Severe Rickettsia typhi Infections, Costa Rica.

Murine typhus is a febrile, fleaborne disease caused by infection with Rickettsia typhi bacteria. Cases can range from mild and nonspecific to fatal. We report 2 cases of murine typhus in Costa Rica, confirming the presence and circulation of R. typhi causing severe disease in the country.

Detection of Severe Murine Typhus by Nanopore Targeted Sequencing, China.

We report a case of murine typhus in China caused by Rickettsia typhi and diagnosed by nanopore targeted sequencing of a bronchoalveolar lavage fluid sample. This case highlights that nanopore targeted sequencing can effectively detect clinically unexplained infections and be especially useful for detecting infections in patients without typical signs and symptoms.

Genetic typing of isolates of Rickettsia typhi.

Murine typhus, which is caused by Rickettsia typhi, has a wide range of clinical manifestations. It has a low mortality rate but may result in meningoencephalitis and interstitial pneumonia in severe cases. Comparisons of complete genome sequences of R. typhi isolates from North Carolina, USA (Wilmington), Myanmar (B9991PP), and Thailand (TH1527) identified only 26 single nucleotide polymorphism (SNP) and 7 insertion-deletion (INDEL) sites in these highly syntenic genomes. Assays were developed to further define the distribution of these variant sites among 15 additional isolates of R. typhi with different histories from Asia, the USA, and Africa. Mismatch amplification mutation assays (MAMA) were validated for 22 SNP sites, while the 7 INDEL sites were analyzed directly on agarose gels. Six SNP types, 9 INDEL types, 11 total types were identified among these 18 isolates. Replicate DNA samples as well as comparisons of isolates with different passage and source histories gave consistent genetic typing profiles. Comparison of the SNP and INDEL markers to R. typhi's nearest neighbor Rickettsia prowazekii demonstrated that the majority of the SNPs represent intra-species variation that arose post divergence of these two species while several INDEL sites also exhibited intraspecies variability among the R. prowazekii genomes that have been completely sequenced. The assays for the presence of these SNP and INDEL sites, particularly the latter, comprise a low technology gel method for consistently distinguishing R. typhi and R. prowazekii as well as for differentiating genetic types of R. typhi.

Molecular Detection of Pathogens in Negative Blood Cultures in the Lao People's Democratic Republic.

Bloodstream infections cause substantial morbidity and mortality. However, despite clinical suspicion of such infections, blood cultures are often negative. We investigated blood cultures that were negative after 5 days of incubation for the presence of bacterial pathogens using specific (Rickettsia spp. and Leptospira spp.) and a broad-range 16S rRNA PCR. From 190 samples, 53 (27.9%) were positive for bacterial DNA. There was also a high background incidence of dengue (90/112 patient serum positive, 80.4%). Twelve samples (6.3%) were positive for Rickettsia spp., including two Rickettsia typhi. The 16S rRNA PCR gave 41 positives; Escherichia coli and Klebsiella pneumoniae were identified in 11 and eight samples, respectively, and one Leptospira species was detected. Molecular investigation of negative blood cultures can identify potential pathogens that will otherwise be missed by routine culture. Patient management would have been influenced in all 53 patients for whom a bacterial organism was identified, and 2.3-6.1% of patients would likely have had an altered final outcome. These findings warrant further study, particularly to determine the cost-benefit for routine use, ways of implementation, and timing of PCR for organisms such as Rickettsia and Leptospira, which are important pathogens in rural Asia.

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.

The Isolation of Orientia tsutsugamushi and Rickettsia typhi from Human Blood through Mammalian Cell Culture: a Descriptive Series of 3,227 Samples and Outcomes in the Lao People's Democratic Republic.

In the Lao People's Democratic Republic (Laos), rickettsial infections, including scrub and murine typhus, account for a significant burden of fevers. The Mahosot Hospital Microbiology Laboratory in Vientiane, Laos, routinely performs rickettsial isolation from hospitalized patients with suspected rickettsioses using mammalian cell culture systems. We review the clinical and laboratory factors associated with successful Orientia tsutsugamushi and Rickettsia typhi isolations from this laboratory over a period of 6 years between 2008 and 2014. The overall isolation success was 7.9% for all samples submitted and 17.3% for samples for which the patient had a positive O. tsutsugamushi or R. typhi rapid diagnostic test (RDT), serology, or PCR. The frequency of successful isolation was highest for samples submitted in November, at the end of the wet season (28.3%). A longer median duration of reported illness, a positive result for a concurrent Orientia or Rickettsia spp. quantitative PCR, and the use of antibiotics by the patient in the week before admission were significantly associated with isolation success (P < 0.05). Buffy coat inoculation and a shorter interval between sample collection and inoculation in the laboratory were associated with a higher frequency of isolation (both P < 0.05). This frequency was highest if cell culture inoculation occurred on the same day as blood sample collection. Factors related to the initial rickettsial bacterial concentration are likely the main contributors to isolation success. However, modifiable factors do contribute to the rickettsial isolation success, especially delays in inoculating patient samples into culture.

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.

Risk1, a Phosphatidylinositol 3-Kinase Effector, Promotes Rickettsia typhi Intracellular Survival.

To establish a habitable intracellular niche, various pathogenic bacteria secrete effectors that target intracellular trafficking and modulate phosphoinositide (PI) metabolism. Murine typhus, caused by the obligate intracellular bacterium Rickettsia typhi, remains a severe disease in humans. However, the mechanisms by which R. typhi effector molecules contribute to internalization by induced phagocytosis and subsequent phagosomal escape into the cytosol to facilitate the intracellular growth of the bacteria remain ill-defined. Here, we characterize a new molecule, Risk1, as a phosphatidylinositol 3-kinase (PI3K) secreted effector and the first bacterial secretory kinase with both class I and III PI3K activities. Inactivation of Risk1 PI3K activities reduced the phosphorylation of phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 3,4,5-trisphosphate within the host, which consequently diminished host colonization by R. typhi During infection, Risk1 targets the Rab5-EEA1-phosphatidylinositol 3-phosphate [PI(3)P] signaling axis to promote bacterial phagosomal escape. Subsequently, R. typhi undergoes ubiquitination and induces host autophagy; however, maturation to autolysosomes is subverted to support intracellular growth. Intriguingly, only enzymatically active Risk1 binds the Beclin-1 core complex and contributes to R. typhi-induced autophagosome formation. In sum, our data suggest that Risk1, with dual class I and class III PI3K activities, alters host PI metabolism and consequently subverts intracellular trafficking to facilitate intracellular growth of R. typhiIMPORTANCERickettsia species are Gram-negative obligate intracellular bacteria that infect a wide range of eukaryotes and vertebrates. In particular, human body louse-borne Rickettsia prowazekii and flea-borne Rickettsia typhi have historically plagued humankind and continue to reemerge globally. The unavailability of vaccines and limited effectiveness of antibiotics late in infection place lethality rates up to 30%, highlighting the need to elucidate the mechanisms of Rickettsia pathogenicity in greater detail. Here, we characterize a new effector, Risk1, as a secreted phosphatidylinositol 3-kinase (PI3K) with unique dual class I and class III activities. Risk1 is required for host colonization, and its vacuolar phosphatidylinositol 3-phosphate generation modulates endosomal trafficking to arrest autophagosomal maturation. Collectively, Risk1 facilitates R. typhi growth by altering phosphoinositide metabolism and subverting intracellular trafficking.

Rise in Murine Typhus in Galveston County, Texas, USA, 2018.

Murine typhus, an undifferentiated febrile illness caused by Rickettsia typhi, is increasing in prevalence and distribution throughout Texas. In 2018, a total of 40 cases of murine typhus were reported in Galveston County. This increase, unprecedented since the 1940s, highlights the importance of awareness by physicians and public health officials.

Oxford Nanopore MinION Sequencing Enables Rapid Whole Genome Assembly of Rickettsia typhi in a Resource-Limited Setting.

The infrastructure challenges and costs of next-generation sequencing have been largely overcome, for many sequencing applications, by Oxford Nanopore Technologies' portable MinION sequencer. However, the question remains open whether MinION-based bacterial whole genome sequencing is by itself sufficient for the accurate assessment of phylogenetic and epidemiological relationships between isolates and whether such tasks can be undertaken in resource-limited settings. To investigate this question, we sequenced the genome of an isolate of Rickettsia typhi, an important and neglected cause of fever across much of the tropics and subtropics, for which only three genomic sequences previously existed. We prepared and sequenced libraries on a MinION in Vientiane, Lao PDR, using v9.5 chemistry, and in parallel, we sequenced the same isolate on the Illumina platform in a genomics laboratory in the United Kingdom. The MinION sequence reads yielded a single contiguous assembly, in which the addition of Illumina data revealed 226 base-substitution and 5,856 indel errors. The combined assembly represents the first complete genome sequence of a human R. typhi isolate collected in the last 50 years and differed from the genomes of existing strains collected over a 90-year time period at very few sites, with no rearrangements. Filtering based on the known error profile of MinION data improved the accuracy of the nanopore-only assembly. However, the frequency of false-positive errors remained greater than true sequence divergence from recorded sequences. Although nanopore-only sequencing cannot yet recover phylogenetic signals in R. typhi, such an approach may be applicable for more diverse organisms.

The temporal dynamics of humoral immunity to Rickettsia typhi infection in murine typhus patients.

OBJECTIVES: This study examined individuals with Rickettsia typhi infection in the Lao People's Democratic Republic (Lao PDR) to (a) investigate humoral immune dynamics; (b) determine the differences in reference diagnostic results and recommend appropriate cut-offs; (c) determine differences in immune response after different antibiotic treatments; and (d) determine appropriate diagnostic cut-off parameters for indirect immunofluorescence assay (IFA). METHODS: Sequential serum samples from 90 non-pregnant, adults were collected at seven time-points (days 0, 7, 14, 28, 90, 180 and 365) as part of a clinical antibiotic treatment trial. Samples were tested using IFA to determine IgM and IgG antibody reciprocal end-point titres against R. typhi and PCR. RESULTS: For all 90 individuals, reciprocal R. typhi IgM and IgG antibody titres ranged from <400 to ≥3200. The median half-life of R. typhi IgM was 126 days (interquartile range 36-204 days) and IgG was 177 days (interquartile range 134-355 days). Overall median patient titres for R. typhi IgM and IgG were significantly different (p < 0.0001) and at each temporal sample collection point (range p < 0.0001 to p 0.0411). Using Bayesian latent class model analysis, the optimal diagnostic cut-off reciprocal IFA titer on patient admission for IgM was 800 (78.6%, 95% CI 71.6%-85.2% sensitivity; 89.9%, 95% CI 62.5%-100% specificity), and for IFA IgG 1600 (77.3%; 95% CI 68.2%-87.6% sensitivity; 99%, 95% CI 95%-100% specificity). CONCLUSIONS: This study suggests suitable diagnostic cut-offs for local diagnostic laboratories and other endemic settings and highlights antibody persistence following acute infection. Further studies are required to validate and define cut-offs in other geographically diverse locations.

Development of multiplex PCR for neglected infectious diseases.

Scrub typhus, murine typhus, and leptospirosis are widely neglected infectious diseases caused by Orientia tsutsugamushi, Rickettsia typhi, and pathogenic Leptospira spp., respectively. Patients usually present with non-specific symptoms and therefore are commonly diagnosed with acute undifferentiated febrile illness. Consequently, patients face delayed treatment and increased mortality. Antibody-based serological test currently used as gold standard has limitations due to insufficient antibody titers, especially in the early phase of infection. In this study, we aimed to develop multiplex PCR to combine 3 primer pairs that target specific genes encoding 56-kDa TSA of O. tsutsugamushi, 17-kDa antigen of R. typhi, and LipL32 of L. Interrogans and evaluate its performance in comparison to the standard serological tests. Using EDTA blood samples of known patients, the sensitivity and specificity of our multiplex PCR was 100% and 70%, respectively. In addition, the assay was able to diagnose the co-infection of scrub typhus and leptospirosis. The assay may be useful in identifying causative agents during the early phase of these diseases, enabling prompt and appropriate treatment.

Murine typhus in Mexico City: report of an imported case.

Murine typhus is endemic in several countries. We herein report an imported case of murine typhus caused by Rickettsia typhi in Mexico City. This is the first report of a case after almost 20 years since the last report. The species was confirmed by DNA sequencing and phylogenetic reconstruction.

Rickettsia typhi as Cause of Fatal Encephalitic Typhus in Hospitalized Patients, Hamburg, Germany, 1940-1944.

We evaluated formalin-fixed paraffin-embedded tissue specimens from 7 patients who died with encephalitic typhus in Hamburg, Germany, during World War II. The archived specimens included only central nervous system tissues >70 years old that had been stored at room temperature. We demonstrated successful detection of Rickettsia typhi DNA by a nested qPCR specific to prsA in 2 patients. These results indicate that R. typhi infections contributed to typhus outbreaks during World War II. Immunohistochemical analyses of brain tissue specimens of R. typhi DNA-positive and -negative specimens showed perivascular B-cell accumulation. Around blood vessels, nodular cell accumulations consisted of CD4-positive and CD8-positive T cells and CD68-positive microglia and macrophages; neutrophils were found rarely. These findings are similar to those of previously reported R. prowazekii tissue specimen testing. Because R. typhi and R. prowazekii infections can be clinically and histopathologically similar, molecular analyses should be performed to distinguish the 2 pathogens.

Rickettsia Lipid A Biosynthesis Utilizes the Late Acyltransferase LpxJ for Secondary Fatty Acid Addition.

Members of the Rickettsia genus are obligate intracellular, Gram-negative coccobacilli that infect mammalian and arthropod hosts. Several rickettsial species are human pathogens and are transmitted by blood-feeding arthropods. In Gram-negative parasites, the outer membrane (OM) sits at the nexus of the host-pathogen interaction and is rich in lipopolysaccharide (LPS). The lipid A component of LPS anchors the molecule to the bacterial surface and is an endotoxic agonist of Toll-like receptor 4 (TLR4). Despite the apparent importance of lipid A in maintaining OM integrity, as well as its inflammatory potential during infection, this molecule is poorly characterized in Rickettsia pathogens. In this work, we have identified and characterized new members of the recently discovered LpxJ family of lipid A acyltransferases in both Rickettsia typhi and Rickettsia rickettsii, the etiological agents of murine typhus and Rocky Mountain spotted fever, respectively. Our results demonstrate that these enzymes catalyze the addition of a secondary acyl chain (C14/C16) to the 3'-linked primary acyl chain of the lipid A moiety in the final steps of the Raetz pathway of lipid A biosynthesis. Since lipid A architecture is fundamental to bacterial OM integrity, we believe that rickettsial LpxJ may be important in maintaining membrane dynamics to facilitate molecular interactions at the host-pathogen interface that are required for adhesion and invasion of mammalian cells. This work contributes to our understanding of rickettsial outer membrane physiology and sets a foundation for further exploration of the envelope and its role in pathogenesis.IMPORTANCE Lipopolysaccharide (LPS) triggers an inflammatory response through the TLR4-MD2 receptor complex and inflammatory caspases, a process mediated by the lipid A moiety of LPS. Species of Rickettsia directly engage both extracellular and intracellular immunosurveillance, yet little is known about rickettsial lipid A. Here, we demonstrate that the alternative lipid A acyltransferase, LpxJ, from Rickettsia typhi and R. rickettsii catalyzes the addition of C16 fatty acid chains into the lipid A 3'-linked primary acyl chain, accounting for major structural differences relative to the highly inflammatory lipid A of Escherichia coli.

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.

Typhus Group Rickettsiosis, Germany, 2010-20171.

Typhus group rickettsiosis is caused by the vectorborne bacteria Rickettsia typhi and R. prowazekii. R. typhi, which causes murine typhus, the less severe endemic form of typhus, is transmitted by fleas; R. prowazekii, which causes the severe epidemic form of typhus, is transmitted by body lice. To examine the immunology of human infection with typhus group rickettsiae, we retrospectively reviewed clinical signs and symptoms, laboratory changes, and travel destinations of 28 patients who had typhus group rickettsiosis diagnosed by the German Reference Center for Tropical Pathogens, Hamburg, Germany, during 2010-2017. Immunofluorescence assays of follow-up serum samples indicated simultaneous seroconversion of IgM, IgA, and IgG or concurrence in the first serum sample. Cytokine levels peaked during the second week of infection, coinciding with organ dysfunction and seroconversion. For 3 patients, R. typhi was detected by species-specific nested quantitative PCR. For all 28 patients, R. typhi was the most likely causative pathogen.

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.

Newly Recognized Pediatric Cases of Typhus Group Rickettsiosis, Houston, Texas, USA.

An increase in typhus group rickettsiosis and an expanding geographic range occurred in Texas, USA, over a decade. Because this illness commonly affects children, we retrospectively examined medical records from 2008-2016 at a large Houston-area pediatric hospital and identified 36 cases. The earliest known cases were diagnosed in 2011.