Prophylactic versus preemptive oral valganciclovir for the management of cytomegalovirus infection in adult renal transplant recipients.

Prophylaxis reduces cytomegalovirus (CMV) disease, but is associated with increased costs and risks for side effects, viral resistance and late onset CMV disease. Preemptive therapy avoids drug costs but requires frequent monitoring and may not prevent complications of asymptomatic CMV replication. Kidney transplant recipients at risk for CMV (D+/R-, D+/R+, D-/R+) were randomized to prophylaxis (valganciclovir 900 mg q.d. for 100 days, n=49) or preemptive therapy (900 mg b.i.d. for 21 days, n=49) for CMV DNAemia (CMV DNA level>2000 copies/mL in >or=1 whole blood specimens by quantitative PCR) assessed weekly for 16 weeks and at 5, 6, 9 and 12 months. More patients in the preemptive group, 29 (59%) than in the prophylaxis group, 14 (29%) developed CMV DNAemia, p=0.004. Late onset of CMV DNAemia (>100 days after transplant) occurred in 11 (24%) randomized to prophylaxis, and none randomized to preemptive therapy. Symptomatic infection occurred in five patients, four (3 D+/R- and 1 D+/R+) in the prophylactic group and one (D+/R-) in the preemptive group. Peak CMV levels were highest in the D+/R- patients. Both strategies were effective in preventing symptomatic CMV. Overall costs were similar and insensitive to wide fluctuations in costs of either monitoring or drug.

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.

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.

Effects of storage temperature and time on qualitative and quantitative detection of cytomegalovirus in blood specimens by shell vial culture and PCR.

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.

Control of cytomegalovirus-associated morbidity in renal transplant patients using intensive monitoring and either preemptive or deferred therapy.

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.

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.

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 IM (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.

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.

Herpes simplex virus type 2 meningitis in the absence of genital lesions: improved recognition with use of the polymerase chain reaction.

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.

Quantitative analysis of cytomegalovirus viremia in lung transplant recipients.

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.

Comparison of heparin and EDTA transport tubes for detection of cytomegalovirus in leukocytes by shell vial assay, pp65 antigenemia assay, and PCR.

The anticoagulants heparin and EDTA were compared for inhibitory effects on the detection of cytomegalovirus from washed leukocytes in specimen transport tubes. Evaluation was made by the centrifugation/shell vial culture technique, the pp65 antigenemia assay, and PCR. For each assay, the results with heparin and EDTA were equivalent.

Comparison of PCR and pp65 antigenemia assay with quantitative shell vial culture for detection of cytomegalovirus in blood leukocytes from solid-organ transplant recipients.

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.

Quantitative cultures of the cell fraction and supernatant of bronchoalveolar lavage fluid for the diagnosis of cytomegalovirus pneumonitis in lung transplant recipients.

Results of quantitative shell vial cultures of cell and supernatant fractions of bronchoalveolar lavage (BAL) fluid from lung transplant recipients were compared with results of transbronchial lung biopsies. Cytomegalovirus (CMV) was present in both the cell and supernatant fractions, including 28 (80%) and 29 (83%), respectively, of 35 BAL samples with corresponding biopsies that showed evidence of CMV pneumonitis and 34 (15%) and 75 (33%), respectively, of 227 BAL samples with corresponding biopsies that did not. Cultures of unseparated BAL fluid had a similar yield to cultures of supernatant. Virus titers of cell fractions and supernatants from BAL samples with corresponding biopsies that were positive for CMV pneumonitis were higher than those from BALs with negative corresponding biopsies. Culture of the unseparated fluid is recommended for simplicity and to maximize detection of CMV. Quantitative results may help in the identification of CMV pneumonitis, but additional studies are needed before quantitative cultures can be recommended for routine use.