J. Stephen Dumler

The Clinical Assessment, Treatment, and Prevention of Lyme Disease, Human Granulocytic Anaplasmosis, and Babesiosis: Clinical Practice Guidelines by the Infectious Diseases Society of America

Evidence-based guidelines for the management of patients with Lyme disease, human granulocytic anaplasmosis (formerly known as human granulocytic ehrlichiosis), and babesiosis were prepared by an expert panel of the Infectious Diseases Society of America. These updated guidelines replace the previous treatment guidelines published in 2000 (Clin Infect Dis 2000; 31[Suppl 1]:1-14). The guidelines are intended for use by health care providers who care for patients who either have these infections or may be at risk for them. For each of these Ixodes tickborne infections, information is provided about prevention, epidemiology, clinical manifestations, diagnosis, and treatment. Tables list the doses and durations of antimicrobial therapy recommended for treatment and prevention of Lyme disease and provide a partial list of therapies to be avoided. A definition of post-Lyme disease syndrome is proposed.

Direct cultivation of the causative agent of human granulocytic ehrlichiosis

BACKGROUND Human granulocytic ehrlichiosis is a potentially fatal tick-borne infection that has recently been described. This acute febrile illness is characterized by myalgias, headache, thrombocytopenia, and elevated serum aminotransferase levels. The disease is difficult to diagnose because the symptoms are non-specific, intraleukocytic inclusions (morulae) may not be seen, and the serologic results are often initially negative. Little is known about the causative agent because it has never been cultivated. METHODS We studied three patients with symptoms and laboratory findings suggestive of human granulocytic ehrlichiosis, including unexplained fever after probable exposure to ticks, granulocytopenia, and thrombocytopenia. Peripheral blood was examined for ehrlichia microscopically and with use of the polymerase chain reaction (PCR). Blood was inoculated into cultures of HL60 cells (a line of human promyelocytic leukemia cells), and the cultures were monitored for infection by Giemsa staining and PCR. RESULTS Blood from the three patients, only one of whom had inclusions suggestive of ehrlichia in neutrophils, was positive for human granulocytic ehrlichiosis on PCR. Blood from all three patients was inoculated into HL60 cell cultures and caused infection, with intracellular organisms visualized as early as 5 days after inoculation and cell lysis occurring within 12 to 14 days. The identity of the cultured organisms was confirmed by immunofluorescence microscopy, PCR analysis, and DNA sequencing. DNA from the infected cells was sequenced in regions of the 16S ribosomal gene reported to differ between the agent of human granulocytic ehrlichiosis and closely related species, including Ehrlichia equi and E. phagocytophila which cause infection in animals. The sequences from all three human isolates were identical and differed from the strain of E. equi studied in having guanine rather than adenine at nucleotide 84. CONCLUSIONS We describe the cultivation of the agent of human granulocytic ehrlichiosis in cell culture. The ability to isolate this organism should lead to a better understanding of the biology, treatment, and epidemiology of this emerging infection.

Human Granulocytic Anaplasmosis and Anaplasma phagocytophilum

Understanding how Anaplasma phagocytophilum alters neutrophils will improve diagnosis, treatment, and prevention of this severe illness.

Gene Sequence-Based Criteria for Identification of New Rickettsia Isolates and Description of Rickettsia heilongjiangensis sp. nov.

ABSTRACT We propose genetic guidelines for the classification of rickettsial isolates at the genus, group, and species levels by using sequences of the 16S rRNA (rrs) gene and four protein-coding genes, the gltA, ompA, and ompB genes and gene D. To be classified as a member of the genus Rickettsia, an isolate should exhibit degrees of rrs and gltA homology with any of the 20 Rickettsia species studied of ≥98.1 and ≥86.5%, respectively. A member of the typhus group should fulfill at least two of the following four criteria: pairwise nucleotide sequence homologies with rrs, gltA, ompB, and gene D of either Rickettsia typhi or Rickettsia prowazekii of ≥99.4, ≥96.6, ≥92.4, and ≥91.6%, respectively. A member of the spotted fever group should either possess the ompA gene or fulfill at least two of the following four criteria: pairwise nucleotide sequence homologies with rrs, gltA, ompB, and gene D of any member of this group of ≥98.8, ≥92.7, ≥85.8, and ≥82.2%, respectively. The existence of a distinct “ancestral” group should be questioned. To be classified as a new Rickettsia species, an isolate should not exhibit more than one of the following degrees of nucleotide similarity with the most homologous validated species: ≥99.8 and ≥ 99.9% for the rrs and gltA genes, respectively, and, when amplifiable, ≥98.8, ≥99.2, and ≥99.3% for the ompA and ompB genes and gene D, respectively. By use of our classification scheme, “Rickettsia heilongjiangii” belongs to a new species for which we officially propose the name Rickettsia heilongjiangensis sp. nov.

Human granulocytic ehrlichiosis.

Human granulocytic ehrlichiosis is a recently recognized tick-borne infectious disease, and to date >600 patients have been identified in the United States and Europe. Most patients have presented with a non-specific febrile illness occurring within 4 weeks after tick exposure or tick bite. The risk for serious illness or death increases with advancing age and delayed onset of therapy. Routine laboratory testing may reveal reduced white blood cell and platelet concentrations and mildly elevated hepatic transaminase activity in peripheral blood. A high index of suspicion is necessary to arrive at a timely clinical diagnosis. Patients suspected of having human granulocytic ehrlichiosis (HGE) should be treated with a tetracycline-class antibiotic while awaiting the outcome of confirmatory laboratory testing.

Human Granulocytic Anaplasmosis

Human granulocytic anaplasmosis, a deer tick-transmitted rickettsial infection caused by Anaplasma phagocytophilum, is a common cause of undifferentiated fever in the northeast and upper Midwest United States. Patients are often initially diagnosed with a mild viral infection, and illness readily resolves in most cases. However, as many as 3% develop life-threatening complications and nearly 1% die from the infection. Although coinfections with Borrelia burgdorferi and Babesia microti occur, there is little evidence to suggest synergism of disease or a role for A phagocytophilum in chronic illness. No vaccine is available.

Nosocomial Transmission of Human Granulocytic Anaplasmosis in China

CONTEXT Human granulocytic anaplasmosis (HGA) is an emerging tick-borne disease in China. A cluster of cases among health care workers and family members following exposure to a patient with fulminant disease consistent with HGA prompted investigation. OBJECTIVE To investigate the origin and transmission of apparent nosocomial cases of febrile illness in the Anhui Province. DESIGN, SETTING, AND PATIENTS After exposure to an index patient whose fatal illness was characterized by fever and hemorrhage at a primary care hospital and regional tertiary care hospitals isolation ward, secondary cases with febrile illness who were suspected of being exposed were tested for antibodies against Anaplasma phagocytophilum and by polymerase chain reaction (PCR) and DNA sequencing for A. phagocytophilum DNA. Potential sources of exposure were investigated. MAIN OUTCOME MEASURE Cases with serological or PCR evidence of HGA were compared with uninfected contacts to define the attack rate, relative risk of illness, and potential risks for exposure during the provision of care to the index patient. RESULTS In a regional hospital of Anhui Province, China, between November 9 and 17, 2006, a cluster of 9 febrile patients with leukopenia, thrombocytopenia, and elevated serum aminotransferase levels were diagnosed with HGA by PCR for A. phagocytophilum DNA in peripheral blood and by seroconversion to A. phagocytophilum. No patients had tick bites. All 9 patients had contact with the index patient within 12 hours of her death from suspected fatal HGA while she experienced extensive hemorrhage and underwent endotracheal intubation. The attack rate was 32.1% vs 0% (P = .04) among contacts exposed at 50 cm or closer, 45% vs 0% (P = .001) among those exposed for more than 2 hours, 75% vs 0% (P < .001) among those reporting contact with blood secretions, and 87.5% vs 0% (P = .004) among those reporting contact with respiratory secretions from the index patient. CONCLUSION We report the identification of HGA in China and likely nosocomial transmission of HGA from direct contact with blood or respiratory secretions.

Antigenic Diversity of Granulocytic Ehrlichia Isolates from Humans in Wisconsin and New York and a Horse in California

The agent of human granulocytic ehrlichiosis (HGE), Ehrlichia phagocytophila, and Ehrlichia equi are very similar. HGE is of variable severity. Genetic and antigenic differences among 3 human isolates (Webster, Spooner, and NY-8) and 1 horse isolate (MRK) were evaluated. The 16S rRNA gene sequences were identical in all human isolates. By use of 5 homologous antisera from these 3 humans and 1 horse and an additional 5 antisera in heterologous reactions, the immunodominant antigens of each isolate were noted to differ in molecular size: 43 kDa in the Webster (Wisconsin) isolate, 46 kDa in the Spooner (Wisconsin) isolate, 42 and 45 kDa in the NY-8 (New York State) isolate, and a 42 kDa doublet in the E. equi MRK isolate from California. Two sera from a Wisconsin patient reacted weakly or not at all with the NY-8 isolate. Antigenic structural diversity exists among otherwise indistinguishable granulocytic ehrlichial isolates.

Anaplasma phagocytophilum AnkA binds to granulocyte DNA and nuclear proteins

Human granulocytic anaplasmosis (HGA) is caused by the obligate intracellular bacterium Anaplasma phagocytophilum. The bacterium infects, survives, propagates in, and alters neutrophil phenotype, indicating unique survival mechanisms. AnkA is the only known A. phagocytophilum component that gains access beyond neutrophil vacuoles and is transported to the infected host cell nucleus. The ability of native and recombinant AnkA to bind DNA and nuclear proteins from host HL‐60 cells was assessed by the use of immunoprecipitation after cis‐diamminedichloroplatinum (cis‐DDP) DNA‐protein crosslinking, by probing uninfected HL‐60 cell nuclear lysates for AnkA binding, and by recovery and sequence analysis of immunoprecipitated DNA. AnkA binds HL‐60 cell DNA as well as nuclear proteins of approximately 86, 53 and 25 kDa, whereas recombinant A. phagocytophilum Msp2 or control proteins do not. DNA immunoprecipitation reveals AnkA binding to a variety of target genes in the human genome, including genes that encode proteins with ATPase, tyrosine phosphatase and NADH dehydrogenase‐like functions. These data indicate that AnkA could exert some effect on cells through binding to protein:DNA complexes in neutrophil nuclei. Whether AnkA binding leads to neutrophil functional alterations, and how such alterations might occur will depend upon definitive identification of binding partners and associated metabolic and biochemical pathways.

Restricted changes in major surface protein-2 (msp2) transcription after prolonged in vitro passage of Anaplasma phagocytophilum

BackgroundAnaplasma phagocytophilum strains often vary in Msp2 expression, a situation assumed to be related to immune evasion. However, Msp2 is also an adhesin, and little is known about the role of endogenous msp2 transcriptional changes in the absence of immune selection. Thus, Msp2 profiles and msp2 transcripts of low passage A. phagocytophilum Webster strain, initially comprised of a single abundant msp2 transcript, were re-examined after ≥ 20 in vitro passages without immune selection.ResultsUsing an Msp2 monoclonal antibody, immunoblots revealed an unchanged dominant band and several weak bands that appeared with passage. Similarly, msp2 transcript diversity changed, with a decrease in the initially abundant low passage transcript and appearance of a newly abundant and several minor msp2 transcripts with high passage. BLASTN search of the A. phagocytophilum HZ strain genome revealed ≥ 52 msp2 paralogs.ConclusionsMsp2 expression and msp2 transcription modulate even without immune selective pressures. However, the limited diversity of msp2 transcripts in the absence of immune pressure suggests selection for Msp2 by specific functions beyond that of immune evasion, in spite of a large genomic reservoir for Msp2 diversity.