Max Q. Arens

Ehrlichia ewingii, a Newly Recognized Agent of Human Ehrlichiosis

BACKGROUND Human ehrlichiosis is a recently recognized tick-borne infection. Four species infect humans: Ehrlichia chaffeensis, E. sennetsu, E. canis, and the agent of human granulocytic ehrlichiosis. METHODS We tested peripheral-blood leukocytes from 413 patients with possible ehrlichiosis by broad-range and species-specific polymerase-chain-reaction (PCR) assays for ehrlichia. The species present were identified by species-specific PCR assays and nucleotide sequencing of the gene encoding ehrlichia 16S ribosomal RNA. Western blot analysis was used to study serologic responses. RESULTS In four patients, ehrlichia DNA was detected in leukocytes by a broad-range PCR assay, but not by assays specific for E. chaffeensis or the agent of human granulocytic ehrlichiosis. The nucleotide sequences of these PCR products matched that of E. ewingii, an agent previously reported as a cause of granulocytic ehrlichiosis in dogs. These four patients, all from Missouri, presented between May and August 1996, 1997, or 1998 with fever, headache, and thrombocytopenia, with or without leukopenia. All had been exposed to ticks, and three were receiving immunosuppressive therapy. Serum samples obtained from three of these patients during convalescence contained antibodies that reacted with E. chaffeensis and E. canis antigens in a pattern different from that of humans with E. chaffeensis infection but similar to that of a dog experimentally infected with E. ewingii. Morulae were identified in neutrophils from two patients. All four patients were successfully treated with doxycycline. CONCLUSIONS These findings provide evidence of E. ewingii infection in humans. The associated disease may be clinically indistinguishable from infection caused by E. chaffeensis or the agent of human granulocytic ehrlichiosis.

MultiCode-PLx System for Multiplexed Detection of Seventeen Respiratory Viruses

ABSTRACT The MultiCode-PLx system (EraGen Biosciences, Inc., Madison, WI) for the detection of respiratory viruses uses an expanded genetic alphabet, multiplex PCR chemistry, and microsphere flow cytometry to rapidly detect and specifically identify 17 different respiratory viruses directly in clinical specimens. The MultiCode-PLx system was tested in parallel with direct fluorescent-antibody (DFA) staining and rapid shell vial culture (R-mix cells; Diagnostic Hybrids, Inc. Athens, OH) with 354 respiratory specimens from adult patients that were submitted to the clinical virology laboratory at the Emory University Hospital. Single-target PCRs were performed with retained samples to confirm the positive results obtained with the MultiCode-PLx system for viruses not covered by DFA and R-mix culture (metapneumovirus, coronaviruses [CoV], parainfluenza viruses 4a and 4b, and rhinoviruses) and to resolve any discrepancies between the DFA and R-mix culture and the MultiCode-PLx results for viruses common to both systems. Respiratory viruses were detected in 77 (21.8%) and 116 (32.7%) specimens by DFA and R-mix culture and with the MultiCode-PLx system, respectively. Among the viruses common to both systems, the MultiCode-PLx system detected significantly more influenza A viruses (P = 0.0026). An additional increased diagnostic yield with the MultiCode-PLx system resulted from the detection of human metapneumovirus (HMPV) in 9 specimens, human CoV (HCoV) in 3 specimens, and human rhinovirus (HRV) in 16 specimens. Also, two mixed viral infections were detected by the MultiCode-PLx system (HCoV OC43 and HRV infections and HMPV and HRV infections), but none were detected by DFA and R-mix culture. Single-target PCRs verified the results obtained with the MultiCode-PLx system for 73 of 81 (90.1%) specimens that had discordant results or that were not covered by DFA and R-mix culture. The MultiCode-PLx system provides clinical laboratories with a practical, rapid, and sensitive means for the massively multiplexed molecular detection of common respiratory viruses.

Analysis and Characterization of Antiviral Drug–Resistant Cytomegalovirus Isolates from Solid Organ Transplant Recipients

The development of cytomegalovirus (CMV) disease and subsequent emergence of drug-resistant strains was examined in a large group of solid organ transplant recipients; drug-resistant CMV was detected in a total of 30 transplant recipients (20 lung, 5 kidney, 4 heart, and 1 liver). Drug resistance was confirmed both phenotypically and genotypically. The sequences of drug-resistant CMV strains from the same patient differed from drug-susceptible baseline sequences only at single sites previously confirmed to confer drug resistance. At least 1 isolate from each patient had a mutation in the UL97 phosphotransferase coding sequence. Mutations in the DNA polymerase gene were found in 6 of 38 sequenced strains. Lung transplant recipients had the highest incidence of drug-resistant virus: of the 30 patients, 28 were CMV-seronegative transplant recipients of CMV-seropositive organs, which strongly supports the premise that drug resistance is most prevalent in that transplant population.

Cross-contamination potential with dental equipment

Some types of reused dental equipment, especially handpieces and their attachments for drilling and cleaning teeth, might be responsible for cross-contamination if patient material were to lodge temporarily in difficult-to-disinfect internal mechanisms. This possibility is worrisome with respect to transmission of hepatitis B and human immunodeficiency viruses (HBV, HIV). Previous cross-contamination studies have relied on laboratory experiments with bacteria or dye tracers. To assess possible risk more thoroughly, we tested 30 new prophylaxis angles and 12 new high-speed handpieces to see whether they would take up and expel contaminants in laboratory and clinical trials. In treatments of three patients, including two infected with HIV, human-specific DNA (beta-globin, HLA DQ alpha) and HIV proviral DNA were detected inside or coming back from the devices. Similarly, when handpieces were operated in contact with blood pooled from HBV-infected patients, HBV DNA was detected in samples taken from inside the equipment and from their attached air/water hoses. When we used bacteriophage phi X174 as a model virus in laboratory tests, many infective viral particles were recovered from internal mechanisms of handpieces, their connecting air/water hoses, and from water spray expelled when the equipment was reused. We recommend that reused high-speed, air-driven handpieces and prophylaxis angles should be cleaned and heat-treated between patients. Further studies are needed to determine ways of eliminating the risks associated with exhaust hoses and air/water input lines.

The impact of ganciclovir-resistant cytomegalovirus infection after lung transplantation.

BACKGROUND Cytomegalovirus (CMV) resistance to ganciclovir has become increasingly common in acquired immunodeficiency syndrome patients but has only rarely been reported in recipients of solid organ transplants. METHODS A retrospective study of ganciclovir susceptibility testing of CMV isolates recovered from lung transplant recipients was performed. Patients with CMV isolates having partial (1 or =3 microg/ml) to ganciclovir determined by plaque reduction assay were included in a case-control study to identify risk factors for ganciclovir resistance. RESULTS Between 2/91 and 5/98, 18 patients (5.2% of patients transplanted) were found to have CMV infections with some degree of ganciclovir resistance (4 partially, 14 fully resistant). More positive viral blood cultures (3.2+/-2.5 vs. 1.6+/-1.4 CMV positive cultures, P=0.02) and more episodes of CMV pneumonitis (0.24+/-0.23 vs. 0.10+/-0.17 episodes/bronchoscopy, P=0.02) occurring before the detection of resistance were seen among resistant patients than controls. Ganciclovir-resistant patients received more antithymocyte globulin during induction (70+/-44 vs. 45+/-39 mg/kg, P=0.03) and received ganciclovir for a greater number of days (79+/-52 vs. 64+/-53 days, P=0.005) before the detection of resistance than controls. Ganciclovir-resistant patients had a shorter survival and an earlier onset of bronchiolitis obliterans syndrome compared with patients in the transplant database at Washington University. CONCLUSIONS Ganciclovir-resistant CMV infection is a serious complication of solid organ transplantation associated with more episodes of viremia, more frequent disease, earlier onset of bronchiolitis obliterans and shorter survival. The use of antithymocyte globulin and prolonged exposure to ganciclovir are risk factors for the development of ganciclovir resistance.

APOBEC3F and APOBEC3G mRNA Levels Do Not Correlate with Human Immunodeficiency Virus Type 1 Plasma Viremia or CD4+ T-Cell Count

ABSTRACT APOBEC3F and APOBEC3G (hA3F and hA3G) are part of an innate mechanism of antiretroviral defense. The human immunodeficiency virus type 1 (HIV-1) accessory protein Vif targets both proteins for proteasomal degradation. Using mRNA from peripheral blood mononuclear cells of 92 HIV-infected subjects not taking antiretroviral therapy and 19 HIV-uninfected controls, we found that hA3F (P < 0.001) and hA3G (P = 0.016) mRNA levels were lower in HIV-infected subjects and were positively correlated with one another (P = 0.003). However, we found no correlation in the abundance of either hA3F or hA3G mRNA with either viral load or CD4 counts in HIV-infected subjects.

Clinical and epidemiologic characterization of WU polyomavirus infection, St. Louis, Missouri.

WU polyomavirus is a recently described polyomavirus found in patients with respiratory infections. Of 2,637 respiratory samples tested in St. Louis, Missouri, 2.7% were positive for WU polyomavirus by PCR, and 71% were coinfected with other respiratory viruses. Persistent human infection with WU polyomavirus is described.

Predominance of Ehrlichia ewingii in Missouri Dogs

ABSTRACT To investigate the species distribution of Ehrlichia present in Missouri dogs, we tested 78 dogs suspected of having acute ehrlichiosis and 10 healthy dogs. Blood from each dog was screened with a broad-range 16S rRNA gene PCR assay that detects known pathogenic species of Ehrlichia and Anaplasma. The species was determined by using species-specific PCR assays and nucleotide sequencing. Ehrlichia antibody testing was performed by using an indirect immunofluorescence assay with Ehrlichia chaffeensis as the antigenic substrate. The broad-range assay detected Ehrlichia or Anaplasma DNA in 20 (26%) of the symptomatic dogs and 2 (20%) of the asymptomatic dogs. E. ewingii accounted for 20 (91%), and E. chaffeensis accounted for 1 (5%) of the positives. Anaplasma phagocytophilum DNA was detected in one dog, and the sequences of regions of the 16S rRNA gene and the groESL operon amplified from the blood of this dog matched the published sequences of this organism. Antibodies reactive with E. chaffeensis were detected in 14 (67%) of the 21 PCR-positive dogs and in 12 (19%) of the 64 PCR-negative dogs. Combining the results of PCR and serology indicated that 33 (39%) of 85 evaluable dogs had evidence of past or current Ehrlichia infection. We conclude that E. ewingii is the predominant etiologic agent of canine ehrlichiosis in the areas of Missouri included in this survey. E. canis, a widely recognized agent of canine ehrlichiosis, was not detected in any animal. The finding of E. ewingii in asymptomatic dogs suggests that dogs could be a reservoir for this Ehrlichia species.

Detection of Viruses in Young Children With Fever Without an Apparent Source

OBJECTIVE: Fever without an apparent source is common in young children. Currently in the United States, serious bacterial infection is unusual. Our objective was to determine specific viruses that might be responsible. METHODS: We enrolled children aged 2 to 36 months with temperature of 38°C or greater without an apparent source or with definite or probable bacterial infection being evaluated in the St Louis Children’s Hospital Emergency Department and afebrile children having ambulatory surgery. Blood and nasopharyngeal swab samples were tested with an extensive battery of virus-specific polymerase chain reaction assays. RESULTS: One or more viruses were detected in 76% of 75 children with fever without an apparent source, 40% of 15 children with fever and a definite or probable bacterial infection, and 35% of 116 afebrile children (P < .001). Four viruses (adenovirus, human herpesvirus 6, enterovirus, and parechovirus) were predominant, being detected in 57% of children with fever without a source, 13% of children with fever and definite or probable bacterial infection, and 7% of afebrile children (P < .001). Thirty-four percent of 146 viral infections were detected only by polymerase chain reaction performed on blood. Fifty-one percent of children with viral infections and no evidence of bacterial infection were treated with antibiotics. CONCLUSIONS: Viral infections are frequent in children with fever without an apparent source. Testing of blood in addition to nasopharyngeal secretions expanded the range of viruses detected. Future studies should explore the utility of testing for the implicated viruses. Better recognition of viruses that cause undifferentiated fever in young children may help limit unnecessary antibiotic use.

Detection of Ehrlichia spp. in the Blood of Wild White-Tailed Deer in Missouri by PCR Assay and Serologic Analysis

ABSTRACT Blood samples collected from wild deer in Missouri in November of 2000 and 2001 were positive by PCR assays for Ehrlichiachaffeensis (50 of 217; 23%), Ehrlichiaewingii (44 of 217; 20%), and Anaplasma species (214 of 217; 99%). Nucleotide sequences of selected amplicons from the assay for anaplasma matched sequences of the white-tailed deer agent. Serologic analysis of 112 deer sampled in 2000 showed a very high prevalence of antibodies to E. chaffeensis (97 of 112; 87%) and a low prevalence of antibodies reactive with Anaplasmaphagocytophila (2 of 112; 2%).