111 research outputs found

    Vaccine-driven evolution of Bordetella pertussis: changes in population structure and strain fitness

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    Whooping cough or pertussis is a highly contagious disease of the respiratory tract, caused by Bordetella pertussis. In The Netherlands mass vaccination against B. pertussis was introduced in 1953 and was very effective in reducing the pertussis incidence and mortality. Although vaccination was initially very effective, the pertussis incidence increased remarkably after 1996 in The Netherlands. Epidemics have been reported in 1996, 1999 and 2001, while in the intermittent years the incidence did not return to levels observed previous to 1996. The goal of this thesis was to find an explanation for the re-emergence of B. pertussis in countries with highly vaccinated populations, in particular The Netherlands. The re-emergence of pertussis might be explained by changes in surveillance and diagnostic criteria, changes in vaccine quality or a decrease in vaccine coverage. However, for The Netherlands these possibilities have been excluded. Waning vaccine-induced immunity and strain adaptations are other possible explanations. Indeed, we observed that the introduction of vaccination affected the B. pertussis population significantly. Notable changes were observed in the population structure of Dutch B. pertussis strains in 2 periods: the 1960s, a decade after the introduction of vaccination and in the 1990s, the period, when pertussis re-emerged in The Netherlands. IS1002-based fingerprint typing showed a change in fingerprint types a decade after the introduction of vaccination. Fingerprint types from the prevaccination period disappeared, while novel fingerprint types emerged. We hypothesized that antigenic divergence between vaccine strains and circulating strains may be the underlying cause of the changes observed with IS1002 fingerprint typing. To address this hypothesis, we studied polymorphism in genes coding for surface proteins. In the 1960s the only mismatch we detected between the vaccine strains and circulating strains was with respect to pertussis toxin subunit 1 (PtxS1). Although we observed divergence between vaccine strains and clinical isolates, suggesting that vaccination selected for strains that were less affected by vaccine-induced immunity, vaccination remained very effective, since mortality decreased 114-fold in the late 1960s compared to the prevaccination period. The second period showing changes in the population structure was observed in the 1990s. In this period we observed divergence between vaccine strains and clinical isolates in the surface associated proteins pertactin (Prn) and tracheal colonization factor (TcfA). Polymorphism was not restricted to genes coding for surface proteins, but was also observed in regulatory genes, such as the promoter region for pertussis toxin, ptxP. The alleles of Prn, TcfA and ptxP, found in the vaccine strains, were replaced by novel variants in the 1990s. Further, animal studies indicate that vaccination has shifted the competitive balance between strains, resulting in expansion of strains that are less affected by vaccine-induced immunity. The data presented in this thesis provide an explanation for the re-emergence of pertussis and a better understanding of how pathogens evolve after the introduction of vaccines

    Studies on Prn Variation in the Mouse Model and Comparison with Epidemiological Data

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    The virulence factor pertactin (Prn) is a component of pertussis vaccines and one of the most polymorphic Bordetella pertussis antigens. After the introduction of vaccination shifts in predominant Prn types were observed and strains with the Prn vaccine type (Prn1) were replaced by strains carrying non-vaccine types (Prn2 and Prn3), suggesting vaccine-driven selection. The aim of this study was to elucidate the shifts observed in Prn variants. We show that, although Prn2 and Prn3 circulated in similar frequencies in the 1970s and 1980s, in the 1990s Prn2 strains expanded and Prn3 strains disappeared, suggesting that in vaccinated populations Prn2 strains are fitter than Prn3 strains. We established a role for Prn in the mouse model by showing that a Prn knock-out (Prn-ko) mutation reduced colonization in trachea and lungs. Restoration of the mutation resulted in a significant increase in colonization compared to the knock-out mutant. The ability of clinical isolates with different Prn variants to colonize the mouse lung was compared. Although these isolates were also polymorphic at other loci, only variation in the promoter for pertussis toxin (ptxP) and Prn were found to contribute significantly to differences in colonization. Analysis of a subset of strains with the same ptxP allele revealed that the ability to colonize mice decreased in the order Prn1>Prn2 and Prn3. Our results are consistent with the predominance of Prn1 strains in unvaccinated populations. Our results show that ability to colonize mice is practically the same for Prn2 and Prn3. Therefore other factors may have contributed to the predominance of Prn2 in vaccinated populations. The mouse model may be useful to assess and predict changes in the B. pertussis population due to vaccination

    Advancing COVID-19 diagnostics:rapid detection of intact SARS-CoV-2 using viability RT-PCR assay

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    Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). Commonly used methods for both clinical diagnosis of SARS-CoV-2 infection and management of infected patients involve the detection of viral RNA, but the presence of infectious virus particles is unknown. Viability PCR (v-PCR) uses a photoreactive dye to bind non-infectious RNA, ideally resulting in the detection of RNA only from intact virions. This study aimed to develop and validate a rapid v-PCR assay for distinguishing intact and compromised SARS-CoV-2. Propidium monoazide (PMAxx) was used as a photoreactive dye. Mixtures with decreasing percentages of intact SARS-CoV-2 (from 100% to 0%) were prepared from SARS-CoV-2 virus stock and a clinical sample. Each sample was divided into a PMAxx-treated part and a non-PMAxx-treated part. Reverse transcription-PCR (RT-PCR) using an in-house developed SARS-CoV-2 viability assay was then applied to both sample sets. The difference in intact SARS-CoV-2 was determined by subtracting the cycle threshold (Ct) value of the PMAxx-treated sample from the non-PMAxx-treated sample. Mixtures with decreasing concentrations of intact SARS-CoV-2 showed increasingly lower delta Ct values as the percentage of intact SARS-CoV-2 decreased, as expected. This relationship was observed in both high and low viral load samples prepared from cultured SARS-CoV-2 virus stock, as well as for a clinical sample prepared directly from a SARS-CoV-2 positive nasopharyngeal swab. In this study, a rapid v-PCR assay has been validated that can distinguish intact from compromised SARS-CoV-2. The presence of intact virus particles, as determined by v-PCR, may indicate SARS-CoV-2 infectiousness.</p

    The supplemental value of mammographic screening over breast MRI alone in BRCA2 mutation carriers

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    Purpose: BRCA2 mutation carriers are offered annual breast screening with MRI and mammography. The aim of this study was to investigate the supplemental value of mammographic screening over MRI screening alone. Methods: In this multicenter study, proven BRCA2 mutation carriers, who developed breast cancer during screening using both digital mammography and state-of-art breast MRI, were identified. Clinical data were reviewed to classify cases in screen-detected and interval cancers. Imaging was reviewed to assess the diagnostic value of mammography and MRI, using the Breast Imaging and Data System (BI-RADS) classification allocated at the time of diagnosis. Results: From January 2003 till March 2019, 62 invasive breast cancers and 23 ductal carcinomas in situ were diagnosed in 83 BRCA2 mutation carriers under surveillance. Overall screening sensitivity was 95.2% (81/85). Four interval cancers occurred (4.7% (4/85)). MRI detected 73 of 85 breast cancers (sensitivity 85.8%) and 42 mammography (sensitivity 49.9%) (p < 0.001). Eight mammography-only lesions occurred. In 1 of 17 women younger than 40 years, a 6-mm grade 3 DCIS, retrospectively visible on MRI, was detected with mammography only in a 38-year-old woman. The other 7 mammography-only breast cancers were diagnosed in women aged 50 years and older, increasing sensitivity in this subgroup from 79.5% (35/44) to 95.5% (42/44) (p ≤ 0.001). Conclusions: In BRCA2 mutation carriers younger than 40 years, the benefit of mammographic screening over MRI was very small. In carriers of 50 years and older, mammographic screening contributed significantly. Hence, we propose to postpone mammographic screening in BRCA2 mutation carriers to at least age 40

    Methicillin-Resistant Staphylococcus aureus in Meat Products, the Netherlands

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    A new methicillin-resistant Staphylococcus aureus (MRSA) clone related to pig and cattle farming was detected in the Netherlands. We investigated the extent of S. aureus presence in meat and found 36 S. aureus strains in 79 samples. Two strains were MRSA; 1 was multilocus sequence type 398, the clone related to farming

    The role of tunneling in enzyme catalysis of C–H activation

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    AbstractRecent data from studies of enzyme catalyzed hydrogen transfer reactions implicate a new theoretical context in which to understand C–H activation. This is much closer to the Marcus theory of electron transfer, in that environmental factors influence the probability of effective wave function overlap from donor to acceptor atoms. The larger size of hydrogen and the availability of three isotopes (H, D and T) introduce a dimension to the kinetic analysis that is not available for electron transfer. This concerns the role of gating between donor and acceptor atoms, in particular whether the system in question is able to tune distance between reactants to achieve maximal tunneling efficiency. Analysis of enzyme systems is providing increasing evidence of a role for active site residues in optimizing the inter-nuclear distance for nuclear tunneling. The ease with which this optimization can be perturbed, through site-specific mutagenesis or an alteration in reaction conditions, is also readily apparent from an analysis of the changes in the temperature dependence of hydrogen isotope effects

    Evaluation of the pharmacokinetics of prednisolone in paediatric patients with acute lymphoblastic leukaemia treated according to Dutch Childhood Oncology Group protocols and its relation to treatment response

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    Glucocorticoids form the backbone of paediatric acute lymphoblastic leukaemia (ALL) treatment. Many studies have been performed on steroid resistance; however, few studies have addressed the relationship between dose, concentration and clinical response. The aim of the present study was to evaluate the pharmacokinetics of prednisolone in the treatment of paediatric ALL and the correlation with clinical parameters. A total of 1028 bound and unbound prednisolone plasma concentrations were available from 124 children (aged 0–18 years) with newly diagnosed ALL enrolled in the Dutch Childhood Oncology Group studies. A population pharmacokinetic model was developed and post hoc area under the curve (AUC) was tested against treatment outcome parameters. The pharmacokinetics of unbound prednisolone in plasma was best described with allometric scaling and saturable binding to proteins. Plasma protein binding decreased with age. The AUC of unbound prednisolone was not associated with any of the disease parameters or treatment outcomes. Unbound prednisolone plasma concentrations correlated with age. No effect of exposure on clinical treatment outcome parameters was observed and does not substantiate individualised dosing. Poor responders, high-risk and relapsed patients showed a trend towards lower exposure compared to good responders. However, the group of poor responders was small and requires further research.</p

    Population Pharmacokinetics and Pharmacodynamics of Ciprofloxacin Prophylaxis in Pediatric Acute Lymphoblastic Leukemia Patients

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    Background. Ciprofloxacin is used as antimicrobial prophylaxis in pediatric acute lymphoblastic leukemia (ALL) to decrease infections with gram-negative bacteria. However, there are no clear guideline

    On the Early Digging of Peanut Fruits

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    1. The flowering period of peanut is about 100 days or more, which differentiates very much the respective maturity of each fruit of peanut, so it is difficult that growers catch its exact yielding time. 2. By the factors of climate, especially temperature, the flowering or maturing period is controlled, and the yielding period is from the middle of September to the end of October. 3. The effective flowering period was until the end of August, and the seeded plant at the beginning of July had its effective flowering period of only about one month, and so the July-seeded plant had only half of the product of the optimum seeded plant at the beginning of May. The seeded plant after July had the common pods, but brought no grain. 4. Immature grains decreased rapidly after about 90 days from the first flowering of each plant, or about 40 days from the maximum flowering time. 5. The plants harvested after the middle of October, which passed over the 110 days from the first flowering of each plant, produced many over-mature grains and germinating grains. 6. The early harvesting time is better than the customary time, and when the immature pods are many, the mature fruits should be harvested and the plants with immature pods should be gathered at the corner of the field and planted temporarily. After they were laid on till the frost time, the Secondly harvest should be done. 7. The optimum harvesting time was about after one month from the end of effective flowering period, or after one month and a half from the maximum of fertile percentage
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