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Bone Marrow Transplantation 03/2007; 39(4):237-8. · 3.75 Impact Factor
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ABSTRACT: PCR was used to amplify a 537-bp region of an Ehrlichia ewingii gene encoding a homologue of the 28-kDa major antigenic protein (P28) of Ehrlichia chaffeensis. The E. ewingii p28 gene homologue was amplified from DNA extracted from whole blood obtained from four humans and one canine with confirmed cases of infection. Sequencing of the PCR products (505 bp) revealed a partial gene with homology to outer membrane protein genes from Ehrlichia and Cowdria spp.: p30 of Ehrlichia canis (< or =71.3%), p28 of E. chaffeensis (< or =68.3%), and map1 of Cowdria ruminantium (67.3%). The peptide sequence of the E. ewingii partial gene product was deduced (168 amino acids) and the antigenicity profile was analyzed, revealing a hydrophilic protein with < or =69.1% identity to P28 of E. chaffeensis, < or =67.3% identity to P30 of E. canis, and < or =63.1% identity to MAP1 of C. ruminantium. Primers were selected from the E. ewingii p28 sequence and used to develop a species-specific PCR diagnostic assay. The p28 PCR assay amplified the expected 215-bp product from DNA that was extracted from EDTA-treated blood from each of the confirmed E. ewingii infections that were available. The assay did not produce PCR products with DNA extracted from E. chaffeensis-, E. canis-, or E. phagocytophila-infected samples, confirming the specificity of the p28 assay for E. ewingii. The sensitivity of the E. ewingii-specific PCR assay was evaluated and determined to detect as few as 38 copies of the p28 gene.
Journal of Clinical Microbiology 11/2001; 39(11):3871-6. · 4.15 Impact Factor
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C D Paddock,
S M Folk,
G M Shore,
L J Machado,
M M Huycke,
L N Slater,
A M Liddell, R S Buller,
G A Storch,
T P Monson,
D Rimland,
J W Sumner,
J Singleton,
K C Bloch,
Y W Tang,
S M Standaert,
J E Childs
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ABSTRACT: The clinical course and laboratory evaluation of 21 patients coinfected with human immunodeficiency virus (HIV) and Ehrlichia chaffeensis or Ehrlichia ewingii are reviewed and summarized, including 13 cases of ehrlichiosis caused by E. chaffeensis, 4 caused by E. ewingii, and 4 caused by either E. chaffeensis or E. ewingii. Twenty patients were male, and the median CD4(+) T lymphocyte count was 137 cells/microL. Exposures to infecting ticks were linked to recreational pursuits, occupations, and peridomestic activities. For 8 patients, a diagnosis of ehrlichiosis was not considered until > or =4 days after presentation. Severe manifestations occurred more frequently among patients infected with E. chaffeensis than they did among patients infected with E. ewingii, and all 6 deaths were caused by E. chaffeensis. Ehrlichiosis may be a life-threatening illness in HIV-infected persons, and the influence of multiple factors, including recent changes in the epidemiology and medical management of HIV infection, may increase the frequency with which ehrlichioses occur in this patient cohort.
Clinical Infectious Diseases 11/2001; 33(9):1586-94. · 9.15 Impact Factor
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ABSTRACT: We evaluated the DiaSorin DNA enzyme immunoassay (DEIA) kit for detection of enteroviral reverse transcription-PCR (RT-PCR) products amplified from cerebrospinal fluid. By use of an optical density of 0.05 as the absorbance cutoff, 35% of 198 specimens were PCR positive, whereas 16% were culture positive. DEIA was rapid and sensitive and can help implement enterovirus RT-PCR in clinical laboratories.
Journal of Clinical Microbiology 12/2000; 38(11):4260-1. · 4.15 Impact Factor
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ABSTRACT: To define clinically relevant reference ("normal") values for cerebrospinal fluid (CSF) protein concentrations in pediatric patients who were evaluated for meningitis by traditional criteria and by enterovirus-polymerase chain reaction (EV-PCR).
A cohort of 906 consecutive pediatric patients to receive CSF analysis at St Louis Children's Hospital, St Louis, Mo, from June 1, 1998, to December 31,1998, was studied for clinical and laboratory data. Age-dependent CSF protein concentrations were then derived from a reference group of 225 patients in whom meningitis and other neurologic diseases were excluded by traditional clinical or laboratory criteria (excluding EV-PCR). Available CSF samples from 132 patients of the reference group were subsequently tested for EV-PCR.
In the reference group, the CSF protein concentration was highest and most variable in neonates, with a maximum of approximately 1.0 g/L. Cerebrospinal fluid protein concentration decreased rapidly to a nadir by 6 months and remained low throughout childhood, rarely exceeding 0.3 g/L and, finally, increasing in adolescence toward adult values. Enterovirus- polymerase chain reaction was positive in CSF of 11% of the reference group, with EV-PCR-positive patients having significantly higher CSF protein concentrations than EV-PCR-negative patients aged between 4 months and 14 years.
Reference values for CSF protein exhibit a characteristic age dependence in pediatric patients. Continued standard use of adult reference values in the pediatric population is inappropriate. The unexpected finding of a positive EV-PCR in patients not diagnosed with meningitis by traditional criteria further emphasizes the importance of selecting the most clinically relevant reference group for age and other variables when defining normal laboratory values. Arch Pediatr Adolesc Med. 2000;154:827-831
Archives of Pediatrics and Adolescent Medicine 09/2000; 154(8):827-31. · 4.14 Impact Factor
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ABSTRACT: Broad-range PCR primers were used to amplify part of the groESL operon of the canine pathogen Ehrlichia ewingii, recently recognized as a human pathogen, and the murine pathogen Ehrlichia muris. Phylogenetic analysis supported the relationships among Ehrlichia species previously determined by comparison of 16S rRNA gene sequences. These sequences provide additional PCR targets for species for which few gene sequences have been determined.
Journal of Clinical Microbiology 08/2000; 38(7):2746-9. · 4.15 Impact Factor
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R S Buller,
M Arens,
S P Hmiel,
C D Paddock,
J W Sumner,
Y Rikhisa,
A Unver,
M Gaudreault-Keener,
F A Manian,
A M Liddell,
N Schmulewitz,
G A Storch
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ABSTRACT: 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.
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.
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.
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.
New England Journal of Medicine 08/1999; 341(3):148-55. · 53.30 Impact Factor
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ABSTRACT: 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.
The Journal of Infectious Diseases 10/1998; 178(3):626-35. · 6.41 Impact Factor
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D C Brennan,
K A Garlock,
G G Singer,
M A Schnitzler,
B J Lippmann, R S Buller,
M Gaudreault-Keener,
J A Lowell,
S Shenoy,
T K Howard,
G A Storch
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ABSTRACT: 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.
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.
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).
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.
Transplantation 12/1997; 64(12):1843-6. · 4.00 Impact Factor
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ABSTRACT: Cytomegalovirus (CMV) infectious titers and DNA levels were determined by quantitative shell vial culture and quantitative-competitive PCR with blood samples from 10 renal transplant recipients with active CMV infection. Blood samples were stored at either room temperature or 4 degrees C and were processed at intervals of 0, 6, 24, 48, and 72 h. All samples were culture and PCR positive at baseline. Whereas the sensitivity of shell vial culture progressively declined, with only 55% positive at 24 h and 10% positive at 48 h, all samples remained PCR positive at all time points. Furthermore, the infectious titer diminished by 83 to 91% by 24 h compared to that at baseline (P < 0.0001), but quantitative DNA levels did not decline over time. Storage temperature had no significant effect on either infectious titer or DNA levels.
Journal of Clinical Microbiology 10/1997; 35(9):2224-8. · 4.15 Impact Factor
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ABSTRACT: The objective of this randomized, prospective study was to compare preemptive to deferred treatment of cytomegalovirus (CMV) infection in high-risk renal transplant recipients. Conducted at a university-affiliated transplant center, the study included 36 renal allograft recipients with donor or recipient CMV-seropositivity who received anti-thymocyte induction therapy. Ganciclovir was administered intravenously for 21 days upon detection of CMV viremia (preemptive, N = 15) or detection of CMV viremia associated with a CMV syndrome (deferred, N = 21). Shell vial culture, conventional culture, and polymerase chain reaction (PCR) were performed upon buffy-coat specimens weekly for 12 to 16 wk. CMV and non-CMV-associated charges were calculated. The comparative sensitivities of PCR, shell vial culture, and conventional culture were 91%, 44%, and 47%, respectively. A delay in specimen processing of > 24 h severely compromised the sensitivity of culture techniques but not that of PCR. Preemptive therapy tended to decrease symptomatic CMV episodes (0.4 versus 0.6 episodes per patient randomized; P = 0.22). One patient in each group had organ involvement, and no patient died. Allograft function and survival were similar. Ganciclovir use was increased in the preemptive group (1.2 versus 0.6 courses per patient randomized; P = 0.02). CMV-associated charges were $10,368 (preemptive) versus $5,752 (deferred); P = 0.13. PCR is superior to conventional monitoring to detect CMV viremia. Culture cannot be considered the "gold standard" for detection of CMV viremia, especially when transport of specimens over distances results in processing delays. Preemptive therapy may reduce symptomatic CMV infections in renal transplant recipients. It was associated with higher CMV-related charges but equivalent overall charges versus deferred treatment with intensive monitoring. Either strategy can achieve control of CMV infection after renal transplantation.
Journal of the American Society of Nephrology 01/1997; 8(1):118-25. · 9.66 Impact Factor
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ABSTRACT: 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.
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.
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).
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.
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.
Clinical and Diagnostic Virology 06/1995; 3(4):317-22.
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ABSTRACT: Herpes simplex virus type 2 (HSV-2) is known to cause aseptic meningitis, which can be recurrent. The diagnosis of HSV-2 infection is suggested when meningitis occurs simultaneously with genital lesions but may be obscure if genital lesions are not present or are not appreciated. Viral culture of the CSF is sometimes positive, but it may also be negative, especially in cases of recurrent disease. We report three cases of HSV meningitis in young women who did not have a history of genital herpetic lesions and for whom genital lesions were not noted on presentation. With use of the polymerase chain reaction (PCR), HSV DNA was detected in CSF from all three patients. The diagnosis of HSV meningitis was further confirmed by a positive culture of CSF in one patient's case and by demonstration of intrathecal synthesis of HSV antibodies in a second patient's case. The use of PCR can improve the recognition of HSV meningitis in adults presenting with aseptic meningitis, even in the absence of herpetic lesions.
Clinical Infectious Diseases 05/1995; 20(4):842-8. · 9.15 Impact Factor
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ABSTRACT: A quantitative culture method was used to test serial blood specimens from 28 lung transplant recipients at risk of cytomegalovirus (CMV) infection (donor [D] or recipient [R] seropositive for CMV). Viremia occurred in 26 (93%) of 28 patients. Highest levels were seen when the donor was seropositive. The median of individual maximum levels was 2.13 infectious centers (ICs/10(5) leukocytes for D+/R- patients (interquartile range [iqr], 0.12-21.77), 1.01 for D+/R+ (iqr, 0.3-2.32), and 0.10 for D-/R+ (iqr, 0.07-0.36; P = .030, Kruskal-Wallis test). Higher levels were seen in patients with biopsy-proven CMV pneumonitis compared with those with negative biopsies (mean, 0.24 [SD 0.51] ICs/10(5) leukocytes vs. 0.01 [SD 0.03]; P = .039, Wilcoxon test) and with symptomatic CMV episodes compared with asymptomatic episodes (median, 0.34 ICs/10(5) [iqr, 0.11-0.61] vs. 0.08 ICs/10(5) [iqr, 0.03-0.13]; P = .045, Wilcoxon test). Further studies are required to determine whether quantification of CMV viremia by this method will be of practical value in the recognition of significant CMV infection in lung transplant recipients.
The Journal of Infectious Diseases 05/1995; 171(4):1006-10. · 6.41 Impact Factor
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ABSTRACT: We designed a polymerase chain reaction method to detect herpes simplex virus (HSV) DNA in spinal fluid from patients with encephalitis. The polymerase chain reaction amplified a 211 base-pair segment of the HSV DNA polymerase gene. Applying this method, we diagnosed HSV type 1 infection in three young children, aged 7 to 13 months, who had atypical forms of the illness. On the basis of magnetic resonance imaging, their disease was diffuse or multifocal in two cases and, in all three, lacked the temporal lobe involvement considered characteristic of HSV encephalitis beyond the neonatal period. Most of the diffuse or multifocal abnormalities detected by magnetic resonance imaging were not apparent by computed tomography. Restriction enzyme analysis of the polymerase chain reaction products from all three patients indicated that their disease was caused by HSV type 1. We conclude that in preschool-age children beyond the neonatal period, the spectrum of HSV encephalitis includes multifocal or diffuse involvement of the brain, which may be detected most efficiently by magnetic resonance imaging. The polymerase chain reaction method has the potential for providing an early diagnosis, but further studies are required to define the sensitivity and specificity of the polymerase chain reaction before it can be used for routine clinical decision making.
Journal of Pediatrics 03/1995; 126(2):234-41. · 4.11 Impact Factor
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ABSTRACT: This study compared PCR and an assay for cytomegalovirus (CMV) pp65 antigenemia (CMV-vue; INCSTAR Corp.) with a quantitative shell vial culture (QSVC) technique for the detection of CMV in serial blood specimens from 46 solid-organ transplant recipients. In a comparison based on 535 specimens tested by PCR and QSVC, CMV was detected by PCR in 41 and by QSVC in 37 of 43 recipients at risk of CMV infection. The mean number of days after transplantation of initial detection of CMV was 29.9 for PCR and 34.0 for QSVC (P = 0.01). The antigenemia assay was performed on 395 specimens, including 304 of those also tested by PCR. In these specimens, CMV was detected by the antigenemia assay, QSVC, and PCR in 30, 32, and 35 (respectively) of 38 patients at risk, with no statistically significant difference in the time to detection. Each of the assays detected CMV in similar proportions of patients with and without clinically significant CMV infection. PCR stayed positive longer after transplantation than the other assays but frequently returned to negative when more than 6 months had elapsed after transplantation. The antigenemia assay and PCR stayed positive longer after institution of antiviral therapy than QSVC. PCR can provide highly sensitive detection of CMV viremia, but a PCR assay for CMV is not yet available in kit form. The pp65 antigenemia assay and shell vial culture are quantifiable and comparable in sensitivity. Either is recommended for rapid detection of CMV in blood specimens from solid-organ transplant recipients.
Journal of Clinical Microbiology 05/1994; 32(4):997-1003. · 4.15 Impact Factor
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ABSTRACT: We used the polymerase chain reaction (PCR) to demonstrate cytomegalovirus (CMV) DNA in the CSF of a patient with the CMV radiculomyelopathy syndrome. To investigate the significance of this finding, we also performed PCR for CMV on CSF samples from 30 patients with human immunodeficiency virus (HIV) infection and neurologic disease, four patients with solid organ transplants including three with active CMV infection, and 10 patients with no clinical suspicion of HIV or CMV infection. There was CMV DNA only in patients with HIV, and it was present more often in patients with evidence of spinal cord dysfunction. Our results suggest that PCR may be useful in the rapid diagnosis of CMV infection of the CNS in patients with HIV and that the radiculomyelopathy syndrome may represent only part of a spectrum of CMV-induced spinal cord dysfunction in these patients.
Neurology 02/1993; 43(1):75-9. · 8.31 Impact Factor
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ABSTRACT: A quantitative modification of the shell vial assay was used to investigate cytomegalovirus viremia in solid-organ transplant recipients. The level of viremia detected in 109 of 407 specimens ranged from 0.02 to 28 infectious foci per 100,000 leukocytes. By using a Poisson model, a technique was developed to determine 95% confidence limits for the measured levels of viremia. These confidence limits were used to determine the level of viremia that could be excluded by culturing a given number of cells. Longitudinal assessment of two transplant recipients revealed different patterns of viremia and demonstrated that significant disease sometimes occurred with low-level viremia. On the basis of the results of the studies, culture of at least 4 x 10(6) leukocytes is recommended for the sensitive detection of cytomegalovirus viremia.
Journal of Clinical Microbiology 11/1992; 30(10):2620-4. · 4.15 Impact Factor
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Transplantation Proceedings 29(1-2):809-11. · 1.00 Impact Factor
