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.

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.

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.

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.

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.

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.

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.

Prenatal Chlamydia trachomatis infection increases the risk of preeclampsia.

The relationship between Chlamydia trachomatis (CT) and preeclampsia was examined longitudinally among 205 cases and 423 normotensive controls nested within the Collaborative Perinatal Project. Antibodies were analyzed at a first prenatal visit (mean 14.2 weeks) and at delivery. Prenatal infections were identified as IgG/IgM seroconversion or a four-fold rise in IgG antibody titers. Although serological evidence of incident prenatal CT infection was uncommon (n=9, 1.4%) in this general pregnant population, infected women were more likely to develop preeclampsia, after adjustment for maternal age, body mass index, smoking status, race and time between blood draws (ORadj 7.2, 95% CI 1.3 - 39.7).

Chlamydia trachomatis infection may increase the risk of preeclampsia.

BACKGROUND: Although the etiology of preeclampsia is not well understood, it has been suggested that excessive systemic inflammation may lead to oxidative stress, promoting the endothelial dysfunction characteristic of preeclampsia. Few prospective studies have examined the role of infection, an immune system stimulator, as a risk factor for preeclampsia. METHODS: We conducted a longitudinal study of the relationships between Chlamydia trachomatis (CT), Chlamydophila pneumoniae (CP), cytomegalovirus (CMV), herpes simplex virus (HSV) and preeclampsia among 509 preeclamptic cases and 336 normotensive controls nested within the Danish National Birth Cohort study. Antibodies were analyzed at a first prenatal visit (mean 17.0weeks) and at a late second/third trimester study visit. Prenatal infections were identified as IgG/IgM seroconversion or a fourfold rise in IgG antibody titers. Multiple regression models were adjusted for maternal age, BMI, smoking status, and time between blood draws. RESULTS: CT infection was associated with preeclampsia (ORadj 1.6, 95% CI 0.7, 3.6), severe preeclampsia (ORadj 1.8, 95% CI 0.6, 5.3), and preeclampsia resulting in preterm birth (ORadj 1.7, 95% CI 0.6-4.9) or birth of a small for gestational age infant (ORadj 2.1, 95% CI 0.6, 7.5), although CT infection was uncommon (n=33, 4.0%) and associations were not statistically significant. CP, CMV, and HSV infection were not associated with preeclampsia. CONCLUSIONS: Women with serological evidence of prenatal CT infection were more likely to develop preeclampsia, although infection was infrequent and confidence intervals were wide. Studies in populations at higher risk for STIs are needed to corroborate this association.

Immunochromatic kits Xpect Legionella and BinaxNOW Legionella for detection of Legionella pneumophila urinary antigen have low sensitivities for the diagnosis of Legionnaires' disease.

Urinary antigen tests are the most widely used methods for diagnosing Legionnaires' disease (LD). However, all available urinary antigen tests have the disadvantage that they have low or no sensitivity for serogroups (sgs) other than Legionella pneumophila sg 1. Recently, Oxoid introduced the Xpect Legionella test for detection of L. pneumophila sg 1 and sg 6. In this study, we have evaluated the Xpect kit together with the BinaxNOW kit and compared them with the BinaxEIA kit. One hundred and fifteen urine samples from 91 patients with laboratory-confirmed LD were examined. Ninety-three samples were from 69 culture-proven cases of which 27 samples were from 23 non-sg 1 cases. At the patient level, the overall sensitivities for the three Legionella urinary antigen kits were 79 % for the BinaxEIA, 47 % for the BinaxNOW and 32 % for the Xpect kit. None of the urine samples from the 10 L. pneumophila sg 6 cases were positive by the Xpect kit whereas samples from four of the patients were positive by the BinaxEIA. Overall, the sensitivities for both immunochromatic assays were poor and they should not be used as the sole method for the diagnosis of LD.

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 and validation of ELISA for detection of antibodies to Legionella pneumophila serogroup 1, 3 and 6 in human sera.

The aim of this study was to determine whether separate measurement of immunoglobulin (Ig) M and G antibodies to Legionella (L.) pneumophila serogroups (sg) 1, 3 and 6 as single antigens can facilitate an early diagnosis of Legionnaires' disease. The developed ELISA was evaluated and compared with an in-house indirect Legionella immunofluorescence antibody test (IFAT) measuring Total Ig. A total of 193 sera from 128 patients with confirmed L. pneumophila infections were used to assess the sensitivity of the developed ELISA. The sensitivity was assessed in different time-periods after onset of symptoms. It was found that the sensitivity of the test increased during the first month of infection, IgM being the most sensitive; ranging from 13% in the first week after onset of symptoms, 45% in the second week to 84% in the third week; in the fourth (and beyond) week a drop to 67% was observed. The IFAT detecting L. pneumophila sg 1-6 had a sensitivity of 11%, 27%, 80% and 59%, respectively, during these time-periods. The test with the lowest sensitivity was the IgG ELISA (0%, 21%, 36% and 52%), but by combining the IgG results with the IgM results, the overall sensitivity of the assay was improved (13%, 48%, 88% and 70%). This study confirms that detection of IgG and IgM antibodies by ELISA is an important diagnostic tool especially during the initial phase of the disease, when supported by other tests like the urinary antigen test, PCR or culture. Furthermore, we showed that the ELISA is suitable for the detection of significant changes in antibody levels in paired serum samples. It was found that the sensitivity was higher for the ELISA assays than for the IFAT. Both the in-house IgM ELISA and the IFAT had a low false positive rate, while a 14% false positive rate was found for the IgG ELISA among serum samples from patients with other infections.

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.

Clinical utility of urinary antigen detection for diagnosis of community-acquired, travel-associated, and nosocomial legionnaires' disease.

The clinical utility of Legionella urinary antigen assays for the diagnosis of Legionnaires' disease was assessed by using samples from 317 culture-proven cases. The sensitivities of the Binax enzyme immunoassay (EIA) and Biotest EIA were found to be 93.7 and 94.4% for travel-associated infection and 86.5 and 76.0% for community-acquired infection but only 44.2 and 45.7% for nosocomially acquired infection, respectively.

[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.

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.

A multicenter evaluation of genotypic methods for the epidemiologic typing of Legionella pneumophila serogroup 1: results of a pan-European study.

OBJECTIVES: To compare genotypic methods for epidemiologic typing of Legionella pneumophila serogroup (sg) 1, in order to determine the best available method within Europe for implementation and standardization by members of the European Working Group on Legionella Infections. METHODS: Coded isolates (114) of L. pneumophila sg 1 comprising one epidemiologically 'unrelated' (79) and one 'related' panel of isolates (35) were sent to 12 laboratories in 11 European countries. Analysis was undertaken in each laboratory using one or more of the following methods: ribotyping, restriction fragment length polymorphism analysis, restriction endonuclease analysis, pulsed-field gel electrophoresis (PFGE), PCR using arbitrary/repeat sequence primers (AP-, AP/rep-PCR), and amplified fragment length polymorphism (AFLP) analysis. Results were analyzed visually or using gel analysis software. Each method was assessed for its: index of discrimination (D), epidemiologic concordance (E), speed of application and ease of use. In addition, phenotypic analysis was performed in two laboratories using monoclonal antibodies (mAbs). RESULTS: The D of each of the genotypic methods ranged from 0.840 for ribotyping to 0.990 for PFGE using Sfil: E ranged from 0.06 for AP- and AP/rep-PCR to 1.00 for ribotyping using Pstl/EcoRI and AFLP: in general, E was inversely related to D. Although offering only limited discrimination (D=0.838), mAb typing was both rapid and highly epidemiologically concordant (E=1.00). CONCLUSIONS: Two methods, PFGE using Sfil and AFLP, were selected for further study. AFLP is rapid and highly epidemiologically concordant (E=1.00), but is not highly discriminatory. This method will be developed as a rapid screening tool. PFGE using Sfil is highly discriminatory but, in the present study, yielded low values of E (0.12-0.71). Attempts will be made to rigorously standardize this method for use as the reference method. Primary screening of isolates by mAb subgrouping is recommended.