A targeted LC-MRM3 proteomic approach for the diagnosis of SARS-CoV-2 infection in nasopharyngeal swabs.

Since its first appearance, SARS-CoV-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 (NCAP). A highly selective multiple reaction monitoring 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 NCAP peptides from nasopharyngeal swabs samples presenting a cycle threshold (Ct) value down to 35 with sensitivity and specificity of 94.2% and 100.0%, without immuno-purification. 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 Ct-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 mass spectrometry.

Epidemiologic and Genomic Analysis of SARS-CoV-2 Delta Variant Superspreading Event in Nightclub, the Netherlands, June 2021.

We report a severe acute respiratory syndrome coronavirus 2 superspreading event in the Netherlands after distancing rules were lifted in nightclubs, despite requiring a negative test or vaccination. This occurrence illustrates the potential for rapid dissemination of variants in largely unvaccinated populations under such conditions. We detected subsequent community transmission of this strain.

National point prevalence study on carriage of multidrug-resistant microorganisms in Dutch long-term care facilities in 2018.

OBJECTIVES: Long-term care facilities (LTCFs) may act as a reservoir of ESBL-producing Enterobacterales (ESBL-E) and carbapenemase-producing Enterobacterales (CPE) for hospitals and the general population. In this study, we estimated the prevalence and molecular epidemiology of rectal carriage with ESBL-E and CPE in residents of Dutch LTCFs between March 2018 and December 2018. METHODS: LTCFs were geographically selected across the country. For each LTCF, a random sample of residents were tested for ESBL-E and CPE in 2018. To identify risk factors for high carriage prevalence and/or individual carriage, characteristics of LTCFs and of a subset of the tested residents were collected. WGS was conducted on isolates from LTCFs with an ESBL-E prevalence of >10% and all CPE isolates to identify institutional clonal transmission. RESULTS: A total of 4420 residents of 159 LTCFs were included. The weighted mean ESBL-E prevalence was 8.3% (95% CI: 6.8-10.0) and no CPE were found. In 53 LTCFs (33%), where ESBL-E prevalence was >10%, MLST using WGS (wgMLST) was performed. This included 264 isolates, the majority being Escherichia coli (n = 224) followed by Klebsiella pneumoniae (n = 30). Genetic clusters were identified in more than half (30/53; 57%) of high ESBL-positive LTCFs. Among the E. coli isolates, blaCTX-M-15 (92/224; 41%) and blaCTX-M-27 (40/224; 18%) were the most prevalent ESBL-encoding genes. For K. pneumoniae isolates, the most common was blaCTX-M-15 (23/30; 80%). CONCLUSIONS: The estimated prevalence of ESBL-E rectal carriage in Dutch LTCFs is 8.3% and resistance is observed mainly in E. coli with predominance of blaCTX-M-15 and blaCTX-M-27. ESBL-E prevalence in LTCFs seems comparable to previously reported prevalence in hospitals and the general population.

Hospitalisation rates differed by city district and ethnicity during the first wave of COVID-19 in Amsterdam, The Netherlands.

BACKGROUND: It is important to gain insight into the burden of COVID-19 at city district level to develop targeted prevention strategies. We examined COVID-19 related hospitalisations by city district and migration background in the municipality of Amsterdam, the Netherlands. METHODS: We used surveillance data on all PCR-confirmed SARS-CoV-2 hospitalisations in Amsterdam until 31 May 2020, matched to municipal registration data on migration background. We calculated directly standardised (age, sex) rates (DSR) of hospitalisations, as a proxy of COVID-19 burden, per 100,000 population by city district and migration background. We calculated standardised rate differences (RD) and rate ratios (RR) to compare hospitalisations between city districts of varying socio-economic and health status and between migration backgrounds. We evaluated the effects of city district and migration background on hospitalisation after adjusting for age and sex using Poisson regression. RESULTS: Between 29 February and 31 May 2020, 2326 cases (median age 57 years [IQR = 37-74]) were notified in Amsterdam, of which 596 (25.6%) hospitalisations and 287 (12.3%) deaths. 526/596 (88.2%) hospitalisations could be matched to the registration database. DSR were higher in individuals living in peripheral (South-East/New-West/North) city districts with lower economic and health status, compared to central districts (Centre/West/South/East) (RD = 36.87,95%CI = 25.79-47.96;RR = 1.82,95%CI = 1.65-1.99), and among individuals with a non-Western migration background compared to ethnic-Dutch individuals (RD = 57.05,95%CI = 43.34-70.75; RR = 2.36,95%CI = 2.17-2.54). City district and migration background were independently associated with hospitalisation. CONCLUSION: City districts with lower economic and health status and those with a non-Western migration background had the highest burden of COVID-19 during the first wave of COVID-19 in Amsterdam.

Low prevalence of current and past SARS-CoV-2 infections among visitors and staff members of homelessness services in Amsterdam at the end of the second wave of infections in the Netherlands.

BACKGROUND: People experiencing homelessness (PEH) may be at increased risk of SARS-CoV-2 infection and severe COVID-19. The Dutch government established emergency shelters and introduced preventive measures for homelessness services. There were no major SARS-CoV-2 outbreaks noticed among PEH during the first two waves of infections. This study aimed to assess the prevalence of current and past infections among PEH and staff by conducting an on-site COVID-19 screening project at homelessness services in Amsterdam, the Netherlands. METHODS: We assessed the proportion of visitors and staff members of four homelessness services at two locations in Amsterdam with positive SARS-CoV-2 qPCR and antibody results (IgG/IgM Rapid Test/Biozek) in May 2021. We also assessed sociodemographic, clinical and lifestyle characteristics, compliance with basic prevention measures and intention to vaccinate against COVID-19 among PEH and staff. RESULTS: A total of 138 visitors and 53 staff members filled out a questionnaire and were tested. Among PEH, the SARS-CoV-2 positivity rate was 0% (0/133;95%CI = 0-1.9) and the antibody positivity rate was 1.6% (2/131;95%CI = 0.8-7.5) among those without prior COVID-19 vaccination. Among staff, these percentages were 3% (1/32;95%CI = 0.1-16.2) and 11% (5/53;95%CI = 3.6-23.6), respectively. Most participants were often compliant with the basic preventive measures 'not shaking hands', 'wearing a face mask' and 'washing hands', but not with 'physical distancing'. High vaccination intent was more common among staff members (55%) than among visitors (42%), while high trust in the governmental COVID-19 policies was more common among visitors (41%) than among staff (30%). CONCLUSIONS: We observed a low prevalence of past and current SARS-CoV-2 infections among PEH, which may be explained by instated shelter policies, limited daily activities of PEH and compliance with prevention measures. Vaccine hesitancy and mistrust among visitors and staff could hinder vaccination uptake, suggesting that interventions towards homelessness services are needed.

Age-dependent patterns of infection and severity explaining the low impact of 2009 influenza A (H1N1): evidence from serial serologic surveys in the Netherlands.

Despite considerable research efforts in specific subpopulations, reliable estimates of the infection attack rates and severity of 2009 influenza A (H1N1) in the general population remain scarce. Such estimates are essential to the tailoring of future control strategies. Therefore, 2 serial population-based serologic surveys were conducted, before and after the 2009 influenza A (H1N1) epidemic, in the Netherlands. Random age-stratified samples were obtained using a 2-stage cluster design. Participants donated blood and completed a questionnaire. Data on sentinel general practitioner-attended influenza-like illness and nationwide hospitalization and mortality were used to assess the severity of infection. The estimated infection attack rates were low in the general population (7.6%, 95% confidence interval: 3.6, 11) but high in children aged 5-19 years (35%, 95% confidence interval: 25, 45). The estimated hospitalization and mortality rates per infection increased significantly with age (5-19 years: 0.042% and 0.00094%, respectively; 20-39 years: 0.12% and 0.0025%; 40-59 years: 0.68% and 0.032%; 60-75 years: >0.81% and >0.068%). The high infection attack rate in children and the very low attack rate in older adults, together with the low severity of illness per infection in children but substantial severity in older adults, produced an epidemic with a low overall impact.

HI responses induced by seasonal influenza vaccination are associated with clinical protection and with seroprotection against non-homologous strains.

Vaccination against influenza induces homologous as well as cross-specific hemagglutination inhibiting (HI) responses. Induction of cross-specific HI responses may be essential when the influenza strain does not match the vaccine strain, or even to confer a basic immune response against a pandemic influenza virus. We carried out a clinical study to evaluate the immunological responses after seasonal vaccination in healthy adults 18-60 years of age, receiving the yearly voluntary vaccination during the influenza season 2006/2007. Vaccinees of different age groups were followed for laboratory confirmed influenza (LCI) and homologous HI responses as well as cross-specific HI responses against the seasonal H1N1 strain of 2008 and pandemic H1N1 virus of 2009 (H1N1pdm09) were determined. Homologous HI titers that are generally associated with protection (i.e. seroprotective HI titers ≥40) were found in more than 70% of vaccinees. In contrast, low HI titers before and after vaccination were significantly associated with seasonal LCI. Cross-specific HI titers ≥40 against drifted seasonal H1N1 were found in 69% of vaccinees. Cross-specific HI titers ≥40 against H1N1pdm09 were also significantly induced, especially in the youngest age group. More specifically, cross-specific HI titers ≥40 against H1N1pdm09 were inversely correlated with age. We did not find a correlation between the subtype of influenza which was circulating at the age of birth of the vaccinees and cross-specific HI response against H1N1pdm09. These data indicate that the HI titers before and after vaccination determine the vaccination efficacy. In addition, in healthy adults between 18 and 60 years of age, young adults appear to be best able to mount a cross-protective HI response against H1N1pdm09 or drifted seasonal influenza after seasonal vaccination.

Utility of the first few100 approach during the 2009 influenza A(H1N1) pandemic in the Netherlands.

BACKGROUND: To guide policy and control measures, decent scientific data are needed for a comprehensive assessment of epidemiological, clinical and virological characteristics of the First Few hundred (FF100) cases. We discuss the feasibility of the FF100 approach during the 2009 pandemic and the added value compared with alternative data sources available. METHODS: The pandemic preparedness plan enabled us to perform a case-control study, assessing patient characteristics and risk factors for experiencing symptomatic influenza A(H1N1)2009 infection and providing insight into transmission. We assessed to what extent timely and novel data were generated compared to other available data sources. RESULTS: In May-December 2009, a total of 68 cases and 48 controls were included in the study. Underlying non-respiratory diseases were significantly more common among cases compared to controls, while a protective effect was found for frequent hand washing. Seroconversion was found for 7/30 controls (23%), and persisting high titers for 4/30 controls (13%). The labour-intensive study design resulted in slow and restricted recruitment. CONCLUSIONS: The findings of our case-control study gave new insights in transmission risks and possible interventions for improved control. Nevertheless, the FF100 approach lacked timeliness and power due to limited recruitment. For future pandemics we suggest pooling data from several countries, to enable collecting sufficient data in a relatively short period.

Course of pandemic influenza A(H1N1) 2009 virus infection in Dutch patients.

The clinical dynamics of influenza A(H1N1) 2009 infections in 61 laboratory-confirmed Dutch cases were examined. An episode lasted a median of 7·5 days of which 2 days included fever. Respiratory symptoms resolved slowly, while systemic symptoms peaked early in the episode and disappeared quickly. Severity of each symptom was rated highest in the first few days. Furthermore, diarrhoea was negatively associated with viral load, but not with faecal excretion of influenza virus. Cases with comorbidities appeared to have higher viral loads than the cases without, suggesting a less effective immune response. These results complement information obtained through traditional surveillance.

Oseltamivir-resistant pandemic A(H1N1) 2009 influenza viruses detected through enhanced surveillance in the Netherlands, 2009-2010.

Enhanced surveillance of infections due to the pandemic A(H1N1) influenza virus, which included monitoring for antiviral resistance, was carried out in the Netherlands from late April 2009 through late May 2010. More than 1100 instances of infection with the pandemic A(H1N1) influenza virus from 2009 and 2010 [A(H1N1) 2009] distributed across this period were analyzed. Of these, 19 cases of oseltamivir-resistant virus harboring the H275Y mutation in the neuraminidase (NA) were detected. The mean 50% inhibitory concentration (IC50) levels for oseltamivir- and zanamivir-susceptible A(H1N1) 2009 viruses were 1.4-fold and 2-fold, respectively, lower than for the seasonal A(H1N1) influenza viruses from 2007/2008; for oseltamivir-resistant A(H1N1) 2009 virus the IC50 was 2.9-fold lower. Eighteen of the 19 patients with oseltamivir-resistant virus showed prolonged shedding of the virus and developed resistance while on oseltamivir therapy. Sixteen of these 18 patients had an immunodeficiency, of whom 11 had a hematologic disorder. The two other patients had another underlying disease. Six of the patients who had an underlying disease died; of these, five had received cytostatic or immunosuppressive therapy. No indications for onward transmission of resistant viruses were found. This study showed that the main association for the emergence of cases of oseltamivir-resistant A(H1N1) 2009 virus was receiving antiviral therapy and having drug-induced immunosuppression or an hematologic disorder. Except for a single case of a resistant virus not linked to oseltamivir therapy, the absence of detection of resistant variants in community specimens and in specimens from contacts of cases with resistant virus suggested that the spread of resistant A(H1N1) 2009 virus was limited. Containment may have been the cumulative result of impaired NA function, successful isolation of the patients, and prophylactic measures to limit exposure.

Pathogenic characteristics of persistent feline enteric coronavirus infection in cats.

Feline coronaviruses (FCoV) comprise two biotypes: feline enteric coronaviruses (FECV) and feline infectious peritonitis viruses (FIPV). FECV is associated with asymptomatic persistent enteric infections, while FIPV causes feline infectious peritonitis (FIP), a usually fatal systemic disease in domestic cats and some wild Felidae. FIPV arises from FECV by mutation. FCoV also occur in two serotypes, I and II, of which the serotype I viruses are by far the most prevalent in the field. Yet, most of our knowledge about FCoV infections relates to serotype II viruses, particularly about the FIPV, mainly because type I viruses grow poorly in cell culture. Hence, the aim of the present work was the detailed study of the epidemiologically most relevant viruses, the avirulent serotype I viruses. Kittens were inoculated oronasally with different doses of two independent FECV field strains, UCD and RM. Persistent infection could be reproducibly established. The patterns of clinical symptoms, faecal virus shedding and seroconversion were monitored for up to 10 weeks revealing subtle but reproducible differences between the two viruses. Faecal virus, i.e. genomic RNA, was detected during persistent FECV infection only in the large intestine, downstream of the appendix, and could occasionally be observed also in the blood. The implications of our results, particularly our insights into the persistently infected state, are discussed.

Transmission of novel influenza A(H1N1) in households with post-exposure antiviral prophylaxis.

BACKGROUND: Despite impressive advances in our understanding of the biology of novel influenza A(H1N1) virus, little is as yet known about its transmission efficiency in close contact places such as households, schools, and workplaces. These are widely believed to be key in supporting propagating spread, and it is therefore of importance to assess the transmission levels of the virus in such settings. METHODOLOGY/PRINCIPAL FINDINGS: We estimate the transmissibility of novel influenza A(H1N1) in 47 households in the Netherlands using stochastic epidemic models. All households contained a laboratory confirmed index case, and antiviral drugs (oseltamivir) were given to both the index case and other households members within 24 hours after detection of the index case. Among the 109 household contacts there were 9 secondary infections in 7 households. The overall estimated secondary attack rate is low (0.075, 95%CI: 0.037-0.13). There is statistical evidence indicating that older persons are less susceptible to infection than younger persons (relative susceptibility of older persons: 0.11, 95%CI: 0.024-0.43. Notably, the secondary attack rate from an older to a younger person is 0.35 (95%CI: 0.14-0.61) when using an age classification of 12 years, and 0.28 (95%CI: 0.12-0.50) when using an age classification of 18 years. CONCLUSIONS/SIGNIFICANCE: Our results indicate that the overall household transmission levels of novel influenza A(H1N1) in antiviral-treated households were low in the early stage of the epidemic. The relatively high rate of adult-to-child transmission indicates that control measures focused on this transmission route will be most effective in minimizing the total number of infections.

[Combating the new influenza A (H1N1) virus. I. Overview of the relevant virological aspects]. / Bestrijding van de nieuwe influenza A (H1N1). I. Overzicht van de relevante virologische aspecten.

In April 2009 a new influenza virus was discovered, which spread from Mexico to the rest of the world. The new influenza A (H1N1) virus is genetically related to swine flu viruses, and differs substantially from circulating human influenza viruses. It is able to spread from person to person. Because it is a completely new virus, there is probably little immunity in the population. The course of the infection is relatively mild, but the virus will mutate and it is not yet certain whether this will affect severity of the influenza. General practitioners have an important role in surveillance and treatment. The Community Health Services must be notified of any patients who are suspected of having the new influenza. Hygiene measures and administration of antiviral drugs to patients and their contacts may slow the spread. A delay in large-scale spread in the Netherlands allows time for the development of vaccines.

Strengthening the diagnostic capacity to detect Bio Safety Level 3 organisms in unusual respiratory viral outbreaks.

BACKGROUND: Experience with a highly pathogenic avian influenza outbreak in the Netherlands (2003) illustrated that the diagnostic demand for respiratory viruses at different biosafety levels (including BSL3), can increase unexpectedly and dramatically. OBJECTIVES: We describe the measures taken since, aimed at strengthening national laboratory surge capacity and improving preparedness for dealing with diagnostic demand during outbreaks of (emerging) respiratory virus infections, including pandemic influenza virus. STUDY DESIGN: Academic and peripheral medical-microbiological laboratories collaborated to determine minimal laboratory requirements for the identification of viruses in the early stages of a pandemic or a large outbreak of avian influenza virus. Next, an enhanced collaborative national network of outbreak assistance laboratories (OAL) was set up. An inventory was made of the maximum diagnostic throughput that this network can deliver in a period of intensified demand. For an estimate of the potential magnitude of this surge demand, historical counts were calculated from hospital- and physician-based registries of patients presenting with respiratory symptoms. RESULTS: Number of respiratory physician-visits ranged from 140,000 to 615,000 per month and hospitalizations ranged from 3000 to 11,500 per month. The established OAL-network provides rapid diagnostic response with agreed quality requirements and a maximum throughput capacity of 1275 samples/day (38,000 per month), assuming other routine diagnostic work needs to be maintained. CONCLUSIONS: Thus surge demand for diagnostics for hospitalized cases (if not distinguishable from other respiratory illness) could be handled by the OAL network. Assessing etiology of community acquired acute respiratory infection however, may rapidly exceed the capacity of the network. Therefore algorithms are needed for triaging for laboratory diagnostics; currently this is not addressed in pandemic preparedness plans.