An International External Quality Assessment Scheme to Assess the Diagnostic Performance of Polymerase Chain Reaction Detection of Acanthamoeba Keratitis.

PURPOSE: The purpose of this study was to assess the variation in methods and to determine whether an External Quality Assessment Scheme (EQAS) for polymerase chain reaction (PCR) detection of Acanthamoeba keratitis is valuable for the diagnostic process. METHODS: A multicenter EQAS was introduced, covering 16 diagnostic laboratories. Using Acanthamoeba castellanii ATCC strain 30010, 3 sets of samples were prepared, containing different amounts of DNA, cysts, or trophozoites. Samples were masked and sent to the participants with instructions for use and a questionnaire concerning the applied methodologies. Special attention in this questionnaire was given to the used pretreatment methods to assess existing variations in these procedures. RESULTS: A large variation in the methodologies and substantial differences in the diagnostic performance were found between participants. In contrast to the DNA samples where all participants had a perfect score, several false negative results were reported for the samples containing cysts or trophozoites. Only 9 participants had an optimal score, whereas one participant reported all samples as negative, one participant reported failures due to inhibition, and the other 5 reported in total 7 false negative results. A clear correlation was noticed between the PCR detection rate and the number of cysts or trophozoites in the sample. CONCLUSIONS: The results indicate that a pretreatment procedure can be a risky step in PCR-based detections of Acanthamoeba , but it improves the sensitivity and reliability, especially of samples containing cysts. Therefore, participation in an EQAS is informative for routine diagnostic laboratories and can assist in improving the laboratory procedures used for the diagnosis of Acanthamoeba keratitis.

In Vitro Effectiveness of Soft Contact Lens Solutions Available on the Dutch Market against Acanthamoeba Species.

Acanthamoeba keratitis is almost universally associated with contact lens (CL) use. Until today, however, CL solution manufacturing protocols lack testing of anti-amoebic activity. This study investigates the effectiveness of CL solutions available on the Dutch market against trophozoites and cysts of Acanthamoeba castellanii and Acanthamoeba polyphaga. Sixteen CL solutions were tested: 13 multiple purpose solutions (MPS), 2 hydrogen peroxidase solutions (HPS) and 1 povidone-iodine-based solution (PIS). The Spearman-Karber (SK) log reduction method and an XTT colorimetric assay were used to evaluate the effectiveness at the manufacturer's minimum recommended disinfection time (MMRDT) and after eight hours. At the MMRDT, one MPS showed an SK mean log reduction (MLR) of >3.0 against A. castellanii trophozoites. Two additional MPS and both HPS reached this threshold after eight hours. The SK MLR values for A. polyphaga trophozoites were between 1 and 3 at all time points. Using the XTT colorimetric assay, only HPS 1 showed >99.9% reduction (equivalent to 3 log reduction) in metabolic activity of A. castellanii trophozoites after eight hours. For A. polyphaga, both HPS and PIS showed a metabolic reduction of >99.9% after eight hours. Cysts were resistant against all solutions. We conclude that following the manufacturer's guidelines, few solutions provide sufficient effectiveness against Acanthamoeba trophozoites and none against cysts. The results underline the importance of adequate hygiene when handling CLs.

Signalling and responding to zoonotic threats using a One Health approach: a decade of the Zoonoses Structure in the Netherlands, 2011 to 2021.

In the Netherlands, the avian influenza outbreak in poultry in 2003 and the Q fever outbreak in dairy goats between 2007 and 2010 had severe consequences for public health. These outbreaks led to the establishment of an integrated human-veterinary risk analysis system for zoonoses, the Zoonoses Structure. The aim of the Zoonoses Structure is to signal, assess and control emerging zoonoses that may pose a risk to animal and/or human health in an integrated One Health approach. The Signalling Forum Zoonoses (SO-Z), the first step of the Zoonoses Structure, is a multidisciplinary committee composed of experts from the medical, veterinary, entomology and wildlife domains. The SO-Z shares relevant signals with professionals and has monthly meetings. Over the past 10 years (June 2011 to December 2021), 390 different signals of various zoonotic pathogens in animal reservoirs and humans have been assessed. Here, we describe the Zoonoses Structure with examples from signals and responses for four zoonotic events in the Netherlands (tularaemia, Brucella canis, West Nile virus, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)). This may serve as an example for other countries on how to collaborate in a One Health approach to signal and control emerging zoonoses.

Pseudo-Outbreak of Bordetella parapertussis Caused by Contaminated Swabs in the Netherlands.

An increase in positive Bordetella parapertussis tests among patients in a teaching hospital in the Netherlands resulted in enhanced infection control and microbiological surveillance. Further analysis revealed that batches of contaminated nasopharyngeal swabs were associated with a pseudo-outbreak, resulting in incorrect diagnoses, antimicrobial treatments, isolation precautions, and public health notifications.

Collaborative Studies for the Detection of Taenia spp. Infections in Humans within CYSTINET, the European Network on Taeniosis/Cysticercosis.

Laboratory tools for diagnosing taeniosis/cysticercosis in non-endemic countries are available; however, there is little data on their performance. To provide information on the sensitivity, specificity, and reproducibility of these tools, inter-laboratory studies were organized within the EU COST-Action CYSTINET (TD1302). Two serological and one coprological Ring Trials (RTs) were organized to test a panel of human-derived sera and stool samples using assays routinely conducted by the participating laboratories to detect Taenia spp. infections. Four Western blots (WBs) and five ELISAs were used by nine laboratories for cysticercosis diagnosis. In the first serological RT, the overall sensitivity was 67.6% (95% CI, 59.1-75.4), whereas specificity was 97% (95% CI, 89.8-99.6). WBs recorded the best accuracy. A second serological RT was organized, to assess the three tests most frequently used during the first RT. Two out of six laboratories performed all the three tests. The overall sensitivity and specificity were 52.8% (95% CI, 42.8-62.7) and 98.1% (95% CI, 93.2-99.7), respectively. Laboratory performance strongly affected test results. Twelve laboratories participated in the coprological RT using conventional microscopy and six laboratories used molecular assays. Traditional diagnosis by microscopy yielded better results than molecular diagnosis. This may have been influenced by the lack of standardization of molecular tests across participating laboratories.

Rapid assessment of regional SARS-CoV-2 community transmission through a convenience sample of healthcare workers, the Netherlands, March 2020.

To rapidly assess possible community transmission in Noord-Brabant, the Netherlands, healthcare workers (HCW) with mild respiratory complaints and without epidemiological link (contact with confirmed case or visited areas with active circulation) were tested for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Within 2 days, 1,097 HCW in nine hospitals were tested; 45 (4.1%) were positive. Of six hospitals with positive HCW, two accounted for 38 positive HCW. The results informed local and national risk management.

Risk Factors for Sporadic Cryptosporidiosis in the Netherlands: Analysis of a 3-Year Population Based Case-Control Study Coupled With Genotyping, 2013-2016.

BACKGROUND: In 2012, cryptosporidiosis cases increased in the Netherlands, but no single source was identified. In April 2013, we began a 3-year population-based case-control study coupled with genotyping to identify risk factors for sporadic cryptosporidiosis. METHODS: Cryptosporidium cases were laboratory confirmed (by microscopy or polymerase chain reaction), and the species (ie, C. hominis or C. parvum) was determined. We analyzed data by study year, combined and by species. We performed single-variable analysis, and variables with a P value of ≤ .10 were included in a multivariable logistic regression model adjusting for age, sex, and season. RESULTS: The study included 609 cases and 1548 frequency-matched controls. C. parvum was the predominant species in the first 2 study years, shifting to C. hominis in the third year. Household person-to-person transmission and eating barbequed food were strongly associated with being a case. Eating tomatoes was negatively associated. When the analysis was stratified by study year, person-to-person transmission was an independent risk factor. Analysis by species identified different risk factors for cases infected with C. parvum and C. hominis. CONCLUSION: This was the first case-control study examining risk factors for sporadic cryptosporidiosis in the Netherlands. Providing information about Cryptosporidium exposure during outdoor activities and improvements in hygiene within households could prevent future sporadic infections.

Diagnosis of intestinal parasites in a rural community of Venezuela: Advantages and disadvantages of using microscopy or RT-PCR.

A cross-sectional study was carried out to determine the prevalence and diagnostic performance of microscopy and real time PCR (RT-PCR) for 14 intestinal parasites in a Venezuelan rural community with a long history of persistent intestinal parasitic infections despite the implementation of regular anthelminthic treatments. A total of 228 participants were included in this study. A multiplex RT-PCR was used for the detection of Dientamoeba fragilis, Giardia intestinalis, Cryptosporidium sp. and a monoplex RT-PCR for Entamoeba histolytica. Furthermore, a multiplex PCR was performed for detection of Ascaris lumbricoides, Strongyloides stercoralis, Necator americanus and Ancylostoma duodenale. Combined microscopy-PCR revealed prevalences of 49.3% for A. lumbricoides, 10.1% for N. americanus (no A. duodenale was detected), 2.0% for S. stercoralis, 40.4% for D. fragilis, 35.1% for G. intestinalis, and 7.9% for E. histolytica/dispar. Significant increases in prevalence at PCR vs. microscopy were found for A. lumbricoides, G. intestinalis and D. fragilis. Other parasites detected by microscopy alone were Trichuris trichiura (25.7%), Enterobius vermicularis (3.4%), Blastocystis sp. (65.8%), and the non-pathogenic Entamoeba coli (28.9%), Entamoeba hartmanni (12.3%), Endolimax nana (19.7%) and Iodamoeba bütschlii (7.5%). Age- but no gender-related differences in prevalences were found for A. lumbricoides, T. trichiura, G. intestinalis, and E. histolytica/dispar. The persistently high prevalences of intestinal helminths are probably related to the high faecal pollution as also evidenced by the high prevalences of non-pathogenic intestinal protozoans. These results highlight the importance of using sensitive diagnostic techniques in combination with microscopy to better estimate the prevalence of intestinal parasites, especially in the case of D. fragilis trophozoites, which deteriorate very rapidly and would be missed by microscopy. In addition, the differentiation between the pathogenic E. histolytica and the non-pathogenic E. dispar can be attained. However, microscopy remains an important diagnostic tool since it can detect other intestinal parasites for which no PCR is available.

Seroepidemiology of human Toxocara and Ascaris infections in the Netherlands.

Toxocara canis, Toxocara cati and Ascaris suum are worldwide-distributed zoonotic roundworms of dogs, cats and pigs, respectively. The epidemiology of these parasites in developed countries is largely unclear. Two countrywide cross-sectional serosurveys were therefore conducted in the Netherlands in 1995/1996 and 2006/2007 to investigate the prevalence, trends and risk factors for human Toxocara and Ascaris infections in the general population. The Netherlands is characterized by high pig production, freedom from stray dogs and virtual absence of autochthonous infections with the human-adapted roundworm Ascaris lumbricoides. Over the 10 years between the two serosurveys, Toxocara seroprevalence decreased significantly from 10.7 % (n = 1159) to 8.0 % (n = 3683), whereas Ascaris seroprevalence increased significantly from 30.4 % (n = 1159) to 41.6 % (n = 3675), possibly reflecting concomitant improvements in pet hygiene management and increased exposure to pig manure-contaminated soil. Increased anti-Toxocara IgGs were associated with increasing age, male gender, contact with soil, ownership of cats, cattle or pigs, hay fever, low education, high income and non-Western ethnic origin. Increased anti-Ascaris IgGs were associated with increasing age, owning pigs, low education, childhood geophagia and non-Dutch ethnic origin. Besides identifying specific groups at highest risk of Toxocara and Ascaris infections, our results suggest that these infections mainly occur through environmental, rather than foodborne, routes, with direct contact with soil or cat and pig ownership being potentially modifiable exposures.

Societal Burden and Correlates of Acute Gastroenteritis in Families with Preschool Children.

Gastrointestinal infection morbidity remains high amongst preschool children in developed countries. We investigated the societal burden (incidence, healthcare utilization, and productivity loss) and correlates of acute gastroenteritis (AGE) in families with preschoolers. Monthly for 25 months, 2000 families reported AGE symptoms and related care, productivity loss, and risk exposures for one preschooler and one parent. Amongst 8768 child-parent pairs enrolled, 7.3% parents and 17.4% children experienced AGE (0.95 episodes/parent-year and 2.25 episodes/child-year). Healthcare utilization was 18.3% (children) and 8.6% (parents), with 1.6% children hospitalized. Work absenteeism was 55.6% (median 1.5 days) and day-care absenteeism was 26.2% (median 1 day). Besides chronic enteropathies, antacid use, non-breastfeeding, and toddling age, risk factors for childhood AGE were having developmental disabilities, parental occupation in healthcare, multiple siblings, single-parent families, and ≤ 12-month day-care attendance. Risk factors for parental AGE were female gender, having multiple or developmentally-disabled day-care-attending children, antimicrobial use, and poor food-handling practices. Parents of AGE-affected children had a concurrent 4-fold increased AGE risk. We concluded that AGE-causing agents spread widely in families with preschool children, causing high healthcare-seeking behaviours and productivity losses. Modifiable risk factors provide targets for AGE-reducing initiatives. Children may acquire some immunity to AGE after one year of day-care attendance.

Intervention strategies to reduce human Toxoplasma gondii disease burden.

Infection with Toxoplasma gondii is acquired through consumption of undercooked infected meat, or by uptake of cat-shed oocysts. Although congenital toxoplasmosis is generally considered to contribute most to the disease burden of T. gondii, ocular disease from acquired infection was recently shown to add substantially to the burden. In addition, toxoplasmosis in immune-compromised individuals usually results from reactivation of an infection acquired earlier in life. Nevertheless, prevention of toxoplasmosis commonly targets mainly pregnant women. We summarize current prevention strategies of congenital toxoplasmosis and evaluate options to improve protection of the general population (including pregnant women). To protect the general population, freezing of meat destined for raw or undercooked consumption is the most readily applicable option, especially when limited to meat from animals originating from nonbiosecure husbandry systems. In the long term, more health benefits are expected from cat vaccination; therefore, development of a cat vaccine and evaluation of its implementation is a research priority.

Gastroenteritis attributable to 16 enteropathogens in children attending day care: significant effects of rotavirus, norovirus, astrovirus, Cryptosporidium and Giardia.

BACKGROUND: Children attending day care experience substantial gastrointestinal morbidity due to circulating seasonal enteropathogens in the day-care environment. The lack of a distinct clinical presentation of gastroenteritis (GE) in these children, in combination with the high diversity of enteropathogenic agents, complicates the assessment of the individual contributions of enteropathogens that may cause GE. We aimed to estimate the proportion of day-care attendees experiencing GE that could be attributed to a range of enteropathogens circulating in day care in the Netherlands in 2010-2013. METHODS: Using time-series data from a national laboratory-based and syndrome-based surveillance system in Dutch day-care centers and generalized estimating equation analysis, we modelled the variation in prevalence of 16 enteropathogens of bacterial (8), viral (5) and parasitic origin (3) circulating in day care to the variation of GE incidence among children attending day care. RESULTS: Rotavirus, norovirus, astrovirus, Giardia and Cryptosporidium were significantly associated with GE morbidity among day-care attendees in our time-series analysis. Together, these enteropathogens accounted for 39% of the GE morbidity: 11% by rotavirus, 10% by norovirus, 8% by Giardia, 7% by astrovirus and 3% by Cryptosporidium. CONCLUSIONS: We demonstrate that circulating viruses and parasites, rather than bacteria, contribute to seasonal GE experienced by children in day care.

High detection rates of enteropathogens in asymptomatic children attending day care.

BACKGROUND: Gastroenteritis morbidity is high among children under the age of four, especially amongst those who attend day care. OBJECTIVE: To determine the prevalence of a range of enteropathogens in the intestinal flora of children attending day care and to relate their occurrence with characteristics of the sampled child and the sampling season. METHODS: We performed three years of enteropathogen surveillance in a network of 29 child day care centers in the Netherlands. The centers were instructed to take one fecal sample from ten randomly chosen children each month, regardless of gastrointestinal symptoms at time of sampling. All samples were analyzed for the molecular detection of 16 enteropathogenic bacteria, parasites and viruses by real-time multiplex PCR. RESULTS: Enteropathogens were detected in 78.0% of the 5197 fecal samples. Of the total, 95.4% of samples were obtained from children who had no gastroenteritis symptoms at time of sampling. Bacterial enteropathogens were detected most often (most prevalent EPEC, 19.9%), followed by parasitic enteropathogens (most prevalent: D. fragilis, 22.1%) and viral enteropathogens (most prevalent: norovirus, 9.5%). 4.6% of samples related to children that experienced symptoms of gastroenteritis at time of sampling. Only rotavirus and norovirus were significantly associated with gastroenteritis among day care attendees. CONCLUSIONS: Our study indicates that asymptomatic infections with enteropathogens in day care attendees are not a rare event and that gastroenteritis caused by infections with these enteropathogens is only one expression of their presence.

How safe is the meat inspection based on artificial digestion of pooled samples for Trichinella in pork? A scenario from wildlife to a human patient in a non-endemic region of Europe.

The occurrence of trichinellosis in a resident of the Netherlands prompted us to examine the likelihood of this originating from infected rats in spite of prevailing biosecurity and testing procedures. In so doing, we sought to calculate the possible risks for trichinellosis in countries deemed non-endemic. The infection risk was determined by simulating a scenario from a reservoir of minimally contaminated wildlife to pigs to humans. Results indicate that humans might become infected even in the event that artificial digestion had been performed on individually tested pig carcasses. Our conclusions justify reconsidering Trichinella control strategies based on the current testing protocol, and emphasize the importance of proper cooking as further insurance against human infection.

Science without meritocracy. Discrimination among European specialists in infectious diseases and clinical microbiology: a questionnaire survey.

OBJECTIVE: In 2009, in a European survey, around a quarter of Europeans reported witnessing discrimination or harassment at their workplace. The parity committee from the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) designed a questionnaire survey to investigate forms of discrimination with respect to country, gender and ethnicity among medical professionals in hospitals and universities carrying out activities in the clinical microbiology (CM) and infectious diseases (ID) fields. DESIGN: The survey consisted of 61 questions divided into five areas (sociodemographic, professional census and environment, leadership and generic) and ran anonymously for nearly 3 months on the ESCMID website. SUBJECTS: European specialists in CM/ID. RESULTS: Overall, we included 1274 professionals. The majority of respondents (68%) stated that discrimination is present in medical science. A quarter of them reported personal experience with discrimination, mainly associated with gender and geographic region. Specialists from South-Western Europe experienced events at a much higher rate (37%) than other European regions. The proportion of women among full professor was on average 46% in CM and 26% in ID. Participation in high-level decision-making committees was significantly (>10 percentage points) different by gender and geographic origin. Yearly gross salary among CM/ID professionals was significantly different among European countries and by gender, within the same country. More than one-third of respondents (38%) stated that international societies in CM/ID have an imbalance as for committee member distribution and speakers at international conferences. CONCLUSIONS: A quarter of CM/ID specialists experienced career and research discrimination in European hospitals and universities, mainly related to gender and geographic origin. Implementing proactive policies to tackle discrimination and improve representativeness and balance in career among CM/ID professionals in Europe is urgently needed.

Glycan microarray profiling of parasite infection sera identifies the LDNF glycan as a potential antigen for serodiagnosis of trichinellosis.

Diagnostic methods for parasite infections still highly depend on the identification of the parasites by direct methods such as microscopic examination of blood, stool and tissue biopsies. Serodiagnosis is often carried out to complement the direct methods; however, few synthetic antigens with sufficient sensitivity and specificity are available. Here we evaluated a glycan microarray approach to select for synthetic glycan antigens that could be used for serodiagnosis of parasitic infections. Using a glycan array containing over 250 different glycan antigens, we identified GalNAcß1-4(Fucα1-3)GlcNAc-R (LDNF) as a glycan antigen that is recognized by antibodies from Trichinella-infected individuals. We synthesized a neoglycoconjugate, consisting of five LDNF molecules covalently coupled to bovine serum albumin (BSA), and used this neoglycoconjugate as an antigen to develop a highly sensitive total-Ig ELISA for serological screening of trichinellosis. The results indicate that glycan microarrays constitute a promising technology for fast and specific identification of parasite glycan antigens to improve serodiagnosis of different parasitic infections, either using an ELISA format, or parasite-specific glycan arrays.

[Decline of echinococcosis in the Netherlands; 1997-2008]. / Afname van echinokokkose in Nederland; 1997-2008.

OBJECTIVE: To establish the prevalence of human echinococcosis in the Netherlands by using data from laboratories carrying out diagnostic procedures and data from pathology registries from 1997-2008. DESIGN: Descriptive. METHOD: Data on serological diagnostic tests for Echinococcus granulosus carried out from 1997 to 2008 were gathered from the National Institute for Public Health and the Environment (RIVM) in Bilthoven and Leiden University Medical Center (LUMC). Additionally, all echinococcosis patients registered on the pathology database of the Dutch pathological anatomy national automated archive (PALGA) were analysed. RESULTS: A total of 7314 serum samples from 5125 patients were examined for antibodies. Cyst material from 39 patients was examined using molecular methods. The number of serum samples sent in annually was stable at 550 to 600. Over the period investigated, 1997-2008, on serological investigation a total of 485 patients were found to have a positive result on IgG-ELISA. Of these, the diagnosis of echinococcosis was confirmed in 445 patients by further serological investigation (on average 37 new patients each year (range: 19-59)) and/or a positive PCR result. Over the duration of the study period the number of new patients decreased from over 40 to fewer than 30 patients per year. Going by the family name, 95.5% of the 445 patients were probably imported cases of disease. CONCLUSION: In the Netherlands, echinococcosis is primarily seen as an imported disease with the majority of patients originating from areas around the Mediterranean Sea where it is endemic. Each year there are nearly 30 confirmed cases.

Toxoplasma gondii infection in animal-friendly pig production systems.

PURPOSE: Consumption of undercooked pork meat products has been considered a major risk factor for contracting toxoplasmosis in humans. Indoor farming and improved hygiene have drastically reduced Toxoplasma infections in pigs over the past decades. Whether introduction of animal-friendly production systems will lead to a reemergence of Toxoplasma infections in pigs is not yet known. Investigating this possibility was the purpose of this study. METHODS: Blood was obtained from pigs raised for slaughter and tested for Toxoplasma antibodies by using latex agglutination and indirect immunofluorescence testing, with confirmation by immunoblotting. RESULTS: None of the slaughter pigs (n = 621) from conventional farms (n = 30) were positive, whereas 38 (2.9%) of 1295 animals from animal-friendly systems tested positive (n = 33 farms; 13 [39%] farms positive). CONCLUSIONS: The following conclusions may be derived from this study: Conventionally (indoors) raised pigs are free from Toxoplasma infection, and (2) animal-friendly production systems may lead to a reemergence of Toxoplasma infections, although many of these farms remain Toxoplasma free. Slaughterhouse monitoring of pigs from animal-friendly production systems combined with on-farm prevention strategies should be applied to ensure safety for consumers of the meat products obtained from these animals.