texas and California where flea-borne rickettsiosis is endemic, resulting in a lack of information from other regions of the country. Between March and August 2016, 222 fleas were collected from 52 client-owned dogs and cats in two urban areas in Oklahoma. Fleas were identified using morphological characteristics then pooled and tested by polymerase chain reaction (PCR) using published primers for gltA, ompB, and 17-kDa. The majority (98.6%) of fleas collected were Ctenocephalides felis (Bouché) followed by Pulex irritans (L) (1.4%). Overall, fleas collected from 30.0% (6/20) cats and 43.8% (14/32) dogs were infected with R. felis. Three C. felis-pools collected from three dogs, two in the Enid area (central Oklahoma) and one in the Elk City area (western Oklahoma), were infected with R. typhi as well as R. felis. 'Candidatus R. senegalensis' was detected in one pool of fleas taken from a cat in Oklahoma City. This is the first evidence that flea-borne Rickettsia species occur in fleas obtained from client-owned dogs and cats in the Great Plains region. The impact of these Rickettsia species on public health in the region needs further investigation.

Acute-phase diagnosis of murine and scrub typhus in Belgian travelers by polymerase chain reaction: a case report.

BACKGROUND: Rickettsiosis is a potential life threatening infectious disease in travelers. Clinical recognition is not always straightforward, as typical manifestations such as rash and/or eschar may be absent. Definite diagnosis is based on seroconversion and therefore often delayed until the convalescent phase of disease. CASE PRESENTATION: In this case report, we describe four patients with severe travel-related rickettsiosis (two patients with murine- and two patients with scrub typhus), in whom acute- phase diagnosis was possible by real-time polymerase chain reaction on serum or blood. CONCLUSIONS: Despite its limitations, we think that polymerase chain reaction can contribute significantly to the early diagnosis and treatment of rickettsial disease in travelers.

Limited Evidence for Rickettsia felis as a Cause of Zoonotic Flea-Borne Rickettsiosis in Southern California.

Over 90% of human flea-borne rickettsioses cases in California are reported from suburban communities of Los Angeles and Orange counties and are presumed to be associated with either Rickettsia typhi or Rickettsia felis infection. Ctenocephalides felis (Bouché) is considered the principal vector for both rickettsiae, and R. felis has largely replaced R. typhi as the presumptive etiologic agent based on the widespread incidence of R. felis in cat flea populations. However, with no evidence to confirm R. felis as the cause of human illness in southern California, coupled with recent findings that showed R. felis to be widespread in cat fleas statewide, we propose that this hypothesis should be reconsidered. Evidence of only limited numbers of R. typhi-infected cat fleas in the environment may indicate a very rare infection and explain why so few cases of flea-borne rickettsioses are reported each year in southern California relative to the population.

Comparative evaluation of two Rickettsia typhi-specific quantitative real-time PCRs for research and diagnostic purposes.

Rickettsioses are caused by intracellular bacteria of the family of Rickettsiaceae. Rickettsia (R.) typhi is the causative agent of endemic typhus. The disease occurs worldwide and is one of the most prevalent rickettsioses. Rickettsial diseases, however, are generally underdiagnosed which is mainly due to the lack of sensitive and specific methods. In addition, methods for quantitative detection of the bacteria for research purposes are rare. We established two qPCRs for the detection of R. typhi by amplification of the outer membrane protein B (ompB) and parvulin-type PPIase (prsA) genes. Both qPCRs are specific and exclusively recognize R. typhi but no other rickettsiae including the closest relative, R. prowazekii. The prsA-based qPCR revealed to be much more sensitive than the amplification of ompB and provided highly reproducible results in the detection of R. typhi in organs of infected mice. Furthermore, as a nested PCR the prsA qPCR was applicable for the detection of R. typhi in human blood samples. Collectively, the prsA-based qPCR represents a reliable method for the quantitative detection of R. typhi for research purposes and is a promising candidate for differential diagnosis.

Molecular evidence of Rickettsia typhi infection in dogs from a rural community in Yucatán, México.

INTRODUCTION: Rickettsia typhi causes murine or endemic typhus, which is transmitted to humans primarily through flea bites contaminated with feces. Synanthropic and domestic animals also contribute to the infection cycle of R. typhi. Cases of murine typhus in humans were reported in the rural community of Bolmay, Yucatán, México, between 2007 and 2010.  OBJECTIVE: To identify the presence of R. typhi and estimate the frequency of infection in dogs from Bolmay, México, a locality with previous reports of murine typhus in humans.  MATERIALS AND METHODS: Whole blood samples were taken from 128 dogs. Total DNA was extracted for use in the polymerase chain reaction (PCR) to amplify fragments of the 17 kDa and omp B genes and confirms the presence of Rickettsia spp. The reaction products were sequenced, and alignment analysis was performed using the BLAST tool.  RESULTS: The frequency of R. typhi infection in dogs was 5.5 % (7/128). The alignment identified 99% and 100% homology to the R. typhi 17 kDa and omp B genes, respectively.  CONCLUSION: We confirmed the presence of R. typhi in dogs in the studied community but at a low frequency. However, there is potential risk of transmission to humans.

Rickettsial Infections among Ctenocephalides felis and Host Animals during a Flea-Borne Rickettsioses Outbreak in Orange County, California.

Due to a resurgence of flea-borne rickettsioses in Orange County, California, we investigated the etiologies of rickettsial infections of Ctenocephalides felis, the predominant fleas species obtained from opossums (Didelphis virginiana) and domestic cats (Felis catus), collected from case exposure sites and other areas in Orange County. In addition, we assessed the prevalence of IgG antibodies against spotted fever group (SFGR) and typhus group (TGR) rickettsiae in opossum sera. Of the 597 flea specimens collected from opossums and cats, 37.2% tested positive for Rickettsia. PCR and sequencing of rickettsial genes obtained from C. felis flea DNA preparations revealed the presence of R. typhi (1.3%), R. felis (28.0%) and R. felis-like organisms (7.5%). Sera from opossums contained TGR-specific (40.84%), but not SFGR-specific antibodies. The detection of R. felis and R. typhi in the C. felis fleas in Orange County highlights the potential risk for human infection with either of these pathogens, and underscores the need for further investigations incorporating specimens from humans, animal hosts, and invertebrate vectors in endemic areas. Such studies will be essential for establishing a link in the ongoing flea-borne rickettsioses outbreaks.