Unraveling the Modes of Transmission of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) During a Nursing Home Outbreak: Looking Beyond the Church Superspreading Event.

BACKGROUND: An outbreak of coronavirus disease 2019 (COVID-19) in a nursing home in the Netherlands, following an on-site church service held on 8 March 2020, triggered an investigation to unravel sources and chain(s) of transmission. METHODS: Epidemiological data were collected from registries and through a questionnaire among church attendees. Symptomatic residents and healthcare workers (HCWs) were tested for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by reverse-transcription polymerase chain reaction and subjected to whole genome sequencing (WGS). Sequences from a selection of people from the same area were included as community reference. RESULTS: After the church service, 30 of 39 attendees (77%) developed symptoms; 14 (11 residents and 3 nonresidents) were tested and were positive for COVID-19. In the following 5 weeks, 62 of 300 residents (21%) and 30 of 640 HCWs (5%) tested positive for COVID-19; 21 of 62 residents (34%) died. The outbreak was controlled through a cascade of measures. WGS of samples from residents and HCWs identified a diversity of sequence types, grouped into 8 clusters. Seven resident church attendees all were infected with distinct viruses, 4 of which belonged to 2 larger clusters in the nursing home. CONCLUSIONS: Although initial investigation suggested the church service as the source of the outbreak, detailed analysis showed a more complex picture, most consistent with widespread regional circulation of the virus in the weeks before the outbreak, and multiple introductions into the nursing home before the visitor ban. The findings underscore the importance of careful outbreak investigations to understand SARS-CoV-2 transmission to develop evidence-based mitigation measures.

Contact investigations for antibiotic-resistant bacteria: a mixed-methods study of patients' comprehension of and compliance with self-sampling requests post-discharge.

BACKGROUND: Contact investigation is an important tool to identify unrecognized patients who are colonized with antibiotic-resistant bacteria. Many Dutch hospitals include already discharged contact patients by sending them a self-sampling request at home, incl. an information letter and sampling materials. Each hospital composes these information letters on their own initiative, however, whether discharged patients comprehend and comply with these requests remains unclear. Therefore, the aim was to provide insight into patients' comprehension of and self-reported compliance with self-sampling requests post-discharge. METHODS: This mixed-methods study was performed in eight Dutch hospitals. First, the Common European Framework of Reference (CEFR) language level of self-sampling request letters was established. Second, a questionnaire about patients' comprehension of the letter, self-reported compliance, and reasons for compliance or non-compliance were sent to patients that received such a request in 2018/2019. Finally, a random selection of questionnaire respondents was interviewed between January and March 2020 to gain additional insights. RESULTS: CEFR levels of 15 letters were established. Four letters were assigned level B1, four letters B1-B2, and seven letters B2. The majority of patients reported good comprehension of the letter they had received. Conversely, some respondents indicated that information about the bacterium (18.4%), the way in which results would be communicated (18.1%), and the self-sampling instructions (9.7%) were (partially) unclear. Furthermore, self-reported compliance was high (88.8%). Reasons to comply were personal health (84.3%), the health of others (71.9%), and general patient safety (96.1%). Compliant patients appeared to have a need for confirmation, wanted to protect family and/or friends, and felt they were providing the hospital the ability to control the transmission of antibiotic-resistant bacteria. Although a limited number of non-compliant patients responded to the questionnaire, it seemed that more patients did not comply with self-sampling requests when they received a letter in a higher CEFR-level (B2) compared to a lower CEFR-level (< B2) (9.8% vs. 2.5%, P = 0.049). CONCLUSIONS: This study showed an overall good comprehension of and high self-reported compliance with self-sampling requests post-discharge. Providing balanced information in self-sampling request letters has the potential to reduce patient's ambiguity and concerns, and can cause increased compliance with self-sampling requests.

Seroprevalence of varicella-zoster virus and predictors for seronegativity in the Amsterdam adult population.

BACKGROUND: In the Netherlands, infection with varicella-zoster virus (VZV) is considered a benign common childhood illness and routine vaccination against VZV is not done. In 1995 it was estimated that 98-100% of the adult Dutch general population is immune, yet the estimate is based on a database in which a relative small number of people of non-Dutch ethnic origin were represented. As the city of Amsterdam has large immigrant communities originating from various subtropical and tropical countries, such as Morocco, Surinam, and Turkey with probably lower VZV transmission, this study aimed to estimate the seroprevalence of VZV IgG antibodies (anti-VZV) among various ethnic groups in Amsterdam, and identify factors associated with seronegative VZV status. METHODS: The study was a cross-sectional survey of the Amsterdam population (2004), and the study sample was stratified by age and ethnicity, with deliberate oversampling of minority ethnic groups. Serum samples obtained from 1,341 residents in 2004 were tested for antibodies to VZV. Basic demographic data (gender, age, country of birth, age at immigration and number of children) were also available. RESULTS: The anti-VZV seroprevalence in the overall Amsterdam population was estimated to be 94% (95% confidence intervals; 92-96%). Regarding ethnic origin, first generation immigrants (Moroccan immigrants 90%, Surinamese or Antillean immigrants 91%, and Turkish 92%), especially those that migrated after the age of 11 years, were more likely to be anti-VZV seronegative compared to those arriving at an earlier age or those born in the Netherlands (97-98%). Both ethnic origin and generation of immigration were positive predictors for IgG seronegativity to VZV (p<0.015). No other predictors for seronegativity were found. CONCLUSION: The results of this study imply that about 4-8% of the general adult Amsterdam population is still susceptible to infection with VZV, and that susceptibility is even higher in some immigrant groups. When assessing the risk of infection after VZV exposure alertness is needed for vulnerable persons like pregnant women, patients with hematological malignancies or organ transplants in particular among first-generation immigrants.

Increased seroprevalence of IgG-class antibodies against cytomegalovirus, parvovirus B19, and varicella-zoster virus in women working in child day care.

BACKGROUND: Primary maternal infection with cytomegalovirus (CMV), parvovirus B19 (B19V), and varicella-zoster virus (VZV) may result in adverse pregnancy outcomes like congenital infection or foetal loss. Women working in child day care have an increased exposure to CMV, B19V, and VZV. By comparing the seroprevalence of IgG-class antibodies against CMV, VZV and B19V in female day care workers (DCW) with the seroprevalence in women not working in day care this study aimed to assess the association between occupation and infection. METHODS: A cross-sectional design was used. Out of a random sample of 266 day care centres, demographic data, data on work history, and blood samples were collected from 285 women from 38 centres. In addition, blood samples and basic demographics from women who participated in a cross-sectional survey of the Amsterdam population (2004) were used. All blood samples were tested for IgG-class antibodies against CMV, B19V, and VZV. RESULTS: Twenty-seven percent of the DCW were still susceptible to B19V or CMV. Working in day care was independently associated with B19V infection in all DCW (prevalence ratio [PR] 1.2; 95 % CI 1.1-1.3), and with CMV infection in DCW of European origin only (PR 1.7; 95 % CI 1.3-2.3). Almost all women born outside Europe tested seropositive for CMV (96 %). All DCW tested seropositive for VZV, compared to only 94 % of the women not working in day care. CONCLUSION: This study confirms the clear association between employment in child day care centres and infection with CMV and B19V. Intervention policies, like screening of new employees and awareness campaigns emphasizing hygienic measures among DCW, should be implemented urgently to improve the maternal health of these women and the health of their offspring.

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.

Real-time PCR with an internal control for detection of all known human adenovirus serotypes.

The "gold standard" for the diagnosis of adenovirus (AV) infection is virus culture, which is rather time-consuming. Especially for immunocompromised patients, in whom severe infections with AV have been described, rapid diagnosis is important. Therefore, an internally controlled AV real-time PCR assay detecting all known human AV serotypes was developed. Primers were chosen from the hexon region, which is the most conserved region, and in order to cover all known serotypes, degenerate primers were used. The internal control (IC) DNA contained the same primer binding sites as the AV DNA control but had a shuffled probe region compared to the conserved 24-nucleotide consensus AV hexon probe region (the target). The IC DNA was added to the clinical sample in order to monitor extraction and PCR efficiency. The sensitivity and the linearity of the AV PCR were determined. For testing the specificity of this PCR assay for human AVs, a selection of 51 AV prototype strains and 66 patient samples positive for other DNA viruses were tested. Moreover, a comparison of the AV PCR method described herein with culture and antigen (Ag) detection was performed with a selection of 151 clinical samples. All 51 AV serotypes were detected in the selection of AV prototype strains. Concordant results from culture or Ag detection and PCR were found for 139 (92.1%) of 151 samples. In 12 cases (7.9%), PCR was positive while the culture was negative. In conclusion, a sensitive, internally controlled nonnested AV real-time PCR assay which is able to detect all known AV serotypes with higher sensitivity than a culture or Ag detection method was developed.

Multiple-locus variable-number tandem repeat analysis of Neisseria meningitidis yields groupings similar to those obtained by multilocus sequence typing.

We identified many variable-number tandem repeat (VNTR) loci in the genomes of Neisseria meningitidis serogroups A, B, and C and utilized a number of these loci to develop a multiple-locus variable-number tandem repeat analysis (MLVA). Eighty-five N. meningitidis serogroup B and C isolates obtained from Dutch patients with invasive meningococcal disease and seven reference strains were analyzed using MLVA and multilocus sequence typing (MLST). MLVA, based on eight VNTR loci with limited variability in the number of repeats, yielded clustering of the strains similar to that obtained by MLST, with congruence between both methods amounting to 69%. The ability to recognize clonal complexes makes MLVA a valuable high-throughput method to serve as a tool complementary to MLST. Four highly variable VNTR loci were used in a second assay to analyze N. meningitidis serogroup C strains collected during an outbreak of meningococcal disease in The Netherlands. Typing based on the latter VNTR loci enabled differentiation of isolates with identical MLST sequence types and grouped epidemiologically related strains.

Cryptococcal breast abscess.

A cryptococcal abscess of the breast is uncommon and may mimic a neoplastic lesion. We describe a patient with an isolated cryptococcal infection of the breast, which was treated with oral fluconazole in combination with surgical excision. With the exception of diabetes mellitus type II, no underlying predisposing illness was identified.

Highly sensitive assay for detection of enterovirus in clinical specimens by reverse transcription-PCR with an armored RNA internal control.

The objective of the present study was the development of a diagnostic reverse transcription (RT)-PCR for the specific detection of enterovirus (EV) RNA in clinical specimens controlled by an internal control (IC) RNA. The IC RNA contains the same primer binding sites as EV RNA but has a different probe region. The IC RNA was packaged into an MS2 phage core particle (armored) and was added to the clinical sample to allow monitoring of both extraction efficiency and RT-PCR efficiency. Serial dilutions of the IC RNA were made, and the detection limit of the RT-PCR was tested in a background of EV RNA-negative cerebrospinal fluid. The sensitivity and specificity of the RT-PCR assay were tested by using all 64 known EV serotypes, several non-EV serotypes, and two Quality Control for Molecular Diagnostics (QCMD) Program EV proficiency panels from 2001 and 2002. In total, 322 clinical specimens were tested by RT-PCR, and to establish the clinical utility of the RT-PCR, a comparison of the results of viral culture and RT-PCR was done with 87 clinical specimens. The lower limit of sensitivity was reached at about 150 copies of IC RNA/ml. All 64 EV serotypes were positive, while all non-EV serotypes were negative. All culture-positive samples of the 2001 QCMD proficiency panel (according to the 50% tissue culture infective doses per milliliter) were positive by RT-PCR. Invalid results, i.e., negativity for both EV RNA and IC RNA, due to inhibition of RT-PCR were observed for 33.3% of the members of the 2002 QCMD proficiency panel and 3.1% of the clinical specimens. Inhibition of RT-PCR could be relieved by the addition of 400 ng of bovine alpha-casein per microl to both the RT reaction mixture and the PCR mixture. With this optimized protocol, the results for all samples of the 2002 QCMD proficiency panel and all clinical specimens except one fecal sample (0.3%) were valid. Evaluation of the clinical samples demonstrated that EV infection could be detected in 12 of 87 samples (13.8%) by RT-PCR, while viral culture was negative. Our data show that the RT-PCR with armored IC RNA offers a very reliable and rapid diagnostic tool for the detection of EV in clinical specimens and that the addition of bovine alpha-casein relieved inhibition of the RT-PCR for 99.7% of clinical specimens.