Decreased Pneumococcal Carriage Among Older Adults in Denmark During the COVID-19 Lockdown.

Background: COVID-19 containment measures reduced the burden of invasive pneumococcal disease. Data on pneumococcal carriage rates among adults during the pandemic are scarce. Methods: Naso- and oropharyngeal swabs and questionnaires were collected during January 2019 to December 2021 from adults ≥64 years of age. Carriage was determined by lytA/piaB PCR. Results: A total of 1556 participants provided paired naso- and oropharyngeal swabs. Their median age was 74 years (IQR, 70-79). Streptococcus pneumoniae DNA was detected in 146 (9.4%) oropharyngeal swabs and 34 (2.2%) nasopharyngeal. The carriage rate decreased from 12.9% (95% CI, 10.1%-16.1%, n = 66/511) prelockdown (January 2019-February 2020) to 4.2% (95% CI, 2.0%-7.5%, n = 10/240) during lockdown (March 2020-February 2021) and increased to 12.1% (95% CI, 9.8%-14.7%, n = 87/719) with the reopening of society (March 2021-December 2021; P = .0009). Conclusions: Pneumococcal carriage prevalence declined significantly during pandemic mitigation measures and rebounded to prepandemic levels as measures were lifted.

Neoehrlichia mikurensis in Danish immunocompromised patients: a retrospective cohort study.

BACKGROUND: The tick-borne bacterium, Neoehrlichia mikurensis (N. mikurensis) can cause severe febrile illness and thromboembolic complications in immunocompromised individuals. We investigated the presence of N. mikurensis DNA in retrospectively collected plasma from a well-characterized cohort of Danish immunocompromised patients. METHODS: Plasma samples from 239 patients with immune dysfunction related to hematological or rheumatological disease or due to immunosuppressive therapy, were retrieved from a transdisciplinary biobank (PERSIMUNE) at Rigshospitalet, Copenhagen, Denmark. Serving as immunocompetent controls, plasma samples from 192 blood donors were included. All samples were collected between 2015 and 2019. Real-time PCR targeting the groEL gene was used to detect N. mikurensis DNA. Sequencing was used for confirmation. Borrelia burgdorferi sensu lato IgG antibodies were detected by ELISA as a proxy of tick exposure. Prevalence was compared using Fisher's exact test. RESULTS: Neoehrlichia mikurensis DNA was detected in 3/239 (1.3%, 95% confidence interval (CI): 0.3 - 3.6%) patients, all of whom primarily had a hematological disease. Follow-up samples of these patients were negative. N. mikurensis DNA was not detected in any of the blood donor samples. IgG antibodies against B. burgdorferi s.l. were detected with similar prevalence in immunocompromised patients and blood donors, i.e., 18/239 (7.5%, 95% CI: 4.8-11.5%) and 11/192 (5.7%, 95%: CI 3.2-10.0%). CONCLUSION: In this study, patients with N. mikurensis were not identified by clinical indication and N. mikurensis may therefore be underdiagnosed in Danish patients. Further investigations are needed to explore the clinical significance and implications of this infection.

Parasitic Intestinal Protists of Zoonotic Relevance Detected in Pigs by Metabarcoding and Real-Time PCR.

Several parasite species are shared between humans and pigs. We explored the application of next-generation sequencing-based metabarcoding supplemented with real-time PCR to fecal DNAs from 259 samples from 116 pigs in Denmark to detect and differentiate single-celled intestinal parasites of zoonotic relevance. Enterocytozoon bieneusi, Balantioides coli, and Giardia duodenalis were observed in 34/37 (92%), 148/259 (57%), and 86/259 (33%) samples, respectively. Entamoeba polecki ST1, E. polecki ST3, and Entamoeba hartmanni were detected in 104/259 (40%), 161/259 (62%), and 8/259 (3%) samples, respectively. Metabarcoding and real-time PCR detected Cryptosporidium in 90/259 (35%) and 239/259 (92%) of the samples, respectively, with Cryptosporidium suis and Cryptosporidium scrofarum observed in nearly equal proportions. Blastocystis subtypes 1, 3, 5, and 15 were found in 72 (28%), 6 (2%), 176 (68%), and 36 (14%) of 259 samples, respectively. Iodamoeba was identified in 1/259 samples (<1%), while none of 37 tested samples was positive for Dientamoeba fragilis. Our results illustrate how metabarcoding exemplifies a 'one-fits-many' approach to detecting intestinal single-celled parasites in feces supplemented with real-time PCR for selected parasites. Using metabarcoding with pathogen-specific assays may help detect emerging and previously underdetected pathogens and further elucidate the role of micro-eukaryotic parasites in human and animal health and disease.

Development of a species-specific real-time PCR test for Chlamydia psittaci and its employment in the investigation of zoonotic transmission from racing pigeons in Denmark.

Species-specific detection of Chlamydia psittaci is challenging and all published PCR tests have so far shown deficiencies in specificity or sensitivity. The present investigation reports on the development of a species-specific real-time PCR assay for C. psittaci. The test is based on an 84 bp indel in a gene of unknown function that is unique to C. psittaci. The Cps-indel84-PCR assay was validated on a wide range of chlamydial and other bacterial strains as well as on clinical samples from animals and humans in two different diagnostic laboratories in Germany and Denmark. Furthermore, the test was employed for investigating samples from racing pigeon flocks in Denmark. The evaluation showed that the Cps-indel84-PCR assay has excellent test characteristics and is a highly reliable method for identifying C. psittaci in clinical samples both from humans and animals.

Point-of-care tests for influenza A and B viruses and RSV in emergency departments - indications, impact on patient management and possible gains by syndromic respiratory testing, Capital Region, Denmark, 2018.

BackgroundPoint-of-care tests (POCT) for influenza A and B viruses and respiratory syncytial virus (RSV) were implemented in emergency departments of all hospitals in the Capital Region of Denmark in 2018.AimTo establish whether POC testing for influenza viruses or RSV is based on a valid respiratory symptom indication, whether changes in patient management based on a positive result are safe and whether syndromic POC testing may benefit patients with influenza or RSV.MethodsSamples from 180 children (< 18 years) and 375 adults tested using POCT between February and July 2018 were retested for 26 respiratory pathogens. Diagnosis, indication for POC testing, hospitalisation time, antimicrobial therapy and readmission or death within one month of testing were obtained from patient records.ResultsA valid indication for POC testing was established in 168 (93.3%) of children and 334 (89.1%) of adults. A positive POCT result significantly reduced antibiotic prescription and median hospitalisation time by 44.3 hours for adults and 14.2 hours for children, and significantly increased antiviral treatment in adults. Risk of readmission or death was not significantly altered by a positive result. Testing for 26 respiratory pathogens established that risk of coinfection is lower with increasing age and that POCT for adults should be restricted to the influenza and RSV season.ConclusionPositive POCT resulted in changed patient management for both children and adults, and was deemed safe. POCT for additional pathogens may be beneficial in children below 5 years of age and outside the influenza and RSV season.

Laboratory Diagnostics of Rickettsia Infections in Denmark 2008-2015.

Rickettsiosis is a vector-borne disease caused by bacterial species in the genus Rickettsia. Ticks in Scandinavia are reported to be infected with Rickettsia, yet only a few Scandinavian human cases are described, and rickettsiosis is poorly understood. The aim of this study was to determine the prevalence of rickettsiosis in Denmark based on laboratory findings. We found that in the Danish individuals who tested positive for Rickettsia by serology, the majority (86%; 484/561) of the infections belonged to the spotted fever group. In contrast, we could confirm 13 of 41 (32%) PCR-positive individuals by sequencing and identified all of these as R. africae, indicating infections after travel exposure. These 13 samples were collected from wound/skin material. In Denmark, approximately 85 individuals test positive for Rickettsia spp. annually, giving an estimated 26% (561/2147) annual prevalence among those suspected of rickettsiosis after tick bites. However, without clinical data and a history of travel exposure, a true estimation of rickettsiosis acquired endemically by tick bites cannot be made. Therefore, we recommend that both clinical data and specific travel exposure be included in a surveillance system of Rickettsia infections.

Characterization of Diarrheagenic Enteroaggregative Escherichia coli in Danish Adults-Antibiotic Treatment Does Not Reduce Duration of Diarrhea.

Enteroaggregative Escherichia coli (EAEC) is frequently isolated from sporadic cases of diarrhea and in outbreaks of gastroenteritis in several regions of the world. The pathophysiology of EAEC continues to be enigmatic, and the efficacy of antibiotic treatment in EAEC-associated diarrhea has been discussed. Since the level of antibiotic resistance is increasing, it is essential to restrict the use of antibiotics to prevent further resistance development. We aimed to investigate EAEC strains in adult Danish patients suffering from diarrhea and from healthy controls. We examined the antibiotic resistance in EAEC strains, the clinical response to antibiotic treatment in EAEC diarrheal cases, and the distribution of virulence genes in diarrheal cases. The EAEC strains were collected from patients suffering from diarrhea in a Danish multicenter study. A medical doctor interviewed the patients by using a questionnaire regarding gastrointestinal symptoms, exposures, and use of antibiotic and over-the-counter antidiarrheal drugs. Follow-up was performed after 3-5 months to inquire about differential diagnosis to gastrointestinal disease. A multiplex polymerase chain reaction characterized virulence genes in diarrheal cases. Finally, the level of antibiotic resistance was examined by using the disc diffusion method. Asymptomatic carriage of EAEC in the adult Danish population was rare, in contrast to findings in healthy Danish children. The duration of diarrhea was not shortened by antibiotic treatment, specifically ciprofloxacin treatment, or by over-the-counter antidiarrheal drugs. Follow-up revealed no pathology in diarrheal patients apart from irritable bowel syndrome in two patients. A high number of patients suffered from long-term diarrhea, which was associated with the enterotoxin EAST-1 and a high virulence factor score. A high level of antibiotic resistance was observed and 58% of the EAEC strains were multidrug resistant. Multidrug resistance was most pronounced in cases of travelers' diarrhea, and it was seen that antibiotic treatment did not reduce the duration of diarrhea.

Evidence of rickettsiae in Danish patients tested for Lyme neuroborreliosis: a retrospective study of archival samples.

BACKGROUND: With a prevalence of 4.7-13% in Danish Ixodes ricinus ticks, Rickettsia helvetica is one of the most frequently detected tick-borne organisms in Denmark. Most reports of human exposure have described asymptomatic seroconversion or a mild, self-limiting flu-like illness but it has also been implicated as a cause of subacute lymphocytic meningitis. Because Borrelia burgdorferi sensu lato (Bbsl) and R. helvetica are both found in the same tick species, potential co-transmission is a possibility. We examined 1) the seroprevalence of anti-rickettsia antibodies in patients investigated for Lyme neuroborreliosis (LNB), and 2) the cerebrospinal fluid (CSF) and sera of same patients for the presence of Rickettsia DNA. METHODS: Ninety-nine sera and 87 CSF samples from patients with intrathecal synthesis of anti-Borrelia antibodies and 101 sera and 103 CSF samples from patients with no detectable intrathecal synthesis were retrospectively examined for this study. Sera were analyzed for antibodies against spotted fever group (SFG) rickettsiae and both the CSF and sera were tested for Rickettsia DNA using a genus-specific real-time PCR. RESULTS: Of the patients tested for LNB, 32% (64/200) had IgG antibodies against SFG rickettsiae. Among patients with confirmed intrathecal synthesis of Borrelia-specific antibodies, 38% (38/99) exhibited IgG antibodies. None of these values were statistically significant when compared with sera from healthy blood donors (p = 0.7 and 0.19). Rickettsia DNA was found in the CSF of 4% (8/190) of patients. CONCLUSION: No statistically significant difference was found in the seroprevalence of anti-rickettsia antibodies in patients tested for LNB and healthy blood donors, indicative of a low rate of exposure in this group of patients. Eight patients showed evidence of Rickettsia DNA in the CSF, five of whom had LNB. However, cycle threshold (Ct) values were high, indicating low concentrations of DNA, and no apparent alteration in the clinical manifestations of LNB were noted in the medical records of these patients.

Molecular epidemiology of Bordetella pertussis in Greece, 2010-2015.

PURPOSE: To determine the predominant strains of Bordetella pertussis in Greece during 2010-2015. METHODOLOGY: Infants and children (n=1150) (15 days to 14 years) of Greek, Roma and immigrant origin with different vaccination statuses were hospitalized in Athens, Greece with suspected pertussis infection. IS481/IS1001 real-time PCR confirmed Bordetella spp./B. pertussis infection in 300 samples. A subset of samples (n=153) were analysed by multi-locus variable number tandem repeat analysis (MLVA) and (n=25) by sequence-based typing of the toxin promotor region (ptxP) on DNA extracted from clinical specimens.Results/Key findings. A complete MLVA profile was determined in 66 out of 153 samples; the B. pertussis MLVA type 27 (n=55) was the dominant genotype and all tested samples (n=25) expressed the ptxP3 genotype. The vaccine coverage in the Greek population was 90 %; however, the study population expressed complete coverage in 2 out of 264 infants (0-11 months) and in 20 out of 36 children (1-14 years). Roma and immigrant minorities represent 7 % of the Greek population, but make up 50 % of the study population, indicating a low vaccine coverage among these groups. CONCLUSIONS: The B. pertussis MT27 and ptxP3 genotype is dominant in Greek, Roma and immigrant infants and children hospitalized in Greece. Thus, the predominant MLVA genotype in Greece is similar to other countries using acellular vaccines.

Temporal trends in Bordetella pertussis populations, Denmark, 1949-2010.

We used multilocus variable-number tandem repeat analysis and multiple antigen sequence typing to characterize isolates of Bordetella pertussis strains circulating in Denmark during periods with and without pertussis vaccination coverage. Our results show substantial shifts in the B. pertussis population over time and a reduction in genetic diversity. These changes might have resulted from the introduction of pertussis vaccines in Denmark and other parts of Europe. The predominant strains currently circulating in Denmark resemble those in other European countries.

Molecular characterization of Salmonella Typhimurium highly successful outbreak strains.

Three large clusters of Salmonella Typhimurium infections in Denmark in 2008 and 2009 were defined by multilocus variable number of tandem repeat analysis (MLVA). One of these proved to be the hereto largest Danish cluster of salmonellosis with 1446 cases. Two smaller clusters with a total of 197 and 89 cases, respectively, were seen concurrently. These clusters shared epidemiological characteristics such as age distribution, geography, and time. To investigate the possible genetic relationship between the cluster strains, these were further characterized by phage typing, pulsed-field gel electrophoresis, and Optical Mapping. Although the MLVA method proved robust and well-performing in detecting and defining clusters, the employment of a second typing method detected an additional fourth cluster among the isolates. The cluster strains were stable throughout the almost 2-year period, even though we detected changes in three of five MLVA loci in a small fraction of isolates. These changes were mainly due to the gain or loss of single repeats. Optical Mapping of the large cluster strain indicated no increased content of virulence genes; however, Optical Mapping did reveal a large insert, a probable prophage, in the main cluster. This probable prophage may give the cluster strain a competitive advantage. The molecular methods employed suggested that the four clusters represented four distinct strains, although they seemed to be epidemiologically linked and shared genotypic characteristics.