CRISPR/Cas9-mediated genome editing of the thymidine kinase gene in a clinical HSV-1 isolate identifies F289S as novel acyclovir-resistant mutation.

Herpes simplex virus type 1 (HSV-1) is a neurotropic alphaherpesvirus that establishes a lifelong infection in sensory neurons of infected individuals, accompanied with intermittent reactivation of latent virus causing (a)symptomatic virus shedding. Whereas acyclovir (ACV) is a safe and highly effective antiviral to treat HSV-1 infections, long-term usage can lead to emergence of ACV resistant (ACVR) HSV-1 and subsequently ACV refractory disease. Here, we isolated an HSV-1 strain from a patient with reactivated herpetic eye disease that did not respond to ACV treatment. The isolate carried a novel non-synonymous F289S mutation in the viral UL23 gene encoding the thymidine kinase (TK) protein. Because ACV needs conversion by viral TK and subsequently cellular kinases to inhibit HSV-1 replication, the UL23 gene is commonly mutated in ACVR HSV-1 strains. The potential role of the F289S mutation causing ACVR was investigated using CRISPR/Cas9-mediated HSV-1 genome editing. Reverting the F289S mutation in the original clinical isolate to the wild-type sequence S289F resulted in an ACV-sensitive (ACVS) phenotype, and introduction of the F289S substitution in an ACVS HSV-1 reference strain led to an ACVR phenotype. In summary, we identified a new HSV-1 TK mutation in the eye of a patient with ACV refractory herpetic eye disease, which was identified as the causative ACVR mutation with the aid of CRISPR/Cas9-mediated genome engineering technology. Direct editing of clinical HSV-1 isolates by CRISPR/Cas9 is a powerful strategy to assess whether single residue substitutions are causative to a clinical ACVR phenotype.

Herpes simplex virus type 1 and type 2 in the Netherlands: seroprevalence, risk factors and changes during a 12-year period.

BACKGROUND: Genital herpes results in considerable morbidity, including risk of neonatal herpes, and is increasingly being caused by Herpes Simplex Virus (HSV) type 1. Possibly children are less often HSV-1 infected, leaving them susceptible until sexual debut. We assessed changes in the Dutch HSV-1 and HSV-2 seroprevalence over time and determinants associated with HSV seropositivity. METHODS: We used data from two population-based seroepidemiological studies conducted in 1995-6 and 2006-7 with a similar study design. Serum samples of 6 months to 44-year-old participants were tested for type-specific HSV antibodies using HerpesSelect® with a cut-off level of >1.10 for seropositivity. Age and sex-specific HSV-1 and HSV-2 seroprevalence was weighted for the Dutch population. Logistic regression was performed to investigate determinants associated with HSV seropositivity. RESULTS: Overall, weighted HSV-1 seroprevalence was significantly lower in 2006-7 [42.7 % 95 % confidence interval (CI) 39.9-45.4] than in 1995-6 (47.7 % 95 % CI 44.8-50.7), especially among 10- to 14-year-olds. Overall, weighted HSV-2 seroprevalence remained stable: 6.8 % in 1995-6 and 6.0 % in 2006-7. Adults who ever had sexual intercourse were more often seropositive for HSV-1 [adjusted Odds Ratio (aOR) 1.69 95 % CI 1.33-2.16] and HSV-2 (aOR 2.35 95 % CI 1.23-4.52). Age at sexual debut was the only sexual risk determinant associated with HSV-1 seropositivity. CONCLUSIONS: Because of the lower HSV-1 seroprevalence in 2006-7 compared to 1995-6, more adults are susceptible to genital HSV-1, including women of reproductive age. Given the higher risk of neonatal herpes when HSV is acquired during pregnancy, prevention and control measures during pregnancy also targeting HSV-1, are important.

Emergence and persistence of macrolide resistance in oropharyngeal flora and elimination of nasal carriage of Staphylococcus aureus after therapy with slow-release clarithromycin: a randomized, double-blind, placebo-controlled study.

To investigate the effect of slow-release (SR) clarithromycin on colonization and the development of resistance in oropharyngeal and nasal flora, a double-blind, randomized, placebo-controlled trial was performed with 8 weeks of follow-up. A total of 296 patients with documented coronary artery disease were randomized in the preoperative outpatient clinic to receive a daily dose of SR clarithromycin (500 mg) (CL group) or placebo tablets (PB group) until the day of surgery. Nose and throat swabs were taken before the start of therapy, directly after the end of therapy, and 8 weeks later. The presence of potential pathogenic bacteria was determined, and if they were isolated, MIC testing was performed. Quantitative culture on media with and without macrolides was performed for the indigenous oropharyngeal flora. In addition, analysis of the mechanism of resistance was performed with the macrolide-resistant indigenous flora. Basic patient characteristics were comparable in the two treatment groups. The average number of tablets taken was 15 (standard deviation = 6.4). From the throat swabs, Haemophilus parainfluenzae was isolated and carriage was not affected in either of the treatment groups. Nasal carriage of Staphylococcus aureus, however, was significantly reduced in the CL group (from 35.3 to 4.3%) compared to the PB group (from 32.4 to 30.3%) (P < 0.0001; relative risk [RR], 7.0; 95% confidence interval [CI], 3.1 to 16.0). Resistance to clarithromycin was present significantly more frequently in H. parainfluenzae in the CL group after treatment (P = 0.007; RR, 1.6; 95% CI, 1.1 to 2.3); also, the percentage of patients with resistance to macrolides in the indigenous flora after treatment was significantly higher in the CL group (31 to 69%) (P < 0.0001; RR, 1.9; 95% CI, 1.4 to 2.5). This persisted for at least 8 weeks. This study shows that besides the effective elimination of nasal carriage of S. aureus, treatment with SR clarithromycin for approximately 2 weeks has a marked and sustained effect on the development of resistance in the oropharyngeal flora for at least 8 weeks.

Genotyping by amplified fragment length polymorphism analysis reveals persistence and recurrence of infection with Streptococcus anginosus group organisms.

Streptococcus anginosus, Streptococcus constellatus, and Streptococcus intermedius, commonly referred to as the Streptococcus anginosus group (SAG), are commensal organisms known for their propensity to cause purulent infections which are difficult to eradicate. In this study, we determined the genetic similarities between SAG isolates consecutively recovered from single patients to assess the duration of infection or colonization. A total of 97 SAG isolates recovered from 30 patients were included; 65 (67.0%) of the isolates were abscess related. The isolates were identified by the 16S rRNA reverse line blot hybridization assay as S. anginosus (n = 34), S. constellatus (n = 55), and S. intermedius (n = 8). Amplified fragment length polymorphism (AFLP) analysis of the SAG isolates produced discriminatory and reproducible patterns. Consecutive SAG isolates with identical AFLP types were found in 27 of 30 (90.0%) patients, and consecutive isolates with only a single AFLP type were demonstrated in 21 (70.0%) patients. The median delay between the times of recovery of the first and last isolates of identical AFLP types from each patient was 36 days, and this delay extended for more than 1 year in patients with both colonizing and abscess-related SAG isolates. In six bacteremic patients, paired blood and nonblood SAG isolates showed identical AFLP types.