A Targeted LC-MRM3 Proteomic Approach for the Diagnosis of SARS-CoV-2 Infection in Nasopharyngeal Swabs.

Since its first appearance, severe acute respiratory syndrome coronavirus 2 quickly spread around the world and the lack of adequate PCR testing capacities, especially during the early pandemic, led the scientific community to explore new approaches such as mass spectrometry (MS). We developed a proteomics workflow to target several tryptic peptides of the nucleocapsid protein. A highly selective multiple reaction monitoring-cubed (MRM3) strategy provided a sensitivity increase in comparison to conventional MRM acquisition. Our MRM3 approach was first tested on an Amsterdam public health cohort (alpha-variant, 760 participants) detecting viral nucleocapsid protein peptides from nasopharyngeal swabs samples presenting a cycle threshold value down to 35 with sensitivity and specificity of 94.2% and 100.0%, without immunopurification. A second iteration of the MS-diagnostic test, able to analyze more than 400 samples per day, was clinically validated on a Leiden-Rijswijk public health cohort (delta-variant, 2536 participants) achieving 99.9% specificity and 93.1% sensitivity for patients with cycle threshold values up to 35. In this manuscript, we also developed and brought the first proof of the concept of viral variant monitoring in a complex matrix using targeted MS.

High prevalence of multidrug resistant Enterobacteriaceae among residents of long term care facilities in Amsterdam, the Netherlands.

INTRODUCTION: The aim of this study was to determine the rate of asymptomatic carriage and spread of multidrug-resistant micro-organisms (MDRO) and to identify risk factors for extended spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E) carriage in 12 long term care facilities (LTCFs) in Amsterdam, the Netherlands. MATERIALS AND METHODS: From November 2014 to august 2015, feces and nasal swabs from residents from LTCFs in Amsterdam, the Netherlands were collected and analyzed for presence of multidrug-resistant Gram-negative bacteria (MDRGN), including ESBL-E, carbapenemase-producing Enterobacteriaceae (CPE), colistin-resistant Enterobacteriaceae and methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). Logistic regression analysis was performed to assess associations between variables and ESBL-carriage. RESULTS: In total, 385 residents from 12 LTCFs (range 15-48 residents per LTCF) were enrolled. The prevalence of carriage of MDRGN was 18.2% (range among LTCFs 0-47%) and the prevalence of ESBL-E alone was 14.5% (range among LTCFs: 0-34%). Of 63 MDRGN positive residents, 50 (79%) were ESBL-E positive of which 43 (86%) produced CTX-M. Among 44 residents with ESBL-E positive fecal samples of whom data on contact precautions were available at the time of sampling, only 9 (20%) were already known as ESBL-E carriers. The prevalence for carriage of MRSA was 0.8% (range per LTCF: 0-7%) and VRE 0%. One CPE colonized resident was found. All fecal samples tested negative for presence of plasmid mediated resistance for colistin (MCR-1). Typing of isolates by Amplified Fragment Length Polymorphism (AFLP) showed five MDRGN clusters, of which one was found in multiple LTCFs and four were found in single LTCFs, suggesting transmission within and between LTCFs. In multivariate analysis only the presence of MDRO in the preceding year remained a risk factor for ESBL-E carriage. CONCLUSIONS: The ESBL-carriage rate of residents in LTCFs is nearly two times higher than in the general population but varies considerably among LTCFs in Amsterdam, whereas carriage of MRSA and VRE is low. The majority (80%) of ESBL-E positive residents had not been detected by routine culture of clinical specimens at time of sampling. Current infection control practices in LTCFs in Amsterdam do not prevent transmission. Both improvement of basic hygiene, and funding for laboratory screening, should allow LTCFs in Amsterdam to develop standards of care to prevent transmission of ESBL-E.

Sexual behaviour and incidence of HIV and sexually transmitted infections among men who have sex with men using daily and event-driven pre-exposure prophylaxis in AMPrEP: 2 year results from a demonstration study.

BACKGROUND: Pre-exposure prophylaxis (PrEP) for HIV might induce risk compensation, defined as increased sexual risk behaviour leading to increased incidence of bacterial sexually transmitted infections (STIs). We examined the incidence of HIV, STIs, and sexual behaviour after initiating daily and event-driven PrEP. METHODS: AMPrEP is a demonstration study that assessed the incidence of HIV and bacterial STIs and sexual behaviour among PrEP users at the STI clinic of the Public Health Service of Amsterdam (Amsterdam, the Netherlands). Eligible adults (aged ≥18 years) were HIV-negative men who have sex with men (MSM) and transgender people who had reported one or more of the following in the previous 6 months: condomless anal sex with casual partners, at least one bacterial STI, use of post-exposure prophylaxis after a sexual risk incident, or an HIV-positive sexual partner with detectable viral load. Participants were given a choice of daily PrEP or event-driven PrEP regimens. Participants were seen every 3 months for HIV and STI testing. Participants also completed self-administered questionnaires about sexual behaviour at each visit. Over the first 2 years, we assessed the number and incidence rate of HIV and chlamydia, gonorrhoea, and syphilis, including STIs diagnosed in between study visits and assessed changes in incidence over time using piecewise exponential survival models. The number of sexual acts and sexual partners were also analysed and changes over time were assessed using negative binomial regression models. This study is ongoing; the data cutoff for this analysis was June 30, 2018. FINDINGS: Between Aug 3, 2015, and May 31, 2016, 376 participants were recruited, of whom nine participants were lost to follow-up, thus 367 participants were included in the analysis. Overall, 365 MSM and two transgender women were included, contributing 681·7 person-years of follow-up. At enrolment, 269 (73%) of 367 participants chose daily PrEP and 98 (27%) participants chose event-driven PrEP. Two individuals in the daily PrEP group became HIV-positive; overall HIV incidence rate was 0·30 per 100 person-years (95% CI 0·07-1·19). 253 participants were diagnosed with one or more STI during the first 2 years (incidence rate 90·4 per 100 person-years). STI incidence did not change over time (adjusted incidence rate ratio [aIRR] 1·00 per 3 month follow-up, 95% CI 0·93-1·07). More than a quarter of all STIs were diagnosed from tests done in between study visits. STI incidence was lower in the event-driven PrEP group than the daily PrEP group (aIRR 0·59, 95% CI 0·46-0·75). The number of condomless anal sex acts with casual partners per 3 months increased, but the number of partners and sex acts remained stable. INTERPRETATION: Although the overall incidence of STIs did not change during 2 years of PrEP use, the incidence of STIs was higher among participants using daily PrEP than those using event-driven PrEP, which is likely a result of differences in sexual behaviour. This finding suggests the need to tailor STI prevention interventions according to behavioural profiles. FUNDING: ZonMw, H-TEAM, Internal GGD research funds, Aidsfonds, Stichting AmsterdamDiner Foundation, Gilead Sciences, Janssen Pharmaceutica, M A C AIDS Fund, and ViiV Healthcare.

Serovar D and E of serogroup B induce highest serological responses in urogenital Chlamydia trachomatis infections.

BACKGROUND: Chlamydia trachomatis is the most prevalent bacterial sexually transmitted infection (STI) worldwide. A strong link between C. trachomatis serogroup/serovar and serological response has been suggested in a previous preliminary study. The aim of the current study was to confirm and strengthen those findings about serological IgG responses in relation to C. trachomatis serogroups and serovars. METHODS: The study population (n = 718) consisted of two patient groups with similar characteristics of Dutch STI clinic visitors. We performed genotyping of serovars and used titre based and quantitative commercially available ELISA kits (medac Diagnostika) to determine specific serum IgG levels. Optical density (OD) values generated by both tests were used to calculate the IgG titres (cut-off 1:50). Analyses were conducted stratified by gender. RESULTS: We observed very significant differences when comparing the median IgG titres of three serogroups, B, C and I: in women for B vs. C: p < 0.0001 (median titres B 200 vs. C <50); B vs. I: p < 0.0001 (200 vs. 50), and in men for B vs. C: p = 0.0006 (150 vs. <50); B vs. I: p = 0.0001 (150 vs. <50); C vs. I was not significant for both sexes. Serovars D and E of serogroup B had the highest median IgG titres compared to the other serovars in both men and women: 200 and 200 vs. ≤ 100 for women and 100 and 200 vs. ≤ 75 for men, respectively. CONCLUSIONS: This study shows that B group serovars induce higher serological responses compared to the C and I group serovars in vivo in both men and women.

Pharmacokinetics of clindamycin in pregnant women in the peripartum period.

The study presented here was performed to determine the pharmacokinetics of intravenously administered clindamycin in pregnant women. Seven pregnant women treated with clindamycin were recruited. Maternal blood and arterial and venous umbilical cord blood samples were obtained. Maternal clindamycin concentrations were analyzed by nonlinear mixed-effects modeling with the NONMEM program. The data were best described by a linear three-compartment model. The clearance and the volume of distribution at steady state were 10.0 liters/h and 6.32 x 10(3) liters, respectively. Monte Carlo simulations were performed to determine the area under the concentration curve (AUC) for the free (unbound) drug (f) in maternal serum for 24 h divided by the MIC (fAUC(0-24)/MIC). At a MIC of 0.5 mg/liter, which is the EUCAST breakpoint, the attainment at the lower 95% confidence interval (CI) was 24.6 if the level of protein binding was 65%, and this value concurred well with the target value of 27. However, for higher degrees of protein binding, as has been described in the literature, the attainment was lower, down to 10.2 for a protein binding level of 85% (lower 95% CI). The concentrations in umbilical cord blood were lower than those in maternal blood. The concentration-time profiles in maternal serum indicate that the level of exposure to clindamycin may be too low in these patients. Together with the lower concentrations in umbilical cord blood, this finding suggests that the current dosing regimen may not be adequate to protect all neonates from group B streptococcal disease.

Timing of group B streptococcus screening in pregnancy: a systematic review.

BACKGROUND: Group B streptococcus (GBS) is an important cause of neonatal sepsis. Guidelines advise to collect cultures at 35-37 weeks' gestation and to administer intrapartum antibiotic prophylaxis in case of GBS-positive cultures, as well as in all preterm deliveries. Improved effectiveness of antenatal cultures might help to further decrease GBS early-onset disease. OBJECTIVE: To determine the best timing of antenatal cultures, which may help establish optimal prevention of perinatal GBS infection in both term and preterm neonates. METHODS: PubMed and EMBASE databases were searched for relevant articles published from 1966 to February 2009. Nine articles were included. Information about study features and predictive values of antenatal cultures were abstracted. RESULTS: Positive predictive values for antenatal GBS cultures ranged from 43 to 100% (mean 69%) and negative predictive values from 80 to 100% (mean 94%). GBS cultures collected in late pregnancy had high positive predictive values for colonization during delivery. The negative predictive value was high and relatively constant regardless of GA. CONCLUSIONS: This systematic review confirms recommendations to screen pregnant women for colonization of GBS at 35-37 weeks' gestation, but one should be aware of the limitations of screening, with 6% of GBS carriers remaining undetected in antenatal cultures.

Pharmacokinetics of amoxicillin in maternal, umbilical cord, and neonatal sera.

The pharmacokinetics of amoxicillin were studied in umbilical cord and neonatal sera relative to maternal concentrations in prevention of neonatal group B streptococcus infection. The subjects were 44 pregnant women receiving amoxicillin as 1 or 2 g as an intravenous infusion. To measure the concentrations, blood samples were obtained from the mother, the arterial and venous umbilical cord, and the neonate. The pharmacokinetics were characterized by a five-compartment model by using nonlinear mixed-effects (population) modeling. The population estimates for the clearance, central volume of distribution, and the two peripheral maternal volumes of distribution were 19.7 +/- 0.99 liters/h, 6.40 +/- 0.61 liters, and 5.88 +/- 0.83 liters (mean +/- standard error), respectively. The volume of distribution of the venous umbilical cord and the neonatal volume of distribution were 3.40 liters and 11.9 liters, respectively. The pharmacokinetic parameter estimates were used to simulate the concentration-time profiles in maternal, venous umbilical cord, and neonatal sera. The peak concentration in the venous umbilical cord serum was 18% of the maternal peak concentration. It was reached 3.3 min after the maternal peak concentration. The concentration-time profile in neonatal serum was determined by the profile in venous umbilical cord serum, which in turn depended on the profile in maternal serum. Furthermore, the simulated concentrations in maternal, venous umbilical cord, and neonatal sera exceeded the MIC for group B streptococcus for more than 90% of the 4-h dosing interval. In a first approximation, the 2-g infusion to the mother appears to be adequate for the prevention of group B streptococcal disease. However, to investigate the efficacy of the prophylaxis, further studies of the interindividual variability in pharmacokinetics are indicated.

The influence of labour on the pharmacokinetics of intravenously administered amoxicillin in pregnant women.

AIMS: Many physiological changes take place during pregnancy and labour. These might change the pharmacokinetics of amoxicillin, necessitating adjustment of the dose for prevention of neonatal infections. We investigated the influence of labour on the pharmacokinetics of amoxicillin. METHODS: Pregnant women before and during labour were recruited and treated with amoxicillin intravenously. A postpartum dose was offered. Blood samples were obtained and amoxicillin concentrations were determined using high-pressure liquid chromatography. The pharmacokinetics were characterized by nonlinear mixed-effects modelling using NONMEM. RESULTS: The pharmacokinetics of amoxicillin in 34 patients was best described by a three-compartment model. Moderate interindividual variability was identified in CL, central and peripheral volumes of distribution. The volume of distribution (V) increased with an increasing amount of oedema. Labour influenced the parameter estimate of peripheral volume of distribution (V(2)). V(2) was decreased during labour, and even more in the immediate postpartum period. For all patients the population estimates (mean +/- SE) for CL and V were 21.1 +/- 4.1 l h(-1) (CL), 8.7 +/- 6.6 l (V(1)), 11.8 +/- 7.7 l (V(2)) and 20.5 +/- 15.4 l (V(3)) respectively. CONCLUSIONS: The peripheral distribution volume of amoxicillin in pregnant women during labour and immediately postpartum is decreased. However, these changes are not clinically relevant and do not warrant deviations from the recommended dosing regimen for amoxicillin during labour in healthy pregnant patients.

Low carriage rate of group B streptococcus in pregnant women in Maputo, Mozambique.

The prevalence of group B streptococcus (GBS) carriage varies strongly with geographical region. A study was done to determine the prevalence of GBS in women in Maputo, Mozambique. The method used was a rectovaginal swab which was taken from women between 35 and 37 weeks of pregnancy who visited the clinic for antenatal consultation. GBS was cultured from 2 out of 113 samples, yielding a prevalence of 1.8% (95% Cl: 0.0-4.0). In conclusion, the prevalence of GBS carriage among pregnant women in Maputo, Mozambique was low.

Amoxicillin pharmacokinetics in pregnant women with preterm premature rupture of the membranes.

OBJECTIVE: This study was undertaken to study the pharmacokinetics of intravenously administered amoxicillin in pregnant women with preterm premature rupture of the membranes (PPROM). STUDY DESIGN: Healthy women with PPROM were recruited and treated with amoxicillin (2 g initially and 1 g subsequently). Blood samples were obtained from the opposite arm and concentrations determined with the use of high-pressure liquid chromatography. Nonlinear mixed-effects modeling was performed in nonlinear mixed effect (population) modeling. RESULTS: The pharmacokinetics of 17 patients was described by a 3-compartment model. Clearance and volume of distribution at steady state were 22.8 L/h and 21.4 L/h, respectively, similar to values in nonpregnant individuals. There was little variability between patients. No relationship was observed between values of individual pharmacokinetic parameters and various covariates. CONCLUSION: The pharmacokinetics of amoxicillin in pregnant patients with PPROM similar to nonpregnant individuals. Given the small interindividual variability in pharmacokinetics, no dose adjustments are required to account for differences between subjects under normal circumstances.

Low rate of carriage of macrolide-resistant group B streptococci in pregnant women in The Netherlands.

OBJECTIVES: To describe prevalence of phenotypic and genotypic macrolide-resistance among GBS isolates in pregnant women and explore the possibility of clonal spread of resistant GBS isolates in a multicultural population. STUDY DESIGN: Antimicrobial resistance patterns of 107 GBS isolates obtained from asymptomatic pregnant women were determined using E-tests. Macrolide resistance genes mef(A), erm(TR) and erm(B) were determined with PCR and a subset of 39 isolates, including the 8 isolates harbouring macrolide resistance genes, was subjected to RAPD analysis to detect clonal spreading. RESULTS: Resistance to erythromycin and clindamycin was found in 8% and 7%, respectively. Macrolide resistance genes mef(A), erm(TR) and erm(B) were found in 1, 2 and 5 isolates, respectively; only five of these eight isolates exhibited both genotypic as well as phenotypic resistance. One genotype occured in 36% of the subset. CONCLUSIONS: Earlier reports on prevalence of phenotypic resistance were confirmed. Among the susceptible isolates one clonal type of GBS was clearly predominant; one of the resistant isolates shared its genotype. When such clonal types acquire resistance traits in the future, GBS disease may become harder to control.

Morbidity related to maternal group B streptococcal infections.

Group B streptococcus is known to be a leading cause of neonatal infection, but less appreciated is the fact that it causes maternal infection also. Maternal group B streptococcal infections during pregnancy and delivery threaten not only the mother, but the child as well. Postpartum infection, such as mastitis, bacteremia, sepsis, meningitis, endometritis, and wound infections are hazards to the mother. We describe the various maternal group B streptococcal infections, their characteristics, associated neonatal morbidity, and prevention and treatment strategies during pregnancy, delivery, and in the postpartum period.

Prevalence of colonisation with group B Streptococci in pregnant women of a multi-ethnic population in The Netherlands.

OBJECTIVE: This study was performed to determine the prevalence of GBS and to identify GBS colonisation risk factors in a multicultural population of pregnant women in The Netherlands. We calculated predictive values of cultures in pregnancy for intrapartum GBS carriage. STUDY DESIGN: From a total of 1702 women visiting several antenatal outpatient departments, rectovaginal swabs were collected at 35-37 weeks' gestation. In 761 women swabs were repeated at time of delivery. Carriage of GBS late in third trimester and at time of delivery was analysed in relation to age, parity, ethnicity and socio-economic status. RESULTS: Twenty-one percent was GBS carrier late in pregnancy. Compared to Europeans, African women were at a higher risk (29%, RR 1.4, CI 1.1-1.7) and Asian women were at lower risk (13%, RR 0.6, CI 0.4-0.8) for GBS carriage. No differences in colonisation were found between women with respect to age, parity or socio-economic background. Positive predictive value of GBS carriage at 35-37 weeks' gestation for carriage at time of parturition was 79% and negative predictive value was 93%. CONCLUSIONS: It was not possible to identify a group of pregnant women at high risk for GBS colonisation. Predictive values of antenatal genital group B streptococci cultures at 35-37 weeks' gestation for intrapartum GBS carriage are lower than previously reported.