A case of ganciclovir-resistant cytomegalovirus (CMV) retinitis in a patient with AIDS: longitudinal molecular analysis of the CMV viral load and viral mutations in blood compartments.

OBJECTIVE: To study the temporal relationships between cytomegalovirus (CMV) viral load and specific UL97 mutations in polymorphonuclear leukocytes (PMNL) and plasma samples from a patient with AIDS who developed ganciclovir-resistant CMV retinitis. METHODS: Sequential PMNL and plasma samples were analysed for determination of the CMV viral load using non-molecular methods and a quantitative polymerase chain reaction (PCR) assay. Screening of the same samples for the most common mutations conferring ganciclovir resistance was performed using nested PCR and restriction enzyme analysis. RESULTS: At the time of progression of CMV retinitis (after 6 months of ganciclovir), a rapid increase in the CMV DNA load was found in both PMNL and plasma samples. This increase paralleled the emergence of a specific mutation (V594) in the same samples and recovery of ganciclovir-resistant blood isolates. In this patient, however, the only tests that substantially predicted the progression of CMV disease were the quantitative PCR assay using PMNL and to a lesser extent the pp65 antigenemia assay. CONCLUSIONS: Quantitative evaluation of the CMV viral load in PMNL using sensitive assays such as PCR appears to be a promising approach for monitoring antiviral therapy in subjects with AIDS. In addition, common mutations conferring ganciclovir resistance can be detected directly in PMNL and plasma samples.

Molecular evolution of influenza A/H3N2 viruses in the province of Québec (Canada) during the 1997-2000 period.

In this study, we compared antigenic (hemagglutination inhibition (HI) assay) and molecular (sequencing of the hemagglutinin (HA1) gene) characterization of influenza isolates collected in the Province of Québec (Canada) during the last three flu seasons (1997-2000). Twenty-three isolates were tested by a standard HI assay and 37 by sequencing of the HA1 gene for their homology to the A/H3N2 vaccine strains A/Wuhan/359/95 (1997-1998) and A/Sydney/5/97 (1998-1999 and 1999-2000). By HI, two isolates were antigenically similar to A/Wuhan/359/95 (both from 1997 to 1998), 16 were similar to A/Sydney/5/97 (1997-2000) and no conclusions could be inferred for the other five isolates due to identical HI titers for the two vaccine strains (n=4) or insufficient viral titer (n=1). Sequence analysis revealed that four isolates from 1997 to 1998 were related to A/Wuhan/359/95 whereas the others (n=4) from 1997 to 1998, as well as all isolates from 1998 to 1999 (n=18) and 1999 to 2000 (n=11) were closer to A/Sydney/5/97. The mean number of amino acid differences for the 33 A/Sydney/5/97-like isolates compared with the homologous vaccine strain was 6.3 (1.9%), 9.2 (2.8%) and 13.6 (4.1%) for those collected in 1997-1998, 1998-1999, and 1999-2000, respectively. Phylogenetic analysis confirmed that a progressive drift occurred among our A/H3N2 influenza isolates over the last three flu seasons. Furthermore, it revealed that isolates collected during the last two flu seasons were in fact more related to A/Panama/2007/99 (2000-2001 vaccine strain) than to A/Sydney/5/97. Our studies suggest that molecular analysis of the HA1 gene should complement the HI assay for a more accurate analysis of influenza A virus drift.

Vitamin E inhibits fish oil-induced hyperlipidemia and tissue lipid peroxidation in hamsters.

Previous research has linked hyperlipidemia with increased serum concentrations of lipid peroxidation products; however, a specific association between diet-induced oxidative stress and hyperlipidemia has not been studied. In the present study, the relationship between tissue lipid peroxidation and hyperlipidemia induced by ingestion of fish oil was examined. In Experiment 1, male Golden Syrian hamsters were fed semipurified diets composed of 1.6 wt% safflower oil plus 15.0 wt% of either butterfat (BF), safflower oil (SAFF), or high-cholesterol menhaden oil [MHO(H-CHOL)] semipurified diets for 27 d. The cholesterol contents of the diets were adjusted to 0.088%. The MHO(H-CHOL)-fed hamsters exhibited higher serum concentrations of total cholesterol, triglycerides, apolipoprotein B, and lipid peroxides when compared to the BF and SAFF diet groups. In a further study (Experiment 2), hamsters were fed for 27 d three dietary treatments: (i) MHO(H-CHOL) with no vitamin E content; (ii) a low-cholesterol menhaden oil containing high concentrations of vitamin E (2.5 mg tocopherol/g oil or dietary concentrations of 375 mg/kg) [MHO(L-CHOL) + E]; and (iii) the MHO(L-CHOL + E) with added cholesterol (595 mg/kg) [MHO(L-CHOL) + CHOL + E] to match the cholesterol content of the MHO(H-CHOL). The MHO(L-CHOL) + E and MHO(L-CHOL) + CHOL + E diet groups showed lower concentrations of serum cholesterol, triglycerides, and hepatic lipid peroxides than the MHO(H-CHOL)-treated group. Moreover, in contrast to the hypercholesterolemia caused by the MHO(H-CHOL) feeding, the MHO(L-CHOL)+ E and MHO(L-CHOL) + CHOL + E diets did not show a serum cholesterol-elevating action. This study supports the hypothesis that oxidative stress in the Syrian hamster could play a causal role in dietary-induced hyperlipidemia which can be inhibited by high vitamin E intake.

Clinical impact of ganciclovir-resistant cytomegalovirus infections in solid organ transplant patients.

Clinical consequences of ganciclovir resistant cytomegalovirus (CMV) infections were studied during 2 large prophylactic trials consisting of 100 days of valganciclovir or ganciclovir prophylaxis in solid organ transplant (SOT) recipients. The first one involved 301 high-risk (CMV donor seropositive/recipient seronegative) SOT recipients excluding lung transplants followed for 12 months, whereas the second one involved 80 lung transplant patients evaluated over 6 months. Among the 7 patients (4 non-lung and 3 lung transplant patients) carrying viruses with known ganciclovir-resistance [corrected] mutations in blood, adverse clinical outcome was only observed in the lung transplant recipients. Additionally, no CMV resistance mutations were observed in non-lung transplant patients receiving valganciclovir.

Quantitative analysis of cytomegalovirus (CMV) viremia using the pp65 antigenemia assay and the COBAS AMPLICOR CMV MONITOR PCR test after blood and marrow allogeneic transplantation.

The performance of a commercially available qualitative PCR test for plasma (AMPLICOR CMV Test; Roche Diagnostics) and a quantitative PCR test for plasma and leukocytes (COBAS AMPLICOR CMV MONITOR Test; Roche Diagnostics) was evaluated with samples from 50 blood or marrow allogeneic transplant recipients who received short courses of sequential ganciclovir therapy (2 weeks intravenously followed by 2 weeks orally) based on a positive cytomegalovirus (CMV) pp65 antigenemia (AG) assay. The number of persons with a positive CMV test was significantly higher for leukocyte-based assays (AG, 67.5%; PCR, 62.5%) compared to both quantitative and qualitative PCR tests of plasma (42.5 and 35%, respectively). One person developed CMV disease during the study despite a negative AG assay; in this particular case, all PCR assays were found to be positive 10 days before his death. There was a trend for earlier positivity after transplantation and more rapid negativity after initiation of ganciclovir for the tests performed on leukocytes. The mean number of CMV copies as assessed by PCR was significantly higher in leukocytes than in plasma (P = 0.02). Overall, excellent agreement (kappa coefficient, >0.75) was found only between the two PCR assays (qualitative and quantitative) based on plasma. These results suggest that either the pp65 AG assay or the COBAS AMPLICOR CMV MONITOR Test using leukocytes could be used to safely monitor CMV viremia in related allogeneic blood or marrow transplant recipients. Such a strategy will result in preemptive treatment for about two-thirds of the persons with a relatively low rate (<33%) of secondary viremic episodes following short courses of ganciclovir therapy.

Colonization of the oral cavity of mice by an unidentified streptococcus.

While studying the oral bacterial biota of mice, we observed an unidentified streptococcus (TG) that eventually became the dominant species of the oral cavities of all other mice in our animal facility. We found that the strain is indigenous to Jackson Laboratory mice but is absent in animals from Charles River Laboratories. TG was also transmitted from artificially contaminated BALB/c mice to the oral cavities of 4 other mouse strains. Streptococcus sp. TG stimulated the secretory and systemic immune systems of artificially contaminated Charles River BALB/c mice but did not provoke clinical symptoms. The increase in antibody level to TG did not prevent its colonization and persistence in these mice. In mice from Jackson Laboratory, the secretory and systemic immune response to TG was significantly lower. In vitro, Streptococcus sp. TG inhibited murine oral lactobacilli and staphylococci, probably due to the production of hydrogen peroxide.

Rapid antiviral effect of inhaled zanamivir in the treatment of naturally occurring influenza in otherwise healthy adults.

The antiviral and clinical effects of inhaled zanamivir (10 mg twice daily for 5 days, started within the first or second day of a flulike illness) were evaluated in a randomized, placebo-controlled trial during the 1997-1998 influenza season in Canada. Pharyngeal secretions were collected with swabs every 12 h during 6 days, and symptoms were self-evaluated twice daily during 14 days. After only 12 h of treatment (1 dose), median virus titers decreased by 1.0 log10 TCID50/mL in the zanamivir group (n=17), compared with a 0. 42-log10 increase in the placebo group (n=10; P=.08). This was associated with a 4.5-day (47.4%) reduction in the median time to alleviation of all significant flu symptoms in the zanamivir recipients (P=.03 after adjusting for the initial virus titer and the time between onset of symptoms and treatment). Resistance to zanamivir was not detected in virus isolates by either phenotypic or genotypic assays.

Inverse correlation between the proportion of salivary bacteria inhibiting Streptococcus mutans and the percentage of untreated carious teeth.

To evaluate the role of inhibitory substances produced by bacteria in the oral cavity, we estimated, by a deferred test on Todd-Hewitt agar enriched with hemin and vitamin K, the proportion of bacteria that inhibited or stimulated the growth of Streptococcus mutans and Porphyromonas gingivalis, from the saliva of 109 patients (54 males and 55 females) attending our dental clinics. The patients, aged from 8 to 75 years old (mean: 31 +/- 18 years), were randomly selected whatever the reason for their visit. The results, evaluated with the Spearman rank test, indicated that there was no statistically significant (P > 0.05) correlation between the proportion of salivary bacteria inhibiting or stimulating P. gingivalis with the Community Periodontal Index of Treatment Needs (CPITN), the number of carious, missing and filled teeth, or with the decayed, missing and filled teeth index (DMFT). Also, no statistically significant correlation was observed between the proportion of salivary bacteria stimulating the growth of S. mutans and the above mentioned health indexes. However, a statistically significant (P < 0.005) negative correlation was found between the percentage of cultivated bacteria that inhibit S. mutans and the percentage of untreated carious teeth as well as with the CPITN. The results thus indicate a possible role for inhibitory substances produced by bacteria in the maintenance of oral health.