Evidence of Orientia spp. Endemicity among Severe Infectious Disease Cohorts, Uganda.

At 3 severe infection cohort sites in Uganda, Orientia seropositivity was common. We identified 4 seroconversion cases and 1 PCR-positive case. These results provide serologic and molecular support for Orientia spp. circulating in sub-Saharan Africa, possibly expanding its endemic range. Orientia infections could cause severe illness and hospitalizations in this region.

Spotted fever rickettsia-induced microvascular endothelial barrier dysfunction is delayed by the calcium channel blocker benidipine.

The tick-borne bacterium Rickettsia parkeri is an obligate intracellular pathogen that belongs to spotted fever group rickettsia (SFGR). The SFG pathogens are characterized by their ability to infect and rapidly proliferate inside host vascular endothelial cells that eventually result in impairment of vascular endothelium barrier functions. Benidipine, a wide range dihydropyridine calcium channel blocker, is used to prevent and treat cardiovascular diseases. In this study, we tested whether benidipine has protective effects against rickettsia-induced microvascular endothelial cell barrier dysfunction in vitro. We utilized an in vitro vascular model consisting of transformed human brain microvascular endothelial cells (tHBMECs) and continuously monitored transendothelial electric resistance (TEER) across the cell monolayer. We found that during the late stages of infection when we observed TEER decrease and when there was a gradual increase of the cytoplasmic [Ca2+], benidipine prevented these rickettsia-induced effects. In contrast, nifedipine, another cardiovascular dihydropyridine channel blocker specific for L-type Ca2+ channels, did not prevent R. parkeri-induced drop of TEER. Additionally, neither drug was bactericidal. These data suggest that growth of R. parkeri inside endothelial cells is associated with impairment of endothelial cell monolayer integrity due to Ca2+ flooding through specific, benidipine-sensitive T- or N/Q-type Ca2+ channels but not through nifedipine-sensitive L-type Ca2+ channels. Further study will be required to discern the exact nature of the Ca2+ channels and Ca2+ transporting system(s) involved, any contributions of the pathogen toward this process, as well as the suitability of benidipine and new dihydropyridine derivatives as complimentary therapeutic drugs against Rickettsia-induced vascular failure.

A Novel Laminin-Binding Protein Mediates Microbial-Endothelial Cell Interactions and Facilitates Dissemination of Lyme Disease Pathogens.

Borrelia burgdorferi conserved gene products BB0406 and BB0405, members of a common B. burgdorferi paralogous gene family, share 59% similarity. Although both gene products can function as potential porins, only BB0405 is essential for infection. Here we show that, despite sequence homology and coexpression from the same operon, both proteins differ in their membrane localization attributes, antibody accessibility, and immunogenicity in mice. BB0406 is required for spirochete survival in mammalian hosts, particularly for the disseminated infection in distant organs. We identified that BB0406 interacts with laminin, one of the major constituents of the vascular basement membrane, and facilitates spirochete transmigration across host endothelial cell barriers. A better understanding of how B. burgdorferi transmigrates through dermal and tissue vascular barriers and establishes disseminated infections will contribute to the development of novel therapeutics to combat early infection.

Rickettsioses as Major Etiologies of Unrecognized Acute Febrile Illness, Sabah, East Malaysia.

Orientia tsutsugamushi, spotted fever group rickettsioses, and typhus group rickettsioses (TGR) are reemerging causes of acute febrile illness (AFI) in Southeast Asia. To further delineate extent, we enrolled patients >4 weeks of age with nonmalarial AFI in Sabah, Malaysia, during 2013-2015. We confirmed rickettsioses (past or acute, IgG titer >160) in 126/354 (36%) patients. We confirmed acute rickettsioses (paired 4-fold IgG titer rise to >160) in 38/145 (26%) patients: 23 O. tsutsugamushi, 9 spotted fever group, 4 TGR, 1 O. tsutsugamushi/spotted fever group, and 1 O. tsutsugamushi/TGR. PCR results were positive in 11/319 (3%) patients. Confirmed rickettsioses were more common in male adults; agricultural/plantation work and recent forest exposure were risk factors. Dizziness and acute hearing loss but not eschars were reported more often with acute rickettsioses. Only 2 patients were treated with doxycycline. Acute rickettsioses are common (>26%), underrecognized, and untreated etiologies of AFI in East Malaysia; empirical doxycycline treatment should be considered.

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.

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.

Development of a Sensitive and Rapid Recombinase Polymerase Amplification Assay for Detection of Anaplasma phagocytophilum.

Human granulocytic anaplasmosis (HGA) is a tick-borne disease caused by the obligate intracellular Gram-negative bacterium Anaplasma phagocytophilum The disease often presents with nonspecific symptoms with negative serology during the acute phase. Direct pathogen detection is the best approach for early confirmatory diagnosis. Over the years, PCR-based molecular detection methods have been developed, but optimal sensitivity is not achieved by conventional PCR while real-time PCR requires expensive and sophisticated instruments. To improve the sensitivity and also develop an assay that can be used in resource-limited areas, an isothermal DNA amplification assay based on recombinase polymerase amplification (RPA) was developed. To do this, we identified a 171-bp DNA sequence within multiple paralogous copies of msp2 within the genome of A. phagocytophilum Our novel RPA assay targeting this sequence has an analytical limit of detection of one genome equivalent copy of A. phagocytophilum and can reliably detect 125 bacteria/ml in human blood. A high level of specificity was demonstrated by the absence of nonspecific amplification using genomic DNA from human or DNA from other closely-related pathogenic bacteria, such as Anaplasma platys, Ehrlichia chaffeensis, Orientia tsutsugamushi, and Rickettsia rickettsii, etc. When applied to patient DNA extracted from whole blood, this new RPA assay was able to detect 100% of previously diagnosed A. phagocytophilum cases. The sensitivity and rapidness of this assay represents a major improvement for early diagnosis of A. phagocytophilum in human patients and suggest a role for better surveillance in its reservoirs or vectors, especially in remote regions where resources are limited.

Death from Transfusion-Transmitted Anaplasmosis, New York, USA, 2017.

We report a death from transfusion-transmitted anaplasmosis in a 78-year-old man. The patient died of septic shock 2 weeks after a perioperative transfusion with erythrocytes harboring Anaplasma phagocytophilum. The patient's blood specimens were positive for A. phagocytophilum DNA beginning 7 days after transfusion; serologic testing remained negative until death.

Diagnosis and Management of Tickborne Rickettsial Diseases: Rocky Mountain Spotted Fever and Other Spotted Fever Group Rickettsioses, Ehrlichioses, and Anaplasmosis - United States.

Tickborne rickettsial diseases continue to cause severe illness and death in otherwise healthy adults and children, despite the availability of low-cost, effective antibacterial therapy. Recognition early in the clinical course is critical because this is the period when antibacterial therapy is most effective. Early signs and symptoms of these illnesses are nonspecific or mimic other illnesses, which can make diagnosis challenging. Previously undescribed tickborne rickettsial diseases continue to be recognized, and since 2004, three additional agents have been described as causes of human disease in the United States: Rickettsia parkeri, Ehrlichia muris-like agent, and Rickettsia species 364D. This report updates the 2006 CDC recommendations on the diagnosis and management of tickborne rickettsial diseases in the United States and includes information on the practical aspects of epidemiology, clinical assessment, treatment, laboratory diagnosis, and prevention of tickborne rickettsial diseases. The CDC Rickettsial Zoonoses Branch, in consultation with external clinical and academic specialists and public health professionals, developed this report to assist health care providers and public health professionals to 1) recognize key epidemiologic features and clinical manifestations of tickborne rickettsial diseases, 2) recognize that doxycycline is the treatment of choice for suspected tickborne rickettsial diseases in adults and children, 3) understand that early empiric antibacterial therapy can prevent severe disease and death, 4) request the appropriate confirmatory diagnostic tests and understand their usefulness and limitations, and 5) report probable and confirmed cases of tickborne rickettsial diseases to public health authorities.

The Tick Protein Sialostatin L2 Binds to Annexin A2 and Inhibits NLRC4-Mediated Inflammasome Activation.

Tick saliva contains a number of effector molecules that inhibit host immunity and facilitate pathogen transmission. How tick proteins regulate immune signaling, however, is incompletely understood. Here, we describe that loop 2 of sialostatin L2, an anti-inflammatory tick protein, binds to annexin A2 and impairs the formation of the NLRC4 inflammasome during infection with the rickettsial agent Anaplasma phagocytophilum Macrophages deficient in annexin A2 secreted significantly smaller amounts of interleukin-1ß (IL-1ß) and IL-18 and had a defect in NLRC4 inflammasome oligomerization and caspase-1 activation. Accordingly, Annexin a2-deficient mice were more susceptible to A. phagocytophilum infection and showed splenomegaly, thrombocytopenia, and monocytopenia. Providing translational support to our findings, better binding of annexin A2 to sialostatin L2 in sera from 21 out of 23 infected patients than in sera from control individuals was also demonstrated. Overall, we establish a unique mode of inflammasome evasion by a pathogen, centered on a blood-feeding arthropod.

Use of Peptide-Based Enzyme-Linked Immunosorbent Assay followed by Immunofluorescence Assay To Document Ehrlichia chaffeensis as a Cause of Febrile Illness in Nicaragua.

Ehrlichia chaffeensis, the etiologic agent of human monocytic ehrlichiosis (HME), has been extensively studied as a cause of acute febrile illness and an emerging tick-borne zoonosis in the United States. Limited data suggest its presence in other regions, including Central and South America but not Nicaragua to date. Diagnosis of E. chaffeensis infection by indirect immunofluorescence assay (IFA) is the reference standard due to its presumed high sensitivity and specificity, but IFA is impractical, variably reproducible, and cumbersome for large epidemiologic studies and for clinical diagnosis in resource-poor regions. We evaluated a high-throughput, objective peptide-based enzyme-linked immunosorbent assay (ELISA) for use alone or in combination with IFA. We found that it performed best as a screening test (sensitivity, 100%; specificity, 84%) to reduce the proportion of serum samples that were required by the more cumbersome and subjective IFA testing to <20%. Using a two-step diagnostic approach (IFA is performed if the ELISA is positive), we identified E. chaffeensis or a serologically and antigenically similar organism as a heretofore unrecognized cause of acute febrile illness in humans in Nicaragua and demonstrated the utility of the peptide ELISA as a screening tool for large-scale clinical studies.

State of the art of diagnosis of rickettsial diseases: the use of blood specimens for diagnosis of scrub typhus, spotted fever group rickettsiosis, and murine typhus.

PURPOSE OF REVIEW: With improved malaria control, acute undifferentiated febrile illness studies in tropical regions reveal a startling proportion of rickettsial illnesses, especially scrub typhus, murine typhus, and spotted fever group rickettsioses. Laboratory diagnosis of these infections evolved little over the past 40 years, but combinations of technologies like PCR and loop-mediated isothermal amplification, with refined rapid diagnostic tests and/or ELISA, are promising for guidance for early antirickettsial treatment. RECENT FINDINGS: The long-term reliance on serological tests - useful only late in rickettsial infections - has led to underdiagnosis, inappropriate therapies, and undocumented morbidity and mortality. Recent approaches integrate nucleic acid amplification and recombinant protein-based serological tests for diagnosing scrub typhus. Optimized using Bayesian latent class analyses, this strategy increases diagnostic confidence and enables early accurate diagnosis and treatment - a model to follow for lagging progress in murine typhus and spotted fever. SUMMARY: A laboratory diagnostic paradigm shift in rickettsial infections is evolving, with replacement of indirect immunofluorescence assay by the more objective ELISA coupled with nucleic acid amplification assays to expand the diagnostic window toward early infection intervals. This approach supports targeted antirickettsial therapy, reduces morbidity and mortality, and provides a robust evidence base for further development of diagnostics and vaccines.

Chromatin-bound bacterial effector ankyrin A recruits histone deacetylase 1 and modifies host gene expression.

Control of host epigenetics is becoming evident as a mechanism by which symbionts and pathogens survive. Anaplasma phagocytophilum, an obligate intracellular bacterium, down-regulates multiple host defence genes where histone deacetylase 1 (HDAC1) binds and histone 3 is deacetylated at their promoters, including the NADPH oxidase component, CYBB. How HDAC1 is targeted to defence gene promoters is unknown. Ankyrin A (AnkA), an A. phagocytophilum type IV secretion system effector, enters the granulocyte nucleus, binds stretches of AT-rich DNA and alters transcription of antimicrobial defence genes, including down-regulation of CYBB. Here we found AnkA binds to a predicted matrix attachment region in the proximal CYBB promoter. Using the CYBB promoter as a model of cis-gene silencing, we interrogated the mechanism of AnkA-mediated CYBB repression. The N-terminus of AnkA was critical for nuclear localization, the central ANK repeats and C-terminus were important for DNA binding, and most promoter activity localized to the central ANK repeats. Furthermore, a direct interaction between AnkA and HDAC1 was detected at the CYBB promoter, and was critical for AnkA-mediated CYBB repression. This novel microbial manipulation of host chromatin and gene expression provides important evidence of the direct effects that prokaryotic nuclear effectors can exert over host transcription and function.

Stat1 negatively regulates immune-mediated injury with Anaplasma phagocytophilum infection.

Human granulocytic anaplasmosis (HGA) is caused by the obligate intracellular bacterium Anaplasma phagocytophilum. Our data previously demonstrated that A. phagocytophilum induces an immunopathologic response by activating IFN-γ production through the Stat1 signaling pathway. In this study, we investigated the broader role of Stat1 signaling in the host response to infection with A. phagocytophilum. In Stat1 knockout (KO) compared with wild-type mice, A. phagocytophilum infection was more highly pathogenic as characterized by the unanticipated development of clinical signs in mice including markedly increased splenomegaly, more severe inflammatory splenic and hepatic histopathology, >100-fold higher blood and splenic bacterial loads, and more elevated proinflammatory cytokine/chemokine responses in serum. CD4(+) and CD8(+) T lymphocyte populations were significantly expanded in spleens of A. phagocytophilum-infected Stat1 KO mice compared with wild-type mice. The leukocyte infiltrates in the livers and spleens of A. phagocytophilum-infected Stat1 KO mice also contained expansions in neutrophil and monocyte/macrophage populations. Importantly, A. phagocytophilum-infected Stat1 KO mice did not demonstrate induction of inducible NO synthase in splenocytes. These results show that Stat1 plays an important role in controlling bacterial loads but also by unexpectedly providing an undefined mechanism for dampening of the immunopathologic response observed with A. phagocytophilum infection.

Short-term malaria reduction by single-dose azithromycin during mass drug administration for trachoma, Tanzania.

Single-dose mass drug administration of azithromycin (AZT) is underway to eliminate trachoma worldwide. Studies in Ethiopia showed a reduction in all-cause childhood deaths after administration. To examine the effect of single-dose AZ MDA on prevalent malaria infections in a large prospective cohort of children and parents in Dodoma Province, Tanzania, we quantified the temporal prevalence of malaria parasitemia by real-time PCR for 6 months after single-dose AZT. In the first month after treatment but not in subsequent months, Plasmodium falciparum infections were reduced by 73% (95% CI 43%-89%) in treatment versus control villages and differences remained significant (p = 0.00497) in multivariate models with village-level random effects. Genetic sequencing of P. falciparum ribosomal L4 protein showed no mutations associated with AZT resistance. AZT mass drug administration caused a transient, 1-month antimalarial effect without selecting for P. falciparum ribosomal L4 resistance mutations in a region with a 10-year history of treating trachoma with this drug.

Detection of new bunyavirus RNA by reverse transcription-loop-mediated isothermal amplification.

Severe fever with thrombocytopenia syndrome (SFTS) is a newly emerging and epidemic infectious disease in central and northeast China. It is caused by New Bunyavirus and carries an average 12% case fatality rate. Early and rapid detection is critical for prevention and control of New Bunyavirus infection, since no vaccine or antiviral drugs are currently available, and prevention requires careful attention to control of the suspected tick vector. In this study, a simple and sensitive reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay was developed for rapid detection of New Bunyavirus. The detection limit of the RT-LAMP assay was approximately 10(3) 50% tissue culture infective doses/ml of New Bunyavirus in culture supernatants, and no cross-reactive amplification of other viruses known to cause similar clinical manifestations was observed. The assay was further evaluated using 138 specimens from clinically suspected SFTS and 40 laboratory-proven hantavirus infection with fever and renal syndrome patients, and the assay exhibited 97% agreement compared to real-time RT-PCR and conventional RT-PCR. Using real-time RT-PCR as the diagnostic gold standard, RT-LAMP was 99% sensitive and 100% specific. The RT-LAMP assay could become a useful alternative in clinical diagnosis of SFTS caused by New Bunyavirus, especially in resource-limited hospitals or rural clinics of China.

Emerging Tick-borne Infections in the Upper Midwest and Northeast United States Among Patients With Suspected Anaplasmosis.

Background: Emerging tick-transmitted illnesses are increasingly recognized in the United States (US). To identify multiple potential tick-borne pathogens in patients from the Upper Midwest and Northeast US with suspected anaplasmosis, we used state-of-the-art methods (polymerase chain reaction [PCR] and paired serology) to test samples from patients in whom anaplasmosis had been excluded. Methods: Five hundred sixty-eight patients without anaplasmosis had optimal samples available for confirmation of alternative tick-borne pathogens, including PCR and/or paired serology (acute-convalescent interval ≤42 days). Results: Among 266 paired serology evaluations, for which the median acute-convalescent sampling interval was 28 (interquartile range, 21-33) days, we identified 35 acute/recent infections (24 [9%] Borrelia burgdorferi; 6 [2%] Ehrlichia chaffeensis/Ehrlichia muris subsp eauclairensis [EC/EME]; 3 [1%] spotted fever group rickettsioses [SFGR], and 2 [<1%] Babesia microti) in 33 (12%) patients. Two had concurrent or closely sequential infections (1 B burgdorferi and EC/EME, and 1 B burgdorferi and SFGR). Using multiplex PCR and reverse-transcription PCR, we identified 7 acute infections (5/334 [1%] Borrelia miyamotoi and 2/334 [1%] B microti) in 5 (1%) patients, including 2 with B microti-B miyamotoi coinfection, but no Borrelia mayonii, SFGR, Candidatus Anaplasma capra, Heartland virus, or Powassan virus infections. Thus, among 568 patients with ruled-out anaplasmosis, 38 (6.7%) had ≥1 agent of tick-borne illness identified, with 33 patients (35 infections) diagnosed by paired serology and 5 additional patients (7 infections) by PCR. Conclusions: By identifying other tick-borne agents in patients in whom anaplasmosis had been excluded, we demonstrate that emerging tick-borne infections will be identified if specifically sought.

Culture and other direct detection methods to diagnose human granulocytic anaplasmosis.

OBJECTIVES: We sought to assess the performance of 3 laboratory tests on blood specimens for direct detection of Anaplasma phagocytophilum, the cause of human granulocytic anaplasmosis (HGA), in patients tested at a single medical institution in New York State. METHODS: Direct tests included microscopic blood smear examination for intragranulocytic inclusions, polymerase chain reaction (PCR), and culture using the HL-60 cell line. The HGA cases testing positive by only 1 direct test were not included, unless HGA was confirmed by acute or convalescent serology using an indirect immunofluorescent assay. RESULTS: From 1997 to 2009, 71 patients with HGA were diagnosed by at least 1 of the 3 direct test methods. For the subgroup of 55 patients who were tested using all 3 methods, culture was positive for 90.9% (50/55) vs 81.8% (45/55) for PCR vs 63.6% (35/55) for blood smear (P =.002). Most cultures (79.3%) were detected as positive within 1 week of incubation. CONCLUSIONS: Although using culture to detect A phagocytophilum is likely not amenable for implementation in most hospital laboratories, in our experience, culture had the highest yield among the direct tests evaluated.

Benidipine impairs innate immunity converting sublethal to lethal infections in a murine model of spotted fever rickettsiosis.

Spotted fever group rickettsiae are tick-borne obligate intracellular bacteria that infect microvascular endothelial cells. Humans and mammalian infection results in endothelial cell barrier dysfunction and increased vascular permeability. We previously demonstrated that treatment of Rickettsia parkeri-infected cells with the calcium channel blocker benidipine significantly delayed vascular barrier permeability. Thus, we hypothesized that benidipine, known to be safe and effective for other clinical processes, could reduce rickettsia-induced vascular permeability in vivo in an animal model of spotted fever rickettsiosis. Based on liver, lung and brain vascular FITC-dextran extravasation studies, benidipine did not reliably impact vascular permeability. However, it precipitated a deleterious effect on responses to control sublethal R. parkeri infection. Animals treated with benidipine alone had no clinical signs or changes in histopathology and splenic immune cell distributions. Benidipine-treated infected animals had marked increases in tissue and blood bacterial loads, more extensive inflammatory histopathologic injury, and changes in splenic architecture and immune cell distributions potentially reflecting diminished Ca2+ signaling, reduced innate immune cell activation, and loss of rickettsial propagation control. Impaired T cell activation by R. parkeri antigen in the presence of benidipine was confirmed in vitro with the use of NKT cell hybridomas. The unexpected findings stand in stark contrast to recent discussions of the benefits of calcium channel blockers for viral infections and chronic infectious or inflammatory diseases. A role for calcium channel blockers in exacerbation of human rickettsiosis and acute inflammatory infections should be evaluated by a retrospective review of patient's outcomes and medications.

Metagenomic next generation sequencing of plasma RNA for diagnosis of unexplained, acute febrile illness in Uganda.

Metagenomic next generation metagenomic sequencing (mNGS) has proven to be a useful tool in the diagnosis and identification of novel human pathogens and pathogens not identified on routine clinical microbiologic tests. In this study, we applied mNGS to characterize plasma RNA isolated from 42 study participants with unexplained acute febrile illness (AFI) admitted to tertiary referral hospitals in Mubende and Arua, Uganda. Study participants were selected based on clinical criteria suggestive of viral infection (i.e., thrombocytopenia, leukopenia). The study population had a median age of 28 years (IQR:24 to 38.5) and median platelet count of 114 x103 cells/mm3 (IQR:66,500 to 189,800). An average of 25 million 100 bp reads were generated per sample. We identified strong signals from diverse virus, bacteria, fungi, or parasites in 10 (23.8%) of the study participants. These included well recognized pathogens like Helicobacter pylori, human herpes virus-8, Plasmodium falciparum, Neisseria gonorrhoeae, and Rickettsia conorii. We further confirmed Rickettsia conorii infection, the cause of Mediterranean Spotted Fever (MSF), using PCR assays and Sanger sequencing. mNGS was a useful addition for detection of otherwise undetected pathogens and well-recognized non-pathogens. This is the first report to describe the molecular confirmation of a hospitalized case of MSF in sub-Saharan Africa (SSA). Further studies are needed to determine the utility of mNGS for disease surveillance in similar settings.