Atypical and fulminant presentations of pneumococcal infections: A case series in a tertiary intensive care unit.

With the introduction of conjugate pneumococcal vaccines, changes in causative serotypes and clinical presentations of Streptococcus pneumoniae infections are occurring. During the 2017-2018 winter, an unusual number of patients with a severe manifestation of pneumococcal disease was admitted to a tertiary care intensive care unit (ICU) in the Netherlands. We describe some of the cases in depth. Given our observed change in infecting serotypes and extreme clinical manifestations of pneumococcal disease, a systematic clinical registry of pneumococcal infections in the ICU may be a valuable addition to pneumococcal disease surveillance.

Surveillance-embedded genomic outbreak resolution of methicillin-susceptible Staphylococcus aureus in a neonatal intensive care unit.

We observed an increase in methicillin-susceptible Staphylococcus aureus (MSSA) infections at a Dutch neonatal intensive care unit. Weekly neonatal MSSA carriage surveillance and cross-sectional screenings of health care workers (HCWs) were available for outbreak tracing. Traditional clustering of MSSA isolates by spa typing and Multiple-Locus Variable number tandem repeat Analysis (MLVA) suggested that nosocomial transmission had contributed to the infections. We investigated whether whole-genome sequencing (WGS) of MSSA surveillance would provide additional evidence for transmission. MSSA isolates from neonatal infections, carriage surveillance, and HCWs were subjected to WGS and bioinformatic analysis for identification and localization of high-quality single nucleotide polymorphisms, and in-depth analysis of subsets of isolates. By measuring the genetic diversity in background surveillance, we defined transmission-level relatedness and identified isolates that had been unjustly assigned to clusters based on MLVA, while spa typing was concordant but of insufficient resolution. Detailing particular subsets of isolates provided evidence that HCWs were involved in multiple outbreaks, yet it alleviated concerns about one particular HCW. The improved resolution and accuracy of genomic outbreak analyses substantially altered the view on outbreaks, along with apposite measures. Therefore, inclusion of the circulating background population has the potential to overcome current issues in genomic outbreak inference.

Density and duration of experimental human pneumococcal carriage.

The density and duration of pneumococcal carriage are considered to affect the likelihood of transmission and invasive disease. Because of its importance in both spreading and causing disease, carriage has been suggested as an endpoint in future vaccine studies. Culture is the current gold standard for detection, but may not be sensitive enough to detect changes at low density. Healthy adult volunteers received an intranasal inoculation of Streptococcus pneumoniae serotype 6B. Pneumococcal density in nasal washes collected at six time-points post-inoculation was determined by culture and quantitative PCR (qPCR). Natural pneumococcal carriers detected at initial screening were followed in parallel. In 331 nasal washes from 79 volunteers, the sensitivity and specificity of pneumococcal detection by qPCR, as compared with culture, were 92.3% and 75.9%. The estimation of pneumococcal density by culture and qPCR was highly correlated (rs  = 0.73, p <0.0001), although qPCR had a lower detection limit. Pneumococcal density fluctuated within a carriage episode, and occasionally fell below the detection limit of both methods. The duration of carriage episodes was underestimated when only one method was used. Similar fluctuations in density were observed in natural carriers. Pneumococcal carriage is a dynamic event. Culture and qPCR are complementary for surveying the density and duration of pneumococcal carriage episodes.

Diagnostic value of serum pneumococcal DNA load during invasive pneumococcal infections.

Detection of pneumococcal DNA in blood could be a fast alternative for blood culture in invasive pneumococcal disease (IPD). In this study we compared the diagnostic value of the serum pneumococcal DNA load between different clinical syndromes in adults with bacteremic pneumococcal infections, also after initiation of antibiotic treatment. Adults hospitalized with a blood culture proven pneumococcal infection between December 2008 and June 2013 were retrospectively included. Pneumococcal DNA loads in corresponding serum samples were determined by qPCR. Data on clinical diagnosis, course of disease and antibiotic treatment were extracted from medical records. For 53 IPD cases eligible stored serum samples were retrieved. The proportion of samples positive in qPCR was lower in uncomplicated pneumonia compared with other clinical syndromes (59.5 % vs. 100 %, p = 0.005). The pneumococcal DNA load was higher in cases other than uncomplicated pneumonia (p = 0.043) as well as in more severe disease (p-values 0.018, 0.029 and 0.003 for PSI Risk Class IV/V, ICU admission and mortality, respectively). Both detection of pneumococcal DNA and distribution of load did not significantly change over the first days of hospitalization despite treatment with appropriate antibiotics. Detection of pneumococcal DNA in serum was more sensitive in clinical syndromes other than uncomplicated pneumonia. Furthermore, the pneumococcal DNA load was associated with the type of IPD and severity of disease. Since the serum pneumococcal DNA load seemed unaffected by antibiotic treatment during the first days of IPD, it may offer an alternative for culture methods after prior antibiotic use.

Epitope mapping and serodiagnosis using Ata- and (Lys)7 peptides.

The general applicability of the new peptide immobilization strategy in which the peptide of interest is N-terminally extended with an acetyl-thio-acetyl group or (poly)-Lys extension during synthesis, has been demonstrated in epitope-mapping experiments and serodiagnosis. Ala-scanning experiments and minimal epitope determination showed that the antigenicity of Ata-extended peptides derived from the human chorionic gonadotropin (hCG) and hepatitis B virus (HBV) amino acid sequence, was superior to the free parent peptides. Further, it could be shown that the choice of the epitope-mapping procedure (peptide in solution or immobilized on a solid support) may lead to a different perception of which residues constitute the epitope. In addition, a time-consuming conjugation process could be circumvented since the ELISA reactivity of BSA-conjugates was comparable to that of Ata-extended peptides. In the serodiagnosis using sera from various HIV-positive individuals, the lysyl-peptide showed a signal/noise ratio 10 times higher than the parent peptide, indicating that sensitivity increased as a result of this N-terminal lysyl tail. In all experiments we observed that antibody detection could be performed at roughly 10 times lower amounts of peptide when N-terminally linked to an Ata-group or lysyl-extension compared to the free parent peptide or the BSA-conjugated equivalent.