Mirjam Kooistra-Smid

Streptococcus pneumoniae DNA Load in Blood as a Marker of Infection in Patients with Community-Acquired Pneumonia

ABSTRACT Direct detection of Streptococcus pneumoniae DNA in blood adds to culture results in the etiological diagnosis of patients with community-acquired pneumonia (CAP). Quantification of the amount of DNA, the bacterial DNA load (BDL), provides a measurement of DNAemia that may increase the understanding of the clinical relevance of S. pneumoniae DNA in blood. We evaluated the S. pneumoniae BDL as a diagnostic tool in adult patients with CAP. The BDL was determined in whole-blood samples collected simultaneously with blood for culture from 45 adult patients with CAP. After DNA extraction, S. pneumoniae DNA was detected with specific real-time PCR amplification, and the BDL was calculated with a standard curve. PCR and microbiological results were compared, and the BDL was related to clinical and laboratory parameters. S. pneumoniae DNA was detected in 10/13 patients with positive blood cultures and in 67% of patients with microbiologically confirmed pneumococcal pneumonia. The positive predictive values of the receiver operating characteristic curves for the BDLs for pneumococcal infection (100%) and pneumococcal bacteremia (69%) were higher than those for the level of C-reactive protein (CRP; 43% and 23%, respectively) and the white blood cell count (WBC; 42% and 35%, respectively); the negative predictive values of these three parameters were in the same range (±90 and ±97%, respectively). The BDL was higher in patients presenting with systemic inflammatory response syndrome and in patients with bacteremia. Positive correlations were observed for the BDL with WBC, CRP level, and length of stay. We conclude that the BDL supports the diagnosis of S. pneumoniae infection in patients with CAP and provides a putative marker of the severity of disease.

Improved detection of microbial DNA after bead-beating before DNA isolation

Detection of microbial DNA in clinical samples by real-time PCR requires a universal extraction method with equally efficient lysis of cell walls of all possible microorganisms. We demonstrated that physical disruption by bead-beating with 0.1mm beads in combination with MagNA Pure DNA III extraction enhances microbial lysis of diverse Gram-positive microorganisms and may be used to optimize DNA extraction protocols in routine clinical diagnostics.

High Detection Rates of Enteropathogens in Asymptomatic Children Attending Day Care

Background Gastroenteritis morbidity is high among children under the age of four, especially amongst those who attend day care. Objective To determine the prevalence of a range of enteropathogens in the intestinal flora of children attending day care and to relate their occurrence with characteristics of the sampled child and the sampling season. Methods We performed three years of enteropathogen surveillance in a network of 29 child day care centers in the Netherlands. The centers were instructed to take one fecal sample from ten randomly chosen children each month, regardless of gastrointestinal symptoms at time of sampling. All samples were analyzed for the molecular detection of 16 enteropathogenic bacteria, parasites and viruses by real-time multiplex PCR. Results Enteropathogens were detected in 78.0% of the 5197 fecal samples. Of the total, 95.4% of samples were obtained from children who had no gastroenteritis symptoms at time of sampling. Bacterial enteropathogens were detected most often (most prevalent EPEC, 19.9%), followed by parasitic enteropathogens (most prevalent: D. fragilis, 22.1%) and viral enteropathogens (most prevalent: norovirus, 9.5%). 4.6% of samples related to children that experienced symptoms of gastroenteritis at time of sampling. Only rotavirus and norovirus were significantly associated with gastroenteritis among day care attendees. Conclusions Our study indicates that asymptomatic infections with enteropathogens in day care attendees are not a rare event and that gastroenteritis caused by infections with these enteropathogens is only one expression of their presence.

Emergence of Escherichia coli encoding Shiga toxin 2f in human Shiga toxin-producing E-coli (STEC) infections in the Netherlands, January 2008 to December 2011

The Shiga toxins of Shiga toxin-producing Escherichia coli (STEC) can be divided into Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2) with several sub-variants. Variant Stx2f is one of the latest described, but has been rarely associated with symptomatic human infections. In the enhanced STEC surveillance in the Netherlands, 198 STEC O157 cases and 351 STEC non-O157 cases, including 87 stx2f STEC isolates, were reported between 2008 and 2011. Most stx2f strains belonged to the serogroups O63:H6 (n=47, 54%), O113:H6 (n=12, 14%) and O125:H6 (n=12, 14%). Of the 87 stx2f isolates, 84 (97%) harboured the E. coli attaching and effacing (eae) gene, but not the enterohaemorrhagic E. coli haemolysin (hly) gene. stx2f STEC infections show milder symptoms and a less severe clinical course than STEC O157 infections. Almost all infections with stx2f (n=83, 95%) occurred between June and December, compared to 170/198 (86%) of STEC O157 and 173/264 (66%) of other STEC non-O157. stx2f STEC infections in the Netherlands are more common than anticipated, and form a distinct group within STEC with regard to virulence genes and the relatively mild disease.

Gastroenteritis Attributable to 16 Enteropathogens in Children Attending Day Care Significant Effects of Rotavirus, Norovirus, Astrovirus, Cryptosporidium and Giardia

Background: Children attending day care experience substantial gastrointestinal morbidity due to circulating seasonal enteropathogens in the day-care environment. The lack of a distinct clinical presentation of gastroenteritis (GE) in these children, in combination with the high diversity of enteropathogenic agents, complicates the assessment of the individual contributions of enteropathogens that may cause GE. We aimed to estimate the proportion of day-care attendees experiencing GE that could be attributed to a range of enteropathogens circulating in day care in the Netherlands in 2010–2013. Methods: Using time-series data from a national laboratory-based and syndrome-based surveillance system in Dutch day-care centers and generalized estimating equation analysis, we modelled the variation in prevalence of 16 enteropathogens of bacterial (8), viral (5) and parasitic origin (3) circulating in day care to the variation of GE incidence among children attending day care. Results: Rotavirus, norovirus, astrovirus, Giardia and Cryptosporidium were significantly associated with GE morbidity among day-care attendees in our time-series analysis. Together, these enteropathogens accounted for 39% of the GE morbidity: 11% by rotavirus, 10% by norovirus, 8% by Giardia, 7% by astrovirus and 3% by Cryptosporidium. Conclusions: We demonstrate that circulating viruses and parasites, rather than bacteria, contribute to seasonal GE experienced by children in day care.

Prevalence and Risk Factors for Colonization With Extended-Spectrum Cephalosporin-Resistant Escherichia coli in Children Attending Daycare Centers: A Cohort Study in the Netherlands.

BACKGROUND The purpose of this study was to determine the prevalence and risk factors for colonization with extended-spectrum cephalosporin-resistant (ESC-R) Escherichia coli in daycare center (DCC)-attending children. METHODS This is a prospective cohort study including 44 DCCs in the Netherlands, combining DCC characteristics and monthly collected stool samples from their attendees, and was performed in 2010-2012. During a 22-month study period, 852 stool samples were collected and screened for ESC-R E coli. Risk factors were studied using logistic regression analysis. RESULTS In DCC-attending children (<4 years old), the overall prevalence of ESC-R E coli was 4.5%, and it was 8% in <1-year-old attendees. Among the 38 children carrying ESC-R E coli, the most common types were blaCMY-2 (26%), blaCTX-M-1 (16%), and chromosomal AmpC type 3 promoter mutants (13%). Extended-spectrum cephalosporin-resistant E coli was less common in DCCs where stricter hygiene protocols were enforced, eg, not allowing ill children to enter the DCC (odds ratio [OR], 0.34; 95% confidence interval [CI], 0.14-0.84), performing extra checks on handwashing of ill children (OR, 0.42; 95% CI, 0.20-0.87), and reporting suspected outbreaks to local health authorities (OR, 0.27; 95% CI, 0.11-0.69). CONCLUSIONS The distribution of ESC-R E coli types in DCCs differs from that of the general population. Extended-spectrum cephalosporin-resistant E coli carriage in DCC-attending children is associated with the hygiene policies enforced in the DCC. Although our results are not conclusive enough to change current DCC practice beyond ensuring compliance with standing policies, they generated hypotheses and defined the degree of ESC resistance among DCC attendees, which may influence empiric antibiotic therapy choices, and tracked the increasing trend in ESC resistance.

Multicenter evaluation of molecular and culture-dependent diagnostics for Shigella species and Entero-invasive Escherichia coli in the Netherlands

An inter-laboratory collaborative trial for the evaluation of diagnostics for detection and identification of Shigella species and Entero-invasive Escherichia coli (EIEC) was performed. Sixteen Medical Microbiological Laboratories (MMLs) participated. MMLs were interviewed about their diagnostic methods and a sample panel, consisting of DNA-extracts and spiked stool samples with different concentrations of Shigella flexneri, was provided to each MML. The results of the trial showed an enormous variety in culture-dependent and molecular diagnostic techniques currently used among MMLs. Despite the various molecular procedures, 15 out of 16 MMLs were able to detect Shigella species or EIEC in all the samples provided, showing that the diversity of methods has no effect on the qualitative detection of Shigella flexneri. In contrast to semi quantitative analysis, the minimum and maximum values per sample differed by approximately five threshold cycles (Ct-value) between the MMLs included in the study. This indicates that defining a uniform Ct-value cut-off for notification to health authorities is not advisable.