Mycoplasma pneumoniae epidemic in Denmark, October to December, 2023.

We report a surge of patients, especially children and adolescents, with respiratory disease caused by Mycoplasma pneumoniae in Denmark since October 2023. While the surge has reached an epidemic level, no impact on hospital capacity has been observed; only 14% (446/3,195) of cases, primarily adults, required hospitalisation. Macrolide resistance was detected in less than 2% of samples tested. Timely monitoring of hospitalisations linked to M. pneumoniae infections has been established to inform the healthcare system, decisionmakers and the public.

Legionella colonisation in hot water systems in care homes from two Danish municipalities.

Legionnaires' disease is a serious health risk among the elderly. Water systems in care homes are therefore of particular interest. We investigated the levels of culturable Legionella in the hot water systems in care homes in two Danish municipalities. Two hundred and sixty-eight water samples from 98 care homes were evaluated. Contents of culturable Legionella counts were calculated, and correlations between temperature and colony-forming units (CFU/L) were analysed. Seventy-seven and 81%, respectively, of the care homes were colonised with Legionella in the two municipalities. Most care homes had less than 1,000 CFU/L, but 13 and 16% had more than 10,000 CFU/L. When including first flush samples, 27% of the care homes in Municipality 1 had Legionella levels above 10,000 CFU/L. Temperatures of ≥50 °C in Municipality 1 and ≥55 °C in Municipality 2 correlated with low levels of Legionella. The content of Legionella colonies was significantly higher in care homes in Municipality 1. However, a significantly higher proportion of taps in Municipality 2 had Legionella colonies. In conclusion, temperatures should be raised to 55 °C to avoid high Legionella levels. Test procedures should be evaluated, and the regular use of taps and routine testing for Legionella should be taken into consideration.

Mycoplasma pneumoniae detections before and during the COVID-19 pandemic: results of a global survey, 2017 to 2021.

BackgroundMycoplasma pneumoniae respiratory infections are transmitted by aerosol and droplets in close contact.AimWe investigated global M. pneumoniae incidence after implementation of non-pharmaceutical interventions (NPIs) against COVID-19 in March 2020.MethodsWe surveyed M. pneumoniae detections from laboratories and surveillance systems (national or regional) across the world from 1 April 2020 to 31 March 2021 and compared them with cases from corresponding months between 2017 and 2020. Macrolide-resistant M. pneumoniae (MRMp) data were collected from 1 April 2017 to 31 March 2021.ResultsThirty-seven sites from 21 countries in Europe, Asia, America and Oceania submitted valid datasets (631,104 tests). Among the 30,617 M. pneumoniae detections, 62.39% were based on direct test methods (predominantly PCR), 34.24% on a combination of PCR and serology (no distinction between methods) and 3.37% on serology alone (only IgM considered). In all countries, M. pneumoniae incidence by direct test methods declined significantly after implementation of NPIs with a mean of 1.69% (SD ± 3.30) compared with 8.61% (SD ± 10.62) in previous years (p < 0.01). Detection rates decreased with direct but not with indirect test methods (serology) (-93.51% vs + 18.08%; p < 0.01). Direct detections remained low worldwide throughout April 2020 to March 2021 despite widely differing lockdown or school closure periods. Seven sites (Europe, Asia and America) reported MRMp detections in one of 22 investigated cases in April 2020 to March 2021 and 176 of 762 (23.10%) in previous years (p = 0.04).ConclusionsThis comprehensive collection of M. pneumoniae detections worldwide shows correlation between COVID-19 NPIs and significantly reduced detection numbers.

Evidence for multiple cases of recurrent Legionella infection: a Danish national surveillance study.

While case reports have documented recurrence of Legionnaires' disease, the frequency of recurrent infections has not been systematically examined at a national level over multiple decades. Between 2000 and 2020 in Denmark, 21 individuals had repeat laboratory-identified Legionella infection, totalling 48 episodes of hospitalisation. The majority of these individuals had underlying comorbidities. In at least 3 of the 21 cases, a different Legionella serogroup was detected during the second episode of infection, which could indicate reinfection from a new source. These results emphasise that Legionella can, and does, reinfect high-risk individuals causing multiple hospitalisations.

Legionella antibiotic susceptibility testing: is it time for international standardization and evidence-based guidance?

Legionella pneumophila, a Gram-negative bacillus, is the causative agent of Legionnaire's disease, a form of severe community-acquired pneumonia. Infection can have high morbidity, with a high proportion of patients requiring ICU admission, and up to 10% mortality, which is exacerbated by the lack of efficacy of typical empirical antibiotic therapy against Legionella spp. The fastidious nature of the entire Legionellaceae family historically required inclusion of activated charcoal in the solid medium to remove growth inhibitors, which inherently interferes with accurate antimicrobial susceptibility determination, an acknowledged methodological shortfall, now rectified by a new solid medium that gives results comparable to those of microbroth dilution. Here, as an international Legionella community (with authors representing various international reference laboratories, countries and clinical stakeholders for diagnosis and treatment of legionellosis), we set out recommendations for the standardization of antimicrobial susceptibility testing methods, guidelines and reference strains to facilitate an improved era of antibiotic resistance determination.

Provincial trends in Legionnaires' disease are not explained by population structure in Denmark, 2015 to 2018.

BackgroundLegionnaires' disease (LD) incidence has been increasing in several European countries since 2011. Currently, Denmark is experiencing one of the highest annual incidences of LD despite its relatively cold climate and homogenous population, and the incidence differs notably across the country.AimWe sought to determine whether provincial differences in LD incidence are attributable to the age and sex distribution of the population, and to characterise the risk of LD by province and age group in Denmark.MethodsUsing national routine surveillance data for domestic LD cases collected between 2015 and 2018, we assessed the incidence of disease by province and year. Poisson regression models were fit to understand the risk of LD by year and province, as well as by 5-year age groups.ResultsIncidence of domestic LD increased 48% between 2015 and 2018 across Denmark. Some provinces continuously had a high incidence of disease, even after adjusting for yearly trends and the underlying population distribution. Variations in the proportion of the population aged 65 years and older were not responsible for the increase in disease in our analysis. Finally, incidence of disease increased with each 5-year age group in both men and women.ConclusionsThe relative differences in incidence between Danish provinces could not be explained by the age and sex distribution of the population, indicating that other factors must be responsible for the varying incidence across the country. These results may help inform trends in other countries in Europe also experiencing an unexplained high incidence of LD.

Mycoplasma pneumoniae infections, 11 countries in Europe and Israel, 2011 to 2016.

BackgroundMycoplasma pneumoniae is a leading cause of community-acquired pneumonia, with large epidemics previously described to occur every 4 to 7 years.AimTo better understand the diagnostic methods used to detect M. pneumoniae; to better understand M. pneumoniae testing and surveillance in use; to identify epidemics; to determine detection number per age group, age demographics for positive detections, concurrence of epidemics and annual peaks across geographical areas; and to determine the effect of geographical location on the timing of epidemics.MethodsA questionnaire was sent in May 2016 to Mycoplasma experts with national or regional responsibility within the ESCMID Study Group for Mycoplasma and Chlamydia Infections in 17 countries across Europe and Israel, retrospectively requesting details on M. pneumoniae-positive samples from January 2011 to April 2016. The Moving Epidemic Method was used to determine epidemic periods and effect of country latitude across the countries for the five periods under investigation.ResultsRepresentatives from 12 countries provided data on M. pneumoniae infections, accounting for 95,666 positive samples. Two laboratories initiated routine macrolide resistance testing since 2013. Between 2011 and 2016, three epidemics were identified: 2011/12, 2014/15 and 2015/16. The distribution of patient ages for M. pneumoniae-positive samples showed three patterns. During epidemic years, an association between country latitude and calendar week when epidemic periods began was noted.ConclusionsAn association between epidemics and latitude was observed. Differences were noted in the age distribution of positive cases and detection methods used and practice. A lack of macrolide resistance monitoring was noted.

Genomic investigation of a suspected outbreak of Legionella pneumophila ST82 reveals undetected heterogeneity by the present gold-standard methods, Denmark, July to November 2014.

Between July and November 2014, 15 community-acquired cases of Legionnaires´ disease (LD), including four with Legionella pneumophila serogroup 1 sequence type (ST) 82, were diagnosed in Northern Zealand, Denmark. An outbreak was suspected. No ST82 isolates were found in environmental samples and no external source was established. Four putative-outbreak ST82 isolates were retrospectively subjected to whole genome sequencing (WGS) followed by phylogenetic analyses with epidemiologically unrelated ST82 sequences. The four putative-outbreak ST82 sequences fell into two clades, the two clades were separated by ca 1,700 single nt polymorphisms (SNP)s when recombination regions were included but only by 12 to 21 SNPs when these were removed. A single putative-outbreak ST82 isolate sequence segregated in the first clade. The other three clustered in the second clade, where all included sequences had < 5 SNP differences between them. Intriguingly, this clade also comprised epidemiologically unrelated isolate sequences from the UK and Denmark dating back as early as 2011. The study confirms that recombination plays a major role in L. pneumophila evolution. On the other hand, strains belonging to the same ST can have only few SNP differences despite being sampled over both large timespans and geographic distances. These are two important factors to consider in outbreak investigations.

Detection of Legionella bozemanae, a new cause of septic arthritis, by PCR followed by specific culture.

Legionella bozemanae is a rare isolate in clinical specimens. We describe a case of joint infection due to L. bozemanae in an immunocompromised patient with dermatomyositis. Without the use of PCR screening or culture on specialized medium, the organism would not have been detected.

A Tale of Four Danish Cities: Legionella pneumophila Diversity in Domestic Hot Water and Spatial Variations in Disease Incidence.

Denmark has one of the highest Legionnaires' disease notification rates within Europe, averaging 4.7 cases per 100,000 population annually (2017 to 2020). The relatively high incidence of disease is not uniform across the country, and approximately 70% of all domestically acquired cases in Denmark are caused by Legionella pneumophila (LP) strains that are considered less virulent. The aim of this study was to investigate if colonization rates, levels of colonization, and/or types of LP present in hot water systems were associated with geographic differences in Legionnaires' disease incidence. Domestic water systems from four cities in Denmark were analyzed via culture and qPCR. Serogrouping and sequence typing was performed on randomly selected isolates. Single nucleotide polymorphism was used to identify clonal relationship among isolates from the four cities. The results revealed a high LP colonization rate from 68% to 87.5% among systems, composed primarily of non-serogroup 1. LP serogroup 1 reacting with the monoclonal antibody (MAb) 3/1 was not identified in any of the systems tested, while MAb 3/1 negative serogroup 1 strains were isolated from 10 systems (9.6%). We hypothesize that a combination of factors influences the incidence rate of LD in each city, including sequence type and serogroup distribution, colonization rate, concentration of Legionella in Pre-flush and Flush samples, and potentially building characteristics such as water temperature measured at the point of use.

Genome analysis of Legionella pneumophila ST23 from various countries reveals highly similar strains.

Legionella pneumophila serogroup 1 (Lp1) sequence type (ST) 23 is one of the most commonly detected STs in Italy where it currently causes all investigated outbreaks. ST23 has caused both epidemic and sporadic cases between 1995 and 2018 and was analysed at genomic level and compared with ST23 isolated in other countries to determine possible similarities and differences. A core genome multi-locus sequence typing (cgMLST), based on a previously described set of 1,521 core genes, and single-nucleotide polymorphisms (SNPs) approaches were applied to an ST23 collection including genomes from Italy, France, Denmark and Scotland. DNAs were automatically extracted, libraries prepared using NextEra library kit and MiSeq sequencing performed. Overall, 63 among clinical and environmental Italian Lp1 isolates and a further seven and 11 ST23 from Denmark and Scotland, respectively, were sequenced, and pangenome analysed. Both cgMLST and SNPs analyses showed very few loci and SNP variations in ST23 genomes. All the ST23 causing outbreaks and sporadic cases in Italy and elsewhere, were phylogenetically related independent of year, town or country of isolation. Distances among the ST23s were further shortened when SNPs due to horizontal gene transfers were removed. The Lp1 ST23 isolated in Italy have kept their monophyletic origin, but they are phylogenetically close also to ST23 from other countries. The ST23 are quite widespread in Italy, and a thorough epidemiological investigation is compelled to determine sources of infection when this ST is identified in both LD sporadic cases and outbreaks.

Detection of Legionella by quantitative-polymerase chain reaction (qPCR) for monitoring and risk assessment.

BACKGROUND: Culture and quantitative polymerase chain reaction (qPCR) assays for the detection of Legionella were compared on samples from a residential area before and after two interventions. A total of 84 samples were collected from shower hoses and taps as first flush samples and at constant temperature. Samples were grouped according to the origin of the sample, a) circulation water b) water from empty apartments c) water from shower hoses. The aims were to investigate the usefulness of qPCR compared to culture for monitoring remedial actions for elimination of Legionella bacteria and as a tool for risk assessment. RESULTS: In water collected from the apartments Legionella spp were detected by qPCR in the concentration range from LOQ to 9.6*105GU/L while L. pneumophila were detected in a range from LOQ to 6.8*105 GU/L. By culturing, the legionellae were detected in the range from below detection limit (> 10 CFU/L) to 1.6*106 CFU/L. In circulating water and in first flush water from shower hoses, culture and qPCR showed the same tendencies. The overall correlation between the bacteria number detected by culture and the two developed qPCR assays (L. spp and L. pneumophila) was relatively poor (r2 = 0.31 for culture and Legionella spp. assay, r2 = 0.20 for culture and L. pneumophila assay). CONCLUSION: Detection by qPCR was suitable for monitoring changes in the concentration of Legionella but the precise determination of bacteria is difficult. Risk assessment by qPCR only on samples without any background information regarding treatment, timing, etc is dubious. However, the rapid detection by qPCR of high concentrations of Legionella - especially Legionella pneumophila - is valuable as an indicator of risk, although it may be false positive compared to culture results. On the other hand, the detection of a low number of bacteria by qPCR is a strong indication for the absence of risk.

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.

Two Legionnaires' disease cases associated with industrial waste water treatment plants: a case report.

BACKGROUND: Finnish and Swedish waste water systems used by the forest industry were found to be exceptionally heavily contaminated with legionellae in 2005. CASE PRESENTATION: We report two cases of severe pneumonia in employees working at two separate mills in Finland in 2006. Legionella serological and urinary antigen tests were used to diagnose Legionnaires' disease in the symptomatic employees, who had worked at, or close to, waste water treatment plants. Since the findings indicated a Legionella infection, the waste water and home water systems were studied in more detail. The antibody response and Legionella urinary antigen finding of Case A indicated that the infection had been caused by Legionella pneumophila serogroup 1. Case A had been exposed to legionellae while installing a pump into a post-clarification basin at the waste water treatment plant of mill A. Both the water and sludge in the basin contained high concentrations of Legionella pneumophila serogroup 1, in addition to serogroups 3 and 13. Case B was working 200 meters downwind from a waste water treatment plant, which had an active sludge basin and cooling towers. The antibody response indicated that his disease was due to Legionella pneumophila serogroup 2. The cooling tower was the only site at the waste water treatment plant yielding that serogroup, though water in the active sludge basin yielded abundant growth of Legionella pneumophila serogroup 5 and Legionella rubrilucens. Both workers recovered from the disease. CONCLUSION: These are the first reported cases of Legionnaires' disease in Finland associated with industrial waste water systems.

Multi-center evaluation of one commercial and 12 in-house real-time PCR assays for detection of Mycoplasma pneumoniae.

Detection of Mycoplasma pneumoniae by real-time PCR is not yet standardized across laboratories. We have implemented a standardization protocol to compare the performance of thirteen commercial and in-house approaches. Despite differences on threshold values of samples, all assays were able to detect at least 20M. pneumoniae genomes per reaction.

Evaluation of a new lateral flow test for detection of Streptococcus pneumoniae and Legionella pneumophila urinary antigen.

Pneumonia is a major cause of morbidity and mortality worldwide. Early diagnosis of the etiologic agent is important in order to choose the correct antibiotic treatment. In this study we evaluated the first commercial combined test for the agents of pneumococcal pneumonia and Legionnaires' disease based on urinary antigen detection, the ImmuView® Streptococcus pneumoniae and Legionella pneumophila Urinary Antigen Test. In this evaluation, the new test had a significantly higher sensitivity than the BinaxNOW® lateral flow tests and the Binax® EIA test. This identifies the ImmuView® S. pneumoniae and L. pneumophila Urinary Antigen Test as a fast and sensitive point of care test for identification of the infectious agent in a major group of patients with pneumonia.

Detection of Legionella pneumophila antigen in urine samples by the BinaxNOW immunochromatographic assay and comparison with both Binax Legionella Urinary Enzyme Immunoassay (EIA) and Biotest Legionella Urin Antigen EIA.

The new BinaxNOW Immunochromatographic (ICT) Assay for the detection of Legionella pneumophila antigens was used to test 535 urine specimens from patients with and without Legionnaires' disease. The specificity, calculated by testing 112 samples from patients with pneumonia of aetiologies other than Legionella infection, and 167 urine specimens from urinary tract infections, was found to be 97.1% if the manufacturer's guidelines were followed. However, it was determined that the 'false positive' results characterised by very weak bands could be discounted by re-examination of the results at 60 min, yielding a specificity of 100%. With this minor modification of the procedure applied to examination of urine samples from 117 patients with legionellosis confirmed by isolation of L. pneumophila and 70 patients who had seroconverted to L. pneumophila serogroup 1, sensitivity was calculated to be 79.7%. In comparison, the sensitivities of the Binax Urinary Antigen Enzyme Immunoassay (EIA) and Biotest Urin Antigen EIA were estimated to be 79.1 and 83.4%, respectively. Eleven cases (5.9%) were positive by BinaxNOW assay but negative by Binax or Biotest EIA, or both. The sensitivities of all assays increased to c. 94% if only diagnosis of cases confirmed by isolation of serogroup 1 L. pneumophila was considered, although the sensitivity for infections caused by L. pneumophila serogroup 1 monoclonal antibody (MAb) subgroup Bellingham was significantly lower than for other MAb subgroups. The Biotest EIA recognised 10 (45%) of the 22 cases not caused by L. pneumophila serogroup 1, whereas the two Binax kits detected only three each. The ICT assay BinaxNOW can be recommended as a rapid specific test for the diagnosis of Legionnaires' diseases caused by L. pneumophila serogroup 1, although very weak bands should be interpreted cautiously.

[Surveillance and prevention of nosocomial Legionella pneumonia]. / Overvågning og forebyggelse af nosokomiel Legionella-pneumoni.

Statens Serum Institut surveys the occurrence of nosocomial pneumonia caused by Legionella. The rate is low compared to other nosocomial infections but carries a high mortality. Verification of the diagnosis and acquisition (nosocomial or community acquired infection) is carried out in each suspected case. The criteria used for classification are described. Preventive measures include protection of susceptible patients as well as maintenance of the hot-water supply providing a minimum temperature of 50 degrees C at any hot tap. Technical solutions to reduce the concentration of Legionella in hospital hot-water supply systems are listed, and a guideline for the control and surveillance of the occurrence is suggested.

[Ornithosis is a diagnostic challenge].

Symptoms of ornithosis are often unspecific and vary in severity. This case report illustrates the challenges in diagnosing ornithosis. It also shows how easy it is to diagnose ornithosis by PCR analysis of a sputum sample and that it is not always possible to base the diagnosis on serology as our patient had negative serology tests even four weeks after hospitalization. The diagnosis was furthermore compounded by an underlying chronic lung disease. To diagnose patients with respiratory problems correctly it is important to analyse lower respiratory samples by PCR along with other diagnostic tests and to get a detailed medical history.

Application of a lateral flow test as an additional serological tool for diagnosis of Legionella infections.

We investigate a new serological lateral flow test for detection of Legionella infection. The sensitivity of the test was compared to existing ELISA methods, using well-defined samples from patients with Legionella infection. The lateral flow test was found to be a good supplement for fast serological diagnosis of legionellosis including Legionnaires' disease.