Arnold Catsburg

Comparison of Pathogen DNA Isolation Methods from Large Volumes of Whole Blood to Improve Molecular Diagnosis of Bloodstream Infections

For patients suffering from bloodstream infections (BSI) molecular diagnostics from whole blood holds promise to provide fast and adequate treatment. However, this approach is hampered by the need of large blood volumes. Three methods for pathogen DNA isolation from whole blood were compared, i.e. an enzymatic method (MolYsis, 1–5 ml), the novel non-enzymatic procedure (Polaris, 1–5 ml), and a method that does not entail removal of human DNA (Triton-Tris-EDTA EasyMAG, 200 µl). These methods were evaluated by processing blood spiked with 0–1000 CFU/ml of Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. Downstream detection was performed with real-time PCR assays. Polaris and MolYsis processing followed by real-time PCRs enabled pathogen detection at clinically relevant concentrations of 1–10 CFU/ml blood. By increasing sample volumes, concurrent lower cycle threshold (Ct) values were obtained at clinically relevant pathogen concentrations, demonstrating the benefit of using larger blood volumes. A 100% detection rate at a concentration of 10 CFU/ml for all tested pathogens was obtained with the Polaris enrichment, whereas comparatively lower detection rates were measured for MolYsis (50–67%) and EasyMAG (58–79%). For the samples with a concentration of 1 CFU/ml Polaris resulted in most optimal detection rates of 70–75% (MolYsis 17–50% and TTE-EasyMAG 20–36%). The Polaris method was more reproducible, less labour intensive, and faster (45 minutes (including Qiagen DNA extraction) vs. 2 hours (MolYsis)). In conclusion, Polaris and MolYsis enrichment followed by DNA isolation and real-time PCR enables reliable and sensitive detection of bacteria and fungi from 5 ml blood. With Polaris results are available within 3 hours, showing potential for improved BSI diagnostics.

Real-Time PCR Detection of Ruminant DNA

To control the spread of bovine spongiform encephalopathy, several DNA methods have been described for the detection of the species origin of meat and bone meal. Most of these methods are based on the amplification of a mitochondrial DNA segment. We have developed a semiquantitative method based on real-time PCR for detection of ruminant DNA, targeting an 88-bp segment of the ruminant short interspersed nuclear element Bov-A2. This method is specific for ruminants and is able to detect as little as 10 fg of bovine DNA. Autoclaving decreased the amount of detectable DNA, but positive signals were observed in feeding stuff containing 10% bovine material if this had not been rendered in accordance with the regulations, i.e., heated at 134 degrees C for 3 instead of 20 min.

Mannose-binding lectin gene polymorphisms in relation to periodontitis

AIM To investigate the correlation of six functional polymorphisms in the MBL gene with MBL plasma levels in relation to periodontitis. MATERIAL AND METHODS A total of 92 periodontitis patients and 70 controls, all of Caucasian origin, were included. Patients and controls were genotyped for the L/H, X/Y, P/Q, A/D, A/B and A/C polymorphisms. Distributions of genotypes, rate of allele carriage and allele frequencies were compared between patients and controls. Patients and controls were subdivided in groups of genotypes. Plasma MBL levels were compared between different genotype groups. RESULTS On the basis of genotyping, three phenotypes with regard to mannose-binding lectin (MBL) production were distinguished: high-producers, low-producers and deficient subjects. No differences in the genotype frequencies were observed between patients and controls. Within patients and controls, subjects with the high-producing genotypes had significantly higher MBL plasma levels than low-producers and deficient subjects (p<0.001). Plasma MBL was higher in low-producer patients compared with low-producer controls (p(adjusted)=0.021). CONCLUSION No association could be observed between MBL gene polymorphisms and susceptibility to periodontitis in Caucasians. However, now that genotyping could distinguish the low producing and deficient subjects from the high-producers, it was observed, for the first time, that MBL acts as a weak acute-phase protein in periodontitis.

Development of a multiplex real-time PCR assay for the rapid diagnosis of neonatal late onset sepsis

The diagnosis of late onset sepsis (LOS), a severe condition with high prevalence in preterm infants, is hampered by the suboptimal sensitivity and long turnaround time of blood culture. Detection of the infecting pathogen directly in blood by PCR would provide a much more timely result. Unfortunately, PCR-based assays reported so far are labor intensive and often lack direct species identification. Therefore we developed a real-time multiplex PCR assay tailored to LOS diagnosis which is easy-to-use, is applicable on small blood volumes and provides species-specific results within 4h. Species-specific PCR assays were selected from literature or developed using bioinformatic tools for the detection of the most prevalent etiologic pathogens: Enterococcus faecalis, Staphylococcus aureus, Staphylococcus spp., Streptococcus agalactiae, Escherichia coli, Pseudomonas aeruginosa, Klebsiella spp. and Serratia marcescens. The PCR assays showed 100% specificity, full coverage of the target pathogens and a limit of detection (LOD) of ≤10CFUeq./reaction. These LOD values were maintained in the multiplex format or when bacterial DNA was isolated from blood. Clinical evaluation showed high concordance between the multiplex PCR and blood culture. In conclusion, we developed a multiplex PCR that allows the direct detection of the most important bacterial pathogens causing LOS in preterm infants.

Mannose-binding lectin in term newborns and their mothers : Genotypic and phenotypic relationship

Functional mannose-binding lectin (f-MBL) plays an important role in the innate neonatal immune system. We studied the origin of f-MBL in umbilical cord blood (UCB) by measuring maternal MBL (n=47), collected before elective cesarean section, and neonatal MBL (n=43) in arterial umbilical cord blood. In a subgroup, arterial and venous UCB MBL levels were measured. In addition, MBL expression was correlated with genetic mutations. The f-MBL levels in term infants were lower than in their mothers (0.70 microg/ml vs 1.11 microg/ml, p<0.01) and maternal and neonatal MBL levels were only weakly correlated (R=0.32, p<0.001), which suggests a fetal origin of f-MBL. Arterial and venous UCB median MBL levels did not differ (0.98 microg/ml vs. 1.40 microg/ml, p=0.20). No homozygous mutations were found. MBL was lower in mothers and infants with a (compound) heterozygous mutation than in those with a wild type. One new (HYPB) and two rare haplotypes (HXPA, LYPD) were reported in our population. Levels of MBL differed depending on the genotype of the mother or the infant. Because the role of MBL in host defense is still unclear, both f-MBL and haplotype should be measured to determine the clinical implications of MBL deficiency in infants.

P1-S1.30 Chlamydia trachomatis prevalence and detection in men attending the urologist's office to get tested for sexually transmitted infections in St Petersburg

Objectives The data about the prevalence sexually transmitted infection (STI) as Chlamydia trachomatis (CT) among the Russian population is limited and controversial. This information is of great scientific and health care interest. The aim of the study was to evaluate the prevalence of genital CT infection among male attendees of the urologists office in STI clinics in St. Petersburg and the role of molecular tests in low-resource settings. Methods The prospective, multicenter study was undertaken throughout urologists offices in St. Petersburg during the January 2007—December 2009 timeframe. Urethral samples from 907 men (mean age 31.7 years), who were seeking to be routinely tested for STIs and with no HIV, gonorrhoea, syphilis and Trichomonas vaginalis detected in the time of study, were collected to be tested for CT infection by culture and in-house RT-PCR assays in St. Petersburg and to be confirmed in Amsterdam. Results The results are presented in the Abstract P1-S1.30 table 1. In total CT infection was found in 6.4% of men tested by RT-PCR. Urethral specimens were tested by culture and RT-PCR assays for CT finding positivity rates of 2.2% (n=466 culture samples) and 7.6% (n=804 RT-PCR samples). Use of only culture test would result in missing up to 60% of CT+ cases (p<0.0001). Symptoms were presented in 48% of CT+ men. CT was less often detected in men reporting previous CT infection, as compared with first CT infection—4.3% vs 7.4% (p=0.0475). Only 14/907 (1.5%) questioned men openly reported being MSM but CT prevalence in this small group was 28.6% (p<0.0001). CT positivity assessed in St. Petersburg by culture and in-house RT-PCR tests was confirmed in Amsterdam by a molecular CE marked CT test. Abstract P1-S1.30 Table 1 Chlamydia trachomatis prevalence, detected by culture and RT-PCR tests, among male attendees of the urologists office in St. Petersburg No men tested Chlamydia trachomatis (CT) detection CT− CT+ CT prevalence In total 907 849 58 6.4% Culture test, only 466 456 10 2.2% RT-PCR test, only 804 747 57 7.6% Culture and RT-PCR, both 376 351 25 6.7% Conclusions Our study showed that—(1) CT prevalence among the Russian population is still high especially in MSM. (2) Risk factor include—being symptomatic at the time of testing (p=0.0043), inconsistent condom use and practicing sex with men (both—p<0.0001). (3) All samples found culture and/or RT-PCR CT+ in Russia were confirmed CT+ using molecular biological techniques in Amsterdam, showing the validity of CT detection in this study in St. Petersburg. At the moment we additionally—A) extent.