Antimicrobial susceptibility profile of clinically relevant Bacteroides, Phocaeicola, Parabacteroides and Prevotella species, isolated by eight laboratories in the Netherlands.

OBJECTIVES: Recently, reports on antimicrobial-resistant Bacteroides and Prevotella isolates have increased in the Netherlands. This urged the need for a surveillance study on the antimicrobial susceptibility profile of Bacteroides, Phocaeicola, Parabacteroides and Prevotella isolates consecutively isolated from human clinical specimens at eight different Dutch laboratories. METHODS: Each laboratory collected 20-25 Bacteroides (including Phocaeicola and Parabacteroides) and 10-15 Prevotella isolates for 3 months. At the national reference laboratory, the MICs of amoxicillin, amoxicillin/clavulanic acid, piperacillin/tazobactam, meropenem, imipenem, metronidazole, clindamycin, tetracycline and moxifloxacin were determined using agar dilution. Isolates with a high MIC of metronidazole or a carbapenem, or harbouring cfiA, were subjected to WGS. RESULTS: Bacteroides thetaiotaomicron/faecis isolates had the highest MIC90 values, whereas Bacteroides fragilis had the lowest MIC90 values for amoxicillin/clavulanic acid, piperacillin/tazobactam, meropenem, imipenem and moxifloxacin. The antimicrobial profiles of the different Prevotella species were similar, except for amoxicillin, for which the MIC50 ranged from 0.125 to 16 mg/L for Prevotella bivia and Prevotella buccae, respectively. Three isolates with high metronidazole MICs were sequenced, of which one Bacteroides thetaiotaomicron isolate harboured a plasmid-located nimE gene and a Prevotella melaninogenica isolate harboured a nimA gene chromosomally.Five Bacteroides isolates harboured a cfiA gene and three had an IS element upstream, resulting in high MICs of carbapenems. The other two isolates harboured no IS element upstream of the cfiA gene and had low MICs of carbapenems. CONCLUSIONS: Variations in resistance between species were observed. To combat emerging resistance in anaerobes, monitoring resistance and conducting surveillance are essential.

Characterization of mobile genetic elements in multidrug-resistant Bacteroides fragilis isolates from different hospitals in the Netherlands.

OBJECTIVES: Five human clinical multidrug-resistant (MDR) Bacteroides fragilis isolates, including resistance to meropenem and metronidazole, were recovered at different hospitals in the Netherlands between 2014 and 2020 and sent to the anaerobic reference laboratory for full characterization. METHODS: Isolates were recovered from a variety of clinical specimens from patients with unrelated backgrounds. Long- and short-read sequencing was performed, followed by a hybrid assembly to study the presence of mobile genetic elements (MGEs) and antimicrobial resistance genes (ARGs). RESULTS: A cfxA gene was present on a transposon (Tn) similar to Tn4555 in two isolates. In two isolates a novel Tn was present with the cfxA gene. Four isolates harbored a nimE gene, located on a pBFS01_2 plasmid. One isolate contained a novel plasmid carrying a nimA gene with IS1168. The tetQ gene was present on novel conjugative transposons (CTns) belonging to the CTnDOT family. Two isolates harbored a novel plasmid with tetQ. Other ARGs in these isolates, but not on an MGE, were: cfiA, ermF, mef(EN2), and sul2. ARGs harboured differed between isolates and corresponded with the observed phenotypic resistance. CONCLUSIONS: Novel CTns, Tns, and plasmids were encountered in the five MDR B. fragilis isolates, complementing our knowledge on MDR and horizontal gene transfer in anaerobic bacteria.

Antibiotic resistance of Escherichia coli isolated from uncomplicated UTI in general practice patients over a 10-year period.

Recommendations of first choice antibiotic therapy need to be based on actual antibiotic susceptibility data. We determined the antibiotic susceptibility of E. coli in uncomplicated UTI among women and compared the results with 2004 and 2009. In 30 sentinel general practitioner practices of Nivel Primary Care database, urine samples were collected from women with symptoms of uncomplicated UTI. Patient characteristics, E. coli susceptibility, and ESBL production were analyzed. Six hundred eighty-nine urine samples were collected; E. coli was the most isolated uropathogen (83%). Antibiotic susceptibility was stable over time except for ciprofloxacin (96% in 2004, 97% in 2009, and 94% in 2014; P < 0.05). The susceptibility to co-amoxiclav was 88%, 87%, and 92% in 2004, 2009, and 2014, respectively. The prevalence of ESBL-producing E. coli increased from 0.1% in 2004 to 2.2% in 2014 (P < 0.05). Regional differences in antibiotic susceptibility for co-trimoxazole were found being the highest in the west (88%) and the lowest in the north (72%, P = 0.021). Ciprofloxacin susceptibility was related to antibiotic use in the past 3 months (97% no use versus 90% use, P = 0.002) and age > 70 years (P = 0.005). In 2014, prescription of fosfomycin increased compared to 2009 (14.3% versus 5.6%) at the expense of co-amoxiclav, co-trimoxazole, and fluoroquinolones (P < 0.05). The susceptibility percentages to most antimicrobial agents tested were stable over 10 years' period although the prevalence of E. coli and ESBLs significantly increased. Performance of a survey with regular intervals is warranted.

An Enzyme-Linked Immunosorbent Spot Assay Measuring Borrelia burgdorferi B31-Specific Interferon Gamma-Secreting T Cells Cannot Discriminate Active Lyme Neuroborreliosis from Past Lyme Borreliosis: a Prospective Study in the Netherlands.

Two-tier serology testing is most frequently used for the diagnosis of Lyme borreliosis (LB); however, a positive result is no proof of active disease. To establish a diagnosis of active LB, better diagnostics are needed. Tests investigating the cellular immune system are available, but studies evaluating the utility of these tests on well-defined patient populations are lacking. Therefore, we investigated the utility of an enzyme-linked immunosorbent spot (ELISpot) assay to diagnose active Lyme neuroborreliosis. Peripheral blood mononuclear cells (PBMCs) of various study groups were stimulated by using Borrelia burgdorferi strain B31 and various recombinant antigens, and subsequently, the number of Borrelia-specific interferon gamma (IFN-γ)-secreting T cells was measured. We included 33 active and 37 treated Lyme neuroborreliosis patients, 28 healthy individuals treated for an early manifestation of LB in the past, and 145 untreated healthy individuals. The median numbers of B. burgdorferi B31-specific IFN-γ-secreting T cells/2.5 × 105 PBMCs did not differ between active Lyme neuroborreliosis patients (6.0; interquartile range [IQR], 0.5 to 14.0), treated Lyme neuroborreliosis patients (4.5; IQR, 2.0 to 18.6), and treated healthy individuals (7.4; IQR, 2.3 to 14.9) (P = 1.000); however, the median number of B. burgdorferi B31-specific IFN-γ-secreting T cells/2.5 × 105 PBMCs among untreated healthy individuals was lower (2.0; IQR, 0.5 to 3.9) (P ≤ 0.016). We conclude that the Borrelia ELISpot assay, measuring the number of B. burgdorferi B31-specific IFN-γ-secreting T cells/2.5 × 105 PBMCs, correlates with exposure to the Borrelia bacterium but cannot be used for the diagnosis of active Lyme neuroborreliosis.

Disagreement between the results from three commercial tests for the detection of Borrelia-specific serum antibodies in the Netherlands associated with antibiotic treatment for Lyme borreliosis: a retrospective study.

The diagnosis of Lyme borreliosis is challenging because of the often non-specific symptoms and persisting antibodies after infection. We investigated the diagnostic characteristics of two enzyme-linked immunosorbent assays (ELISAs) and an immunoblot for the detection of Borrelia-specific serum antibodies using different test strategies in individuals with and without antibiotic treatment for Lyme borreliosis. This retrospective study included healthy individuals, patients with active Lyme neuroborreliosis and patients treated for Lyme neuroborreliosis. Two ELISAs were compared: the C6 ELISA and the SERION ELISA. Equivocal and positive results were confirmed by immunoblot. We included 174 healthy individuals, of whom 27 (15.5%) were treated for Lyme borreliosis in the past, 36 patients were treated for Lyme neuroborreliosis and 27 patients had active Lyme neuroborreliosis. All the active Lyme neuroborreliosis patients were reactive in both ELISAs (100% sensitivity); less reactivity was seen in the other three groups (range 17.7% to 69.4%). The concordance between the ELISA results was high in active Lyme neuroborreliosis patients (26/27; 96.3%) and healthy individuals (131/147; 89.1%), but lower in treated healthy individuals (18/27; 66.7%) and treated Lyme neuroborreliosis patients (18/36; 50.0%) (p ≤ 0.005). This study showed that antibiotic treatment against Lyme borreliosis was strongly associated with discordant ELISA and test strategy results (odds ratio: 10.52; p < 0.001 and 9.98; p = 0.014, respectively) suggesting antibiotic treatment influences the pace at which the various antibodies directed to the different antigens used in both ELISAs wane. Among treated neuroborreliosis patients, the SERION ELISA stayed positive for a longer period after infection compared to the C6 ELISA. This should be taken into consideration when requesting and/or interpreting Lyme serology.

Detection and epidemiology of carbapenemase producing Enterobacteriaceae in the Netherlands in 2013-2014.

Laboratory detection of carbapenemase-producing Enterobacteriaceae (CPE) is complicated. Screening with MIC values below clinical breakpoints followed by genotypic confirmation is recommended. We evaluated the application of recommended CPE screening and confirmation methods and provide an overview of CPE epidemiology in E. coli and K. pneumoniae in the Netherlands. Data on E. coli and K. pneumoniae isolates with elevated meropenem (>0.25 mg/L) and/or imipenem (>1 mg/L) MIC values in 2013-2014 were selected from the Infectious Disease Surveillance Information System for Antibiotic Resistance. Laboratories were requested to provide additional results of any confirmatory testing performed. Confirmation of elevated carbapenem MIC values using gradient testing was performed in 59.8 % of eligible isolates. Confirmatory testing showed elevated MIC values in 8 % of E. coli and 32 % of K. pneumoniae isolates. The overall proportion of confirmed non-susceptible E. coli and K. pneumoniae was 0.01 % and 0.16 %, respectively. Genotypic confirmation was performed in 61.0 % of isolates with confirmed elevated carbapenem MIC values. A carbapenemase gene was identified in 47 % of E. coli and 65 % of K. pneumoniae isolates. OXA-48, NDM and KPC were the most frequently found carbapenemase genes. The majority (62 %) of CPE isolates was detected through targeted screening. CPE are a rare finding in the Netherlands. Adherence to the national guideline is suboptimal and differs between laboratories, implying a risk of inadequate CPE detection. Since accurate identification of CPE is the first step in prevention of CPE spread, successful implementation of guidelines for testing and reporting of CPE is essential.

Difficulties in diagnosing terminal ileitis due to Yersinia pseudotuberculosis.

We report three patients with terminal ileitis and positive fecal cultures with Yersinia pseudotuberculosis. From one patient, a virulence plasmid (pYV)-negative Y. pseudotuberculosis was isolated, which represents the second finding of a pYV-negative isolate associated with human disease. All patients were treated with ciprofloxacin and fully recovered. Since conventional culture methods for yersiniosis are gradually replaced with molecular tests not recognizing Y. pseudotuberculosis, we recommend to include a specific culture medium or to apply a specific polymerase chain reaction (PCR) assay on fecal samples from patients suspected of terminal ileitis.

Time-course of antibody responses against Coxiella burnetii following acute Q fever.

Large outbreaks of Q fever in The Netherlands have provided a unique opportunity for studying longitudinal serum antibody responses in patients. Results are presented of a cohort of 344 patients with acute symptoms of Q fever with three or more serum samples per patient. In all these serum samples IgM and IgG against phase 1 and 2 Coxiella burnetii were measured by an immunofluorescence assay. A mathematical model of the dynamic interaction of serum antibodies and pathogens was used in a mixed model framework to quantitatively analyse responses to C. burnetii infection. Responses show strong heterogeneity, with individual serum antibody responses widely different in magnitude and shape. Features of the response, peak titre and decay rate, are used to characterize the diversity of the observed responses. Binary mixture analysis of IgG peak levels (phases 1 and 2) reveals a class of patients with high IgG peak titres that decay slowly and may represent potential chronic cases. When combining the results of mixture analysis into an odds score, it is concluded that not only high IgG phase 1 may be predictive for chronic Q fever, but also that high IgG phase 2 may aid in detecting such putative chronic cases.

False aminoglycoside resistance in Enterobacterales and non-fermenters by an automated testing system: a descriptive study.

IMPORTANCE: Antimicrobial sensitivity data are important to guide antimicrobial therapy. In microbiological laboratories, routine sensitivity measurements are typically performed with automated testing systems such as VITEK2 and Phoenix. Using data from the Dutch national surveillance system for antimicrobial resistance over a 6-year period, we found that the measured minimum inhibitory concentrations for aminoglycosides in Enterobacterales and non-fermenters were too high for the Phoenix system. In addition, we observed a yearly increase in resistance for several species measured by Phoenix. These findings might have consequences for clinical treatment of patients with sepsis.

Etiology of acute gastroenteritis in children requiring hospitalization in the Netherlands.

Infectious gastroenteritis causes a considerable burden of disease worldwide. Costs due to gastroenteritis are dominated by the hospitalized cases. Effective control of gastroenteritis should be targeted at the diseases with the highest burden and costs. For that, an accurate understanding of the relative importance of the different bacterial, viral, and parasitic pathogens is needed. The objective of the present study was to determine the incidence and etiology of gastroenteritis requiring hospital admission in the Netherlands. Six hospitals enrolled patients admitted with gastroenteritis for approximately one year over the period May 2008 to November 2009. Participants provided questionnaires and a fecal sample, and the hospital filled out a clinical questionnaire. In total, 143 children hospitalized for gastroenteritis and 64 matched controls were included in the study. Overall incidence of gastroenteritis requiring hospitalization was estimated at 2.92 per 1,000 children aged 0-17 years per year, with the highest incidence in children under the age of 5 years. The full diagnostic panel of pathogens could be studied in fecal samples of 96 cases. One or more pathogens were found in 98% of these cases. Co-infections were observed relatively often (40%). Viruses were detected in 82% of the samples, with rotavirus being most common (56%), bacteria in 32% and parasites in 10%. The present study emphasizes the importance of viral pathogens, especially rotavirus, in hospitalizations of children with gastroenteritis. Policies to reduce (costs of) hospitalizations due to gastroenteritis should therefore be first targeted at rotavirus.

The application of an enzyme-linked immunosorbent assay or an immunofluorescent assay test leads to different estimates of seroprevalence of Coxiella burnetii in the population.

The diagnosis and epidemiological studies of Q fever depend on serology. Among the main methods employed are the enzyme-linked immunosorbent assay (ELISA) and the immunofluorescent assay test (IFAT). We show that two commercial assays representing the two methods with two different cut-off titres can lead to significant differences in diagnostic and seroprevalence estimates. This in turn emphasizes the need for a standardized gold method to compare the various assays; whether this standard is 'in-house' or commercially obtained.

Low seroprevalence of Q fever in The Netherlands prior to a series of large outbreaks.

The Netherlands has experienced large community outbreaks of Q fever since 2007. Sera and questionnaires containing epidemiological data from 5654 individuals were obtained in a nationwide seroprevalence survey used to evaluate the National Immunization Programme in 2006-2007. We tested these sera for IgG phase-2 antibodies against Coxiella burnetii with an ELISA to estimate the seroprevalence and to identify determinants for seropositivity before the Q fever outbreaks occurred. Overall seroprevalence was 1·5% [95% confidence interval (CI) 1·3-1·7]. Corrected for confirmation with immunofluorescence results in a subset, the estimated seroprevalence was 2·4%. Seropositivity ranged from 0·48% (95% CI 0·00-0·96) in the 0-4 years age group to 2·30% (95% CI 1·46-3·15) in the 60-79 years age group. Keeping ruminants, increasing age and being born in Turkey were independent risk factors for seropositivity. The low seroprevalence before the start of the outbreaks supports the hypothesis that The Netherlands has been confronted with a newly emerging Q fever problem since spring 2007.

Aetiology of acute gastroenteritis in adults requiring hospitalization in The Netherlands.

SUMMARY Infectious gastroenteritis causes a considerable burden of disease worldwide. Effective control should be targeted at diseases with the highest burden and costs. Therefore, an accurate understanding of the relative importance of the different microorganisms is needed. The objective of this study was to determine the incidence and aetiology of gastroenteritis in adults requiring hospital admission in The Netherlands. Five hospitals enrolled patients admitted with gastroenteritis for about 1 year during the period May 2008 to November 2009. Participants completed questionnaires and provided a faecal sample. The hospital completed a clinical questionnaire. In total, 44 adults hospitalized for gastroenteritis were included in the study. The cases had serious symptoms, with 31% subsequently developing kidney failure. One or more pathogens were found in 59% of cases. Overall, rotavirus (22%) was the most common infection. Co-infections were observed relatively often (22%). This study emphasizes that rotavirus can also cause serious illness in adults.

Shifting priorities in the aftermath of a Q fever epidemic in 2007 to 2009 in The Netherlands: from acute to chronic infection.

From 2007 to 2009, the Netherlands faced large seasonal outbreaks of Q fever, in which infected dairy goat farms were identified as the primary sources. Veterinary measures including vaccination of goats and sheep and culling of pregnant animals on infected farms seem to have brought the Q fever problem under control. However, the epidemic is expected to result in more cases of chronic Q fever among risk groups in the coming years. In the most affected area, in the south of the country, more than 12% of the population now have antibodies against Coxiella burnetii. Questions remain about the follow-up of acute Q fever patients, screening of groups at risk for chronic Q fever, screening of donors of blood and tissue, and human vaccination. There is a considerable ongoing research effort as well as enhanced veterinary and human surveillance.

Outbreak of Salmonella Thompson in the Netherlands since July 2012.

An ongoing outbreak of salmonellosis due to Salmonella Thompson is affecting the Netherlands. Between 2 August and 19 October 2012, 866 cases were confirmed. Their median age was 44 years (range: 0-95 years), 63% were female and 36% were hospitalised. A matched case-control study suggested smoked salmon as the vehicle. Salmonella Thompson was confirmed in four of nine batches of smoked salmon from one producer. A recall of all concerned smoked salmon products was executed starting end of September.

Large differences between test strategies for the detection of anti-Borrelia antibodies are revealed by comparing eight ELISAs and five immunoblots.

We investigated the influence of assay choice on the results in a two-tier testing algorithm for the detection of anti-Borrelia antibodies. Eighty-nine serum samples from clinically well-defined patients were tested in eight different enzyme-linked immunosorbent assay (ELISA) systems based on whole-cell antigens, whole-cell antigens supplemented with VlsE and assays using exclusively recombinant proteins. A subset of samples was tested in five immunoblots: one whole-cell blot, one whole-cell blot supplemented with VlsE and three recombinant blots. The number of IgM- and/or IgG-positive ELISA results in the group of patients suspected of Borrelia infection ranged from 34 to 59%. The percentage of positives in cross-reactivity controls ranged from 0 to 38%. Comparison of immunoblots yielded large differences in inter-test agreement and showed, at best, a moderate agreement between tests. Remarkably, some immunoblots gave positive results in samples that had been tested negative by all eight ELISAs. The percentage of positive blots following a positive ELISA result depended heavily on the choice of ELISA-immunoblot combination. We conclude that the assays used to detect anti-Borrelia antibodies have widely divergent sensitivity and specificity. The choice of ELISA-immunoblot combination severely influences the number of positive results, making the exchange of test results between laboratories with different methodologies hazardous.

Antibiotic therapy for acute Q fever in The Netherlands in 2007 and 2008 and its relation to hospitalization.

Data about the effectiveness of different antibiotic regimens for the treatment of acute Q fever from clinical studies is scarce. We analysed the antibiotic treatment regimens of acute Q fever patients in 2007 and 2008 in The Netherlands and assessed whether hospitalization after a minimum of 2 days antibiotic therapy was related to the initial antibiotic therapy. Clinical data on antibiotic treatment and risk factors of acute Q fever patients were obtained from general practitioner medical records and self-reported by patients. For the 438 study patients, doxycycline was the most commonly prescribed initial antibiotic in both study years. After adjustments for confounding factors, doxycycline (200 mg/day), moxifloxacin, as well as other possibly effective antibiotics [including other new fluoroquinolones and doxycycline (100 mg/day)] showed significant lower risks for hospitalization compared to ß-lactam antibiotics and azithromycin (reference group), with the lowest risk for doxycycline (200 mg/day) (odds ratio 0·04, 95% confidence interval 0·01-0·22). These data support current guidelines that recommend doxycycline as the first choice antibiotic for treating acute Q fever.

Biphasic kinetics of peripheral blood T cells after triple combination therapy in HIV-1 infection: a composite of redistribution and proliferation.

The origin of CD4+ T cells reappearing in the blood following antiretroviral therapy in human immunodeficiency virus type-1 (HIV-1) infection is still controversial. Here we show, using mathematical modeling, that redistribution of T cells to the blood can explain the striking correlation between the initial CD4+ and CD8+ memory T-cell repopulation and the observation that 3 weeks after the start of treatment memory CD4+ T-cell numbers reach a plateau. The increase in CD4+ T cells following therapy most likely is a composite of initial redistribution, accompanied by a continuous slow repopulation with newly produced naive T cells.

Pertussis disease burden in the household: how to protect young infants.

BACKGROUND: We conducted a population-based, nation-wide, prospective study to identify who introduced pertussis into the household of infants aged 6 months admitted to the hospital for pertussis in the Netherlands. METHODS: During the period 2006-2008, a total of 560 household contacts of 164 hospitalized infants were tested by polymerase chain reaction, culture, and serological examination to establish Bordetella pertussis infection. Clinical symptoms and vaccination history were obtained by a questionnaire submitted during sample collection and 4-6 weeks afterwards. RESULTS: Overall, 299 household contacts (53%) had laboratory-confired pertussis; 159 (53%) had symptoms compatible with typical pertussis infection, and 42 (14%) had no symptoms. Among children vaccinated with a whole-cell vaccine, 17 (46%) of 37 had typical pertussis 1-3 years after completion of the primary series, compared with 9 (29%) of 31 children who had been completely vaccinated with an acellular vaccine. For 96 households (60%), the most likely source of infection of the infant was established, being a sibling (41%), mother (38%), or father (17%). CONCLUSIONS: If immunity to pertussis in parents is maintained or boosted, 35%-55% of infant cases could be prevented. Furthermore, we found that, 1-3 years after vaccination with whole-cell or acellular vaccine, a significant percentage of children are again susceptible for typical pertussis. In the long term, pertussis vaccines and vaccination strategies should be improved to provide longer protection and prevent transmission.

A review of diagnostic tests for congenital syphilis in newborns.

Congenital syphilis (CS) can occur when a mother is inadequately treated or not treated at all for an active Treponema pallidum infection. Symptoms of CS are often subtle and non-specific, and it is estimated that up to 60% of affected infants are asymptomatic at birth, making the diagnosis dependent on laboratory findings. Despite decades of experience with CS, problems still arise in its diagnosis because laboratory test results for children at risk for CS can be inconclusive and no single diagnostic test can be used to diagnose CS. The development of diagnostic tests such as enzyme immunoassays, immunoblotting and polymerase chain reaction (PCR) has increased the sensitivity and specificity of diagnoses, but the detection of specific IgM is currently the most sensitive serological method, and the presence of specific IgM should be considered as evidence of a congenital T. pallidum infection. Suspected cases can also be confirmed or excluded by serial post-partum tests of antibody kinetics. The authors note strongly that it is considered unethical not to treat a baby at risk of contracting CS, even without a definitive diagnosis. In this review, we describe the various microbiological methods-and their shortcomings-used in the laboratory diagnosis of CS.