Monique Gaudreault-Keener

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.

Epstein-Barr virus DNA in peripheral blood leukocytes of patients with posttransplant lymphoproliferative disease.

We tested the hypotheses that Epstein-Barr virus (EBV) DNA levels in peripheral blood leukocytes (PBL) of transplant recipients with posttransplant lymphoproliferative disease (PTLD) (1) exceed those of patients without PTLD, (2) rise with or before clinical detection of the disease, and (3) fall with effective therapy. Using the polymerase chain reaction (PCR) and an endpoint dilution technique, we compared EBV DNA levels in sequential specimens from 5 patients with PTLD, 16 solid organ transplant recipients without PTLD, and 5 young adults with primary infectious mononucleosis (IM), and in single specimens from 21 healthy seropositive subjects. EBV DNA levels in the first two groups rose with induction of immunosuppression despite prophylactic acyclovir. Markedly elevated levels of EBV DNA were seen in 4 of 5 patients with PTLD at or before clinical diagnosis. The peak levels in these patients exceeded those of transplant recipients without PTLD (P = 0.02) and healthy adults with EM (P = 0.02). EBV DNA levels fell dramatically with effective therapy. Four of 21 healthy seropositive subjects demonstrated low levels of EBV DNA, similar to levels seen late in the course of patients with IM. We conclude that a semiquantitative PCR assay for EBV DNA in PBL can assist in the detection of PTLD and in monitoring the effect of therapy.

Prophylactic oral ganciclovir compared with deferred therapy for control of cytomegalovirus in renal transplant recipients.

BACKGROUND Treatment with prophylactic oral acyclovir, intravenous ganciclovir, or immunoglobulins to prevent cytomegalovirus (CMV) infection and disease in renal transplantation is associated with variable efficacy and significant expense. We studied control of CMV in renal transplant recipients using either prophylactic oral ganciclovir or deferred therapy with intensive monitoring with polymerase chain reaction (PCR) analysis. METHODS Forty-two recipients were followed for 6 months after transplantation. Ganciclovir (1000 mg p.o. t.i.d.; n=19) or acyclovir (200 mg p.o. b.i.d.; n=23) was begun at transplantation and continued for 12 weeks. PCR for CMV was performed on buffy-coat specimens every week for 15 weeks and at months 5 and 6. RESULTS No patients in the ganciclovir group, compared with 14 of 23 patients (61%) in the deferred-therapy group (P<0.0001), developed CMV disease during the first 12 weeks. In the ganciclovir group, 4 of 19 patients (21%) subsequently experienced 5 episodes, whereas 14 patients in the deferred-therapy group experienced 18 episodes (P=0.013 for subjects and P=0.026 for episodes). The time to disease was also delayed in the ganciclovir group compared with the deferred-therapy group (133+/-17 days vs. 51+/-7 days; P<0.0001). Oral ganciclovir also prevented CMV viremia during prophylaxis (2/19 patients [11%] vs. 23/23 patients [100%]). Time to CMV viremia was delayed in the ganciclovir group; however, 13/19 patients (68%) ultimately showed PCR evidence for CMV viremia (P=0.005). CONCLUSIONS An initial 12-week course of oral ganciclovir prevents CMV disease and infection in renal transplant recipients during prophylaxis, and the benefits persist after discontinuation.

Quantitative Polymerase Chain Reaction to Predict Occurrence of Symptomatic Cytomegalovirus Infection and Assess Response to Ganciclovir Therapy in Renal Transplant Recipients

Cytomegalovirus (CMV) DNA levels were measured by quantitative-competitive polymerase chain reaction (PCR) in weekly leukocyte samples from 50 renal transplant recipients, including 23 with symptomatic and 27 with asymptomatic CMV infection. Peak and week 4 CMV DNA levels were higher in symptomatic subjects (P = .07 and .02, respectively). In a logistic regression model, the logarithm of the week 4 level independently predicted symptomatic infection (odds ratio, 1.78 for a 1 log10 increase; 95% confidence interval, 1.14-2.78; P = .01). All subjects whose week 4 level exceeded 1000 copies/100,000 leukocytes developed symptoms. In subjects with adequate samples for analysis, CMV levels declined exponentially with ganciclovir treatment, with an average half-life of 3.3 days. Levels exceeding 10,000 copies were associated with prolonged time to clearing of CMV DNA. Potential clinical applications of quantitative CMV PCR include predicting occurrence of symptomatic first episodes after transplantation and individualizing duration of antiviral therapy.

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 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%).

Detection of respiratory viruses and the associated chemokine responses in serious acute respiratory illness

Background A specific diagnosis of a lower respiratory viral infection is often difficult despite frequent clinical suspicion. This low diagnostic yield may be improved by use of sensitive detection methods and biomarkers. Methods The prevalence, clinical predictors and inflammatory mediator profile of respiratory viral infection in serious acute respiratory illness were investigated. Sequential bronchoalveolar lavage (BAL) fluids from all patients hospitalised with acute respiratory illness over 12 months (n=283) were tested for the presence of 17 respiratory viruses by multiplex PCR assay and for newly discovered respiratory viruses (bocavirus, WU and KI polyomaviruses) by single-target PCR. BAL samples also underwent conventional testing (direct immunoflorescence and viral culture) for respiratory virus at the clinicians discretion. 27 inflammatory mediators were measured in a subset of the patients (n=64) using a multiplex immunoassay. Results 39 respiratory viruses were detected in 37 (13.1% of total) patients by molecular testing, including rhinovirus (n=13), influenza virus (n=8), respiratory syncytial virus (n=6), human metapneumovirus (n=3), coronavirus NL63 (n=2), parainfluenza virus (n=2), adenovirus (n=1) and newly discovered viruses (n=4). Molecular methods were 3.8-fold more sensitive than conventional methods. Clinical characteristics alone were insufficient to separate patients with and without respiratory virus. The presence of respiratory virus was associated with increased levels of interferon γ-inducible protein 10 (IP-10) (p<0.001) and eotaxin-1 (p=0.017) in BAL. Conclusions Respiratory viruses can be found in patients with serious acute respiratory illness by use of PCR assays more frequently than previously appreciated. IP-10 may be a useful biomarker for respiratory viral infection.

Inhibitory Interactions between BK and JC Virus among Kidney Transplant Recipients

BK and JC polyomaviruses can reactivate after transplantation, causing renal dysfunction and graft loss. The incidence of JC reactivation after renal transplant is not well understood. Here, we characterized JC reactivation using samples collected during the first year after transplantation from 200 kidney recipients. We detected BK and JC viruses in the urine of 35 and 16% of transplant recipients, respectively. The median viral load in the urine was 400 times higher for BK virus than JC virus. The presence of BK viruria made concurrent JC viruria less likely: JC viruria was detected in 22% of non-BK viruric recipients compared with 4% of BK viruric recipients (P=0.001). The co-detection rate was 1.5%, which is less than the expected 5.6% if reactivation of each virus was independent (P=0.001). We did not observe JC viremia, JC nephropathy, or progressive multifocal leukoencephalopathy. The onset of JC viruria was associated with donor, but not recipient, JC-specific antibody in a titer-dependent fashion and inversely associated with donor and recipient BK-specific antibody. Donor and recipient JC seropositivity did not predict BK viruria or viremia. In conclusion, among renal transplant recipients, infection with one polyomavirus inversely associates with infection with the other.

Progression from Sustained BK Viruria to Sustained BK Viremia with Immunosuppression Reduction Is Not Associated with Changes in the Noncoding Control Region of the BK Virus Genome

Changes in the BK virus archetypal noncoding control region (NCCR) have been associated with BK-virus-associated nephropathy (BKVAN). Whether sustained viremia, a surrogate for BKVAN, is associated with significant changes in the BK-NCCR is unknown. We performed PCR amplification and sequencing of (1) stored urine and (2) plasma samples from the time of peak viremia from 11 patients with sustained viremia who participated in a 200-patient clinical trial. The antimetabolite was withdrawn for BK viremia and reduction of the calcineurin inhibitor for sustained BK viremia. DNA sequencing from the 11 patients with sustained viremia revealed 8 insertions, 16 transversions, 3 deletions, and 17 transitions. None were deemed significant. No patient developed clinically evident BKVAN. Our data support, at a genomic level, the effectiveness of reduction of immunosuppression for prevention of progression from viremia to BKVAN.

Direct quantitative comparison of shell vial and conventional culture for detection of CMV viremia

BACKGROUND Centrifugation shell vial (SV) and conventional tube culture (TC) are the most common methods for detecting cytomegalovirus (CMV) viremia. Studies have indicated that SV is more sensitive than TC but at least one report suggested that TC was more sensitive. Because CMV in the blood is primarily associated with infected leukocytes, the number of leukocytes inoculated into the different culture systems could affect the sensitivities of the two systems. OBJECTIVES To compare the sensitivities of SV and TC for detection of CMV viremia by inoculating equal numbers of leukocytes into paired SV cultures and TC cultures. STUDY DESIGN Leukocytes from transplant recipients were isolated and counted. Equal numbers of leukocytes were then inoculated into each of two MRC-5 SV and into each of two MRC-5 TC. SV was considered positive when either one or both vials were positive, and TC was considered positive when either one or both tubes showed evidence of CMV cytopathic effect (CPE). RESULTS From a total of 434 specimens tested, 85 (19.6%) were positive by SV or TC. CMV was detected by SV in 75 (88%) of the positive specimens, compared to TC which was positive in 40 (47%) of the positive specimens. CONCLUSIONS When equal numbers of leukocytes were inoculated into each system, SV had significantly greater sensitivity than TC for detecting CMV viremia. However, a small number of episodes of viremia were detected only by TC. Therefore, both methods should be used for maximum sensitivity.