Oral maribavir for treatment of refractory or resistant cytomegalovirus infections in transplant recipients.

BACKGROUND: Despite advances in cytomegalovirus (CMV) prophylaxis and therapy, some transplant recipients still develop refractory CMV infections. Maribavir (MBV), an investigational benzimidazole antiviral agent, acts by a mechanism different from that of existing anti-CMV drugs. Previous Phase I and II studies have demonstrated a favorable safety profile for MBV, but its utility in treatment of complex CMV syndromes is unknown. METHODS: Between June and December 2008, MBV was released for use under individual emergency investigational new drug applications requested by treating physicians and approved by the US Food and Drug Administration and local institutional review boards. Six patients (5 solid organ transplant recipients and 1 hematopoietic stem cell transplant recipient) who had failed to respond to other therapies and/or had known ganciclovir-resistant CMV were treated with MBV at a starting oral dose of 400 mg twice daily. RESULTS: Patients were treated for a median of 207 days (range, 15-376). Four of 6 patients had no detectable CMV DNAemia within 6 weeks of starting MBV therapy. One patient, who had an initial viral load of 1.8 million copies/mL, developed MBV resistance mutations. One patient, who had low serum levels of MBV, had persistent CMV DNAemia and viruria without developing genotypic or phenotypic resistance to MBV. One patient cleared CMV DNAemia, but died of pneumonia and multiorgan failure. No significant adverse effects attributable to MBV were observed. CONCLUSIONS: MBV deserves further systematic evaluation as treatment for CMV infection that is resistant and/or refractory to standard therapies, but its optimal dose, duration of therapy, and use in combinations versus as a single agent have yet to be determined.

Repression of the HSV-1 latency-associated transcript (LAT) promoter by the early growth response (EGR) proteins: involvement of a binding site immediately downstream of the TATA box.

During herpes simplex virus (HSV) latency, in neurons of the nervous system, a single family of viral transcripts (the Latency-Associated Transcripts or LATs) are synthesized. Within the LAT promoter region, we have identified a consensus sequence for the EGR proteins in an unusual position immediately downstream of the TATA box. The early growth response (EGR) proteins are rapidly induced in cells by stimuli which also induce HSV to reactivate from latency. In order to determine if EGR proteins play any role in control of LAT transcription, we have analyzed the interactions between EGR proteins and the LAT promoter. Gel retardation and DNase I protection assays demonstrated that EGR1 zinc finger protein bound specifically to the LAT promoter region EGR consensus sequence. To determine if EGR proteins could modulate transcription through the LAT promoter, cotransfection assays were performed using chloramphenicol acetyltransferase (CAT) reporter constructs driven by either the wild-type LAT promoter or a LAT promoter with a mutated EGR binding site. Contransfection of the wild-type LAT promoter construct with EGR expression plasmids resulted in inhibition of the basal level of CAT activity with EGR-2 but not EGR-1 or 3. However, normal levels of CAT activity were observed in cotransfections using the mutant LAT promoter CAT construct suggesting that repression was mediated by the binding of EGR-2 proteins to the LAT promoter. Furthermore, data from combination binding assays using EGR1 and TATA binding protein (TBP) in vitro support the hypothesis that binding of EGR proteins to the LAT promoter prevents binding of TBP and thus suppresses transcription. These results may provide a link between stress responses in neurons of the CNS which activate the EGR family of proteins and HSV reactivation from latency due to the same stress response.

A ganciclovir-resistant clinical isolate of human cytomegalovirus exhibiting cross-resistance to other DNA polymerase inhibitors.

Clinical isolates of human cytomegalovirus (HCMV) were screened for susceptibility to ganciclovir by plaque-reduction assay and in situ ELISA. A pretreatment isolate of HCMV obtained from the bronchial brushing of a heart transplant recipient contained both ganciclovir-susceptible and -resistant virus. Ganciclovir-susceptible (P8) and -resistant (D16) strains were further isolated by plaque purification. Both strains phosphorylated ganciclovir at levels similar to the ganciclovir-susceptible strain AD169. D16 was also resistant to phosphonoformic acid and to (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)adenine and cytosine. These data suggest that the resistance of D16 to these drugs results from a mutation in the viral DNA polymerase gene.

Efficacy of intraperitoneal antibiotics in the treatment of severe fecal peritonitis.

A study was performed with rabbits to examine the efficacy of treatments for fecal peritonitis and, specifically, to determine whether it is beneficial to include antibiotics in the saline used to irrigate the peritoneum. A standardized inoculum of human stool suspension was placed in the peritoneal cavity of the rabbits. Fifty-six rabbits were studied to compare the effect of treatments begun 2 hours after peritoneal soiling. The administration of no treatment resulted in 100% mortality (14 of 14). Parenteral cefotetan 25 mg/kg intramuscularly (IM) twice a day (BID) with no other treatment reduced mortality to 50% (p less than 0.05). Cefotetan 25 mg/kg IM BID plus irrigation of the peritoneum with plain saline further reduced mortality to 21% (3 of 14, p less than 0.05). Cefotetan 25 mg/kg IM BID plus irrigation of the peritoneum with saline containing cefotetan 1.0 mg/mL reduced mortality to 14% (2 of 14, p = not significant). These treatments also produced a progressive decrease in the number of intraperitoneal abscesses from 24.0 +/- 2.1 (mean +/- SEM) in the animals receiving no treatment to 9.7 +/- 1.2 abscesses in the animals receiving peritoneal irrigation with saline containing cefotetan (p less than 0.001). A second experiment then was performed specifically to examine the efficacy of intraperitoneal antibiotics. A lethal fecal inoculum was determined in rabbits receiving conventional therapy, i.e., parenteral antibiotics (cefotetan) and irrigation of the peritoneum with plain saline. With two hours delay before treatment, cefotetan 25 mg/kg IM BID and irrigation with plain saline produced 80% mortality (11 of 14). Cefotetan 25 mg/kg IM BID plus cefotetan 1.0 mg/mL in the saline washout reduced mortality to 21% (3 of 14, p = 0.003) and markedly reduced the number of intraperitoneal abscesses from 13.4 +/- 0.7 in the animals receiving irrigation with plain saline to 8.1 +/- 0.8 in the animals receiving irrigation with saline containing cefotetan (p less than 0.0001). Thus, intraperitoneal irrigation with antibiotics was highly effective. Serum antibiotic levels drawn 30 minutes after irrigation were 112.7 +/- 22.4 micrograms/mL in animals that received irrigation with plain saline, and 101.7 +/- 15.2 micrograms/mL in animals that received irrigation with saline containing cefotetan. These serum levels were not significantly different. With 6 hours delay before treatment, all therapy was less effective. Cefotetan 25 mg/kg IM BID and irrigation with plain saline resulted in 100% mortality (14 of 14). With 6 hours delay, cefotetan 25 mg/kg IM BID and irrigation with saline containing cefotetan reduced mortality to 80% (11 of 14).(ABSTRACT TRUNCATED AT 400 WORDS)

In situ ELISA for the evaluation of antiviral compounds effective against human cytomegalovirus.

An in situ ELISA was developed as an improved procedure over the plaque reduction assay for antiviral susceptibility testing of HCMV. Unlike the plaque reduction assay, the ELISA can be completed at 4-5 days post-infection. The effective dose (ED50) of ganciclovir (GCV), acyclovir (ACV), phosphonoacetic acid (PAA), or phosphonoformic acid (PFA), was determined using HCMV strain AD169. The resistance profiles of two laboratory-derived GCV-resistant mutants of HCMV strain AD169 and seven clinical isolates were determined using the ELISA. The ELISA results were confirmed by the plaque reduction assay. The ED50 for GCV with the AD169 control ranged from 3.1 to 6.2 microM with a mean inhibitory concentration of 5.4 +/- 1.4 microM. Six of the clinical isolates were susceptible to GCV (ED50 = 3.1-6.2 microM). The seventh isolate had an ED50 of 50 microM and was resistant to GCV. This ELISA assay is reproducible and relatively simple to perform. The ELISA endpoints are clearly determined and the assay works well with a variety of antiviral compounds.

The value of continuous 72-hour peritoneal lavage for peritonitis.

Studies were performed in 120 rabbits to determine whether 72-hour peritoneal lavage is beneficial or harmful in the treatment of peritonitis. Results showed that against a high concentration fecal inoculum (90 percent mortality), peritoneal lavage containing gentamicin and clindamycin reduced mortality to 10 to 20 percent (p less than 0.05). Parenteral antibiotics alone and lavage not containing antibiotics did not decrease mortality. By contrast, against a low fecal inoculum (30 percent mortality), peritoneal lavage containing gentamicin and clindamycin did not alter mortality. However, lavage not containing antibiotics increased mortality to 70 to 80 percent (p less than 0.05). These data demonstrate that continuous peritoneal lavage may be helpful in the treatment of peritonitis provided the lavage solution contains antibiotics and may be harmful if it does not contain antibiotics.

Activity of ciprofloxacin against multiply resistant strains of Pseudomonas aeruginosa, Staphylococcus epidermidis, and group JK corynebacteria.

The susceptibilities of multiply resistant clinical isolates of Pseudomonas aeurginosa to ciprofloxacin, norfloxacin, and several beta-lactam and aminoglycoside antibiotics were evaluated. Ciprofloxacin also was compared with methicillin and vancomycin against methicillin-resistant Staphylococcus epidermidis and group JK corynebacteria. Ciprofloxacin exhibited the lowest MICs and MBCs for 90% of the isolates among all of the antibiotics tested against P. aeruginosa. In addition, ciprofloxacin exhibited excellent bactericidal activity against the gram-positive organisms. Clinical trials are necessary to confirm the in vitro results and monitor for emergence of resistance.

In vitro comparison of amifloxacin and six other antibiotics against aminoglycoside-resistant Pseudomonas aeruginosa.

The in vitro activity of the synthetic fluoroquinolone amifloxacin was compared with those of six other antibiotics: ampicillin, aztreonam, cefotaxime, cephalexin, cinoxacin, and gentamicin. Amifloxacin had the lowest 50% MIC of any of the antibiotics tested against aminoglycoside-resistant Pseudomonas aeruginosa, 4 micrograms/ml.