Phenotypic testing of patient herpes simplex virus type 1 and 2 isolates for acyclovir resistance by a novel method based on real-time cell analysis.

BACKGROUND: Acyclovir (ACV) is the most commonly used drug for herpes simplex virus (HSV) infection therapy. Prolonged antiviral therapy or prophylaxis in immunocompromised patients may promote the development of drug-resistant strains. Due to the high polymorphism in genes involved in drug resistance, phenotypic methods, although work-intensive, are still required to test drug susceptibility. Real-time cell analysis (RTCA) based methods could offer a rapid and less labor-intensive alternative for phenotypic testing of ACV resistance. OBJECTIVE: To investigate the utility of a new RTCA based assay (RTCAA) to test acyclovir susceptibility of HSV clinical isolates. STUDY DESIGN: Four reference strains and 93 clinical isolates (60 HSV-1 and 33 HSV-2) were tested by RTCAA. In the presence of ACV concentrations from 2.2 to 140.8 µM, Vero cells were infected with different virus dilutions. IC50 values were calculated by dose-response curve (DRC) with area-under-curve (AUC) method. The reference strains and 22 clinical isolates were additionally tested by dye-uptake assay, and IC50 values of both methods were compared. RESULTS: IC50 values from RTCAA and dye-uptake assays were positively correlated (Spearman's rho = 0.897, p < 0.001) and quantitatively agreed (Bland-Altman plot). Based on a cut-off of 4 µM for HSV-1 and 13 µM for HSV-2, 87 isolates were classified as ACV-sensitive and 6 isolates as ACV-resistant. The reference strains showed the expected results of ACV susceptibility. CONCLUSION: RTCAA agrees well with the dye-uptake assay. Compared with other phenotypic methods, RTCAA requires less manipulation, reduces the workload and the turnaround time, and appears to be an objective and reliable method to test ACV susceptibility.

Respiratory viruses in healthy infants and infants with cystic fibrosis: a prospective cohort study.

RATIONALE: Acute viral respiratory tract infections in children with cystic fibrosis (CF) are known causes of disease exacerbation. The role of viral infections during infancy is, however, less known, although early infancy is thought to be a crucial period for CF disease development.We prospectively assessed symptomatic and asymptomatic viral detection in the first year of life in infants with CF and healthy controls. METHODS: In a prospective cohort study, we included 31 infants with CF from the Swiss Cystic Fibrosis Infant Lung Development Cohort and 32 unselected, healthy infants from the Basel Bern Infant Lung Development Cohort and followed them throughout the first year of life. Respiratory symptoms were assessed by weekly telephone interviews. Biweekly nasal swabs were analysed for 10 different viruses and two atypical bacteria with real-time seven duplex PCR (CF=561, controls=712). MEASUREMENTS AND RESULTS: Infants with CF and healthy controls showed similar numbers of swabs positive for virus (mean 42% vs 44%; OR 0.91, 95% CI 0.66 to 1.26, p=0.6). Virus-positive swabs were less often accompanied by respiratory symptoms in infants with CF (17% vs 23%; OR 0.64, 95% CI 0.43 to 0.95, p=0.026). This finding was pronounced for symptomatic human rhinovirus detection (7% vs 11%; OR 0.52, 95% CI 0.31 to 0.9, p=0.02). CONCLUSIONS: Viral detection is not more frequent in infants with CF and respiratory symptoms during viral detection occur even less often than in healthy controls. It is likely an interplay of different factors such as local epithelial properties and immunological mechanisms that contribute to our findings.

Interactions of Respiratory Viruses and the Nasal Microbiota during the First Year of Life in Healthy Infants.

Traditional culture techniques have shown that increased bacterial colonization is associated with viral colonization; however, the influence of viral colonization on the whole microbiota composition is less clear. We thus aimed to understand the interaction of viral infections and the nasal microbiota in early life to appraise their roles in disease development. Thirty-two healthy, unselected infants were included in this prospective longitudinal cohort study within the first year of life. Biweekly nasal swabs (n = 559) were taken, and the microbiota was analyzed by 16S rRNA pyrosequencing, and 10 different viruses and 2 atypical bacteria were characterized by real-time PCR (combination of seven duplex samples). In contrast to asymptomatic human rhinovirus (HRV) colonization, symptomatic HRV infections were associated with lower alpha diversity (Shannon diversity index [SDI]), higher bacterial density (PCR concentration), and a difference in beta diversities (Jaccard and Bray-Curtis index) of the microbiota. In addition, infants with more frequent HRV infections had a lower SDI at the end of the study period. Overall, changes in the microbiota associated with symptomatic HRV infections were characterized by a loss of microbial diversity. The interaction between HRV infections and the nasal microbiota in early life might be of importance for later disease development and indicate a potential approach for future interventions. IMPORTANCE Respiratory viral infections are very frequent in infancy and of importance in acute and chronic disease development. Infections with human rhinovirus (HRV) are, e.g., associated with the later development of asthma. We found that only symptomatic HRV infections were associated with acute changes in the nasal microbiota, mainly characterized by a loss of microbial diversity. Infants with more frequent symptomatic HRV infections had a lower bacterial diversity at the end of the first year of life. Whether the interaction between viruses and the microbiota is one pathway contributing to asthma development will be assessed in the follow-ups of these children. Independent of that, measurements of microbial diversity might represent a potential marker for risk of later lung disease or monitoring of early life interventions.