Legionella Pneumophila Antigen Research Articles

Graphene oxide/Cu–MOF-based electrochemical immunosensor for the simultaneous detection of Mycoplasma pneumoniae and Legionella pneumophila antigens in water

The combination of copper–metal organic framework (Cu–MOF) with graphene oxide (GO) has received growing interest in electrocatalysis, energy storage and sensing applications. However, its potential as an electrochemical biosensing platform remains largely unexplored. In this study, we introduce the synthesis of GO/Cu–MOF nanocomposite and its application in the simultaneous detection of two biomarkers associated with lower respiratory infections, marking the first instance of its use in this capacity. The physicochemical properties and structural elucidation of this composite were studied with the support of XRD, FTIR, SEM and electrochemical techniques. The immunosensor was fabricated by drop casting the nanocomposite on dual screen-printed electrodes followed by functionalization with pyrene linker. The covalent immobilization of the monoclonal antibodies of the bacterial antigens of Mycoplasma pneumoniae (M. pneumoniae; M. p.) and Legionella pneumophila (L. pneumophila; L. p.) was achieved using EDC-NHS chemistry. The differential pulse voltammetry (DPV) signals of the developed immunosensor platform demonstrated a robust correlation across a broad concentration range from 1 pg/mL to 100 ng/mL. The immunosensor platform has shown high degree of selectivity against antigens for various respiratory pathogens. Moreover, the dual immunosensor was successfully applied for the detection of M. pneumoniae and L. pneumophila antigens in spiked water samples showing excellent recovery percentages. We attribute the high sensitivity of the immunosensor to the enhanced electrocatalytic characteristics, stability and conductivity of the GO–MOF composite as well as the synergistic interactions between the GO and MOF. This immunosensor offers a swift analytical response, simplicity in fabrication and instrumentation, rendering it an appealing platform for the on-field monitoring of pathogens in environmental samples.

Detection of Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila in children with acute respiratory infection

Objective To analyze the characteristics of Mycoplasma pneumo-niae(MP), Chlamydia pneumo-niae (CP), Legionella pneumophila (LP) in hospitalized children with acute respiratory tract infection. Methods Serum and urine were collected from 5275 children (aged from 3 days to 16 years old) admitted with acute respiratory tract infection from Jan. to Dce. 2012. The levels of MP-IgM, CP-IgM in serum, and LP antigen in urine were detected by passive agglutination, enzyme-linked immunosorbent assay, and immunochromatographic assay respectively. The epidemiology of positive cases were analyzed. Results 1. Among 5275 children with acute respiratory tract infection, 1537 cases (29.1%) were detected with at least 1 type of the pathogens: MP infection was 20.1% (1060 cases), CP infection was 8.7%(45 cases), and LP positive rate was 0.3%(18 cases), mix infection rate was 2.56%(135 cases).2. The prevalence of MP, CP and LP infections in the different months and quarters were significantly different(all P<0.01). The positive rate of MP was highest in November while it was lowest in May. The positive rate of CP was highest in November while it was lowest in July. The positive rate of LP was highest in December, no infection case was found in February, May and the time during July to November.3. In all, the occurrence of positive results of MP, CP, and LP in children were higher in winter than that in other seasons. The infection of December was more than that in the other month. Meanwhile, infection of MP, CP and LP were mostly occured in children over age of 5. In addition, the positive rates were higher in male than that in female. Furthermore, children with bronchial pneumonia had higher positive rates of infection compared to children with acute respiratory tract infection. Conclusion Pathogen detection is an important basis for the diagnosis of children with the infection of MP, CP, LP. Key words: Acute respiratory tract infection; Mycoplasma pneumoniae; Chlamydia pneumoniae; Legionella pneumophila; Child

Legionellosis presenting as singultus and external ophthalmoplegia

We report a 71-year-old man with legionellosis, who presented with abducens nerve palsy, singultus, confusion, memory impairment, ataxia, and hyporeflexia. Legionella pneumonia was diagnosed on the basis of detection of Legionella pneumophila antigen in the urine. The cerebrospinal fluid was negative for the antigen and antibody, but an oligoclonal band was detected, and the IgG index was elevated. It was speculated that an undetermined immune-mediated mechanism had contributed to the development of the neurological manifestations.

Clinical impact of combined viral and bacterial infection in patients with community-acquired pneumonia

New methods for identifying respiratory pathogens have led to several reports of a high yield of mixed infections in patients with community-acquired pneumonia (CAP). The clinical impact of these findings has, however, not been fully evaluated. We aimed to compare patients with a pure bacterial etiology with those with findings of both bacteria and virus regarding severity of illness and length of hospital stay. Adults with CAP admitted to Karolinska University Hospital were studied prospectively (N = 184). Microbiological methods included cultures from blood, sputum and nasopharyngeal secretions; sputum samples analyzed with quantitative real-time polymerase chain reaction for Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis; nasopharyngeal specimens analyzed with polymerase chain reaction and serology for Mycoplasma pneumoniae, Chlamydophila pneumoniae and viruses common in the respiratory tract; and urine antigen assays for detecting pneumococcal and Legionella pneumophila antigens. The pneumonia severity index (PSI) was used to assess the severity of illness. The likelihood of getting a score corresponding to PSI classes IV or V was higher in patients with findings of both bacteria and virus than in those with a bacterial pathogen alone (odds ratio 4.98, 95% confidence interval 2.09-11.89; p < 0.001). The median length of hospital stay was 7 days among patients with mixed infections and 4 days among those with a bacterial etiology alone (p = 0.018). Patients infected with a virus and a bacterial pathogen more often develop severe CAP and have a longer hospitalization than those with a bacterial etiology alone.

Cytomegalovirus Viremia, Pneumonitis, and Tocilizumab Therapy

Cytomegalovirus Viremia, Pneumonitis, and Tocilizumab Therapy

Legionnaires’ disease in a neonatal unit of a private hospital, Cyprus, December 2008: preliminary outbreak report

We report an outbreak of Legionnaires disease in neonates, affecting 11 newborn babies. The case fatality rate is currently 27%. The outbreak has been confirmed by detection of Legionella pneumophila antigen in eight of the 11 cases. Tests are in progress to determine the source of infection.

Severe form of Legionnaires' disease in an immunocompetent patient

Legionnaires' disease (LD) is a pneumonia caused by Legionella pneumophila (LP). The disease occurs more often in immunocompromised persons and can be manifested by severe pneumonia, multiple organ failure and has a high mortality. Immunocompetent patient, male, 53-year old, with severe form of LB had fever, cough, weakness and diarrhea as the initial symptoms of the disease. Laboratory results showed increased number of leukocytes, increased values of acute phase proteins, liver enzymes and hyponatremia. Computed tomography of the chest showed the marked inflammatory lessions on both sides. Pathohistological analysis of the samples retrieved by bronchoscopy pointed to a pneumonia, and diagnosis of LD was confirmed by positive urine test for LP antigen. Later, the disease was complicated by acute adult respiratory distress syndrome (ARDS). Treatment with antibiotics (erythromycin, rifampicin, azithromycin) combined with ARDS treatment led to a clinical recovery of the patient together with complete resolution of inflammatory lesions seen on chest radiography. In severe pneumonias it is necessary to consider LD in differential diagnosis, perform tests with aim of detecting LP and apply adequate antibiotic treatment in order to accomplish positive outcome of the therapy and prevent complications.

Legionnaires' Disease with Hypoperfusion in the Cerebellum and Frontal Lobe on Single Photon Emission Computed Tomography

A 59-year-old man was admitted for further investigation of headache. Neurological examination revealed memory loss, disorientation, and bilateral intention tremor. Legionella pneumophila antigen was detected in the urine. Brain magnetic resonance diffusion-weighted images showed marked hyperintensity in the splenium of the corpus callosum without other abnormalities. Single photon emission CT with Tc-99m hexamethyl-propyleneamine oxime showed multi-focal hypoperfusion in the brain, involving mainly the cerebellum and frontal lobe. This is the first report demonstrating cerebellar and frontal lobe hypoperfusion without corresponding MRI abnormalities in a patient with central nervous system Legionnaires' disease.

Detection of Legionella pneumophila in water samples by quantitative culture and an antigen detection assay

We evaluated the new Legionella pneumophila antigen detection assay Binax EquateTM for quantitative determination of legionellae in potable water samples. Seventy-seven water samples from different sources were investigated by Binax EquateTM and quantitative culture. Our culture assay is able to detect 20 to 40 cfu per 100 ml water. The rates of detection of legionellae were 1% (1 of 77) for the antigen detection assay and 25% (19 of 77) by culture. We were able to detect antigen in one water sample with 28 cfu per ml L. pneumophila serogroup 1. In in-vitro experiments the antigen assay had a sensitivity of about 333 cfu per ml when the bacteria were added directly to the test tubes and about 1000 cfu per ml when a simulated water sample was investigated. None of the water samples positive for L. pneumophila serogroup 2 to 14 was positive in the Binax EquateTM. The new antigen assay proved to be a valuable tool for investigating heavy L. pneumophila Serogroup 1 contamination in potable water systems but lacks sufficient sensitivity to be used in the surveillance of water supplies.

Assessment of a new test to detect Legionella urinary antigen for the diagnosis of Legionnaires’ Disease

Given that the rate of mortality by Legionella pneumonia increases in incorrectly treated patients, rapid diagnosis and early antibiotic treatment are needed. We have assessed the performance of a new enzyme immunoassay (EIA) test (Bartels Inc. Trinity Biotech Company, Wicklow, Ireland) to detect Legionella pneumophila antigen in urine comparing it to Binax EIA (Binax, Portland, Maine). We also evaluated the capability of both EIAs to detect extracted soluble antigens of Legionella strains. Using nonconcentrated urine samples (NCU) the sensitivity of Bartels EIA was 74.1% (66/89) and the sensitivity of Binax EIA was 51.7% (46/89). The sensitivity of both EIA tests were 91.5% (54/59) using concentrated urine samples (CU). Specificity of both EIA tests was 100% in NCU and CU. Bartels EIA was able to detect all serogroup L. pneumophila antigens, achieving a higher sensitivity in the case of L. pneumophila serogroup 1 soluble antigen. The new EIA was found to be a useful test for the rapid diagnosis of Legionella pneumonia, being a better alternative to the Binax EIA if NCU is used.

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.

De novo synthesis of Legionella pneumophila antigens during intracellular growth in phagocytic cells.

Legionella pneumophilia is a gram-negative rod which is able to multiply within phagocytic cells. The process of phagocytosis leads to a rapid environmental change that might require a coordinate regulation of gene expression to ensure intracellular survival. Since there is little information on up- and downregulation of genes during the early phases of phagocytosis, we radiolabeled intracellular L. pneumophila at different times after phagocytosis by macrophages of the Mono Mac 6 cell line and immunoprecipitated antigens with antilegionella sera or monoclonal antibodies. We could identify two antigens which were upregulated, one of which was the Mip protein, three antigens which were downregulated, and three antigens which were not detectable in extracellularly grown L. pneumophila. The Mip protein was stained most intensively 4 to 8 h after intracellular infection, suggesting that it is needed during intracellular multiplication rather than initiation of infection. A 44-kDa antigen which was not detectable during extracellular growth was most prominent from 2 to 4 h postinfection when Mono Mac 6 cells were used as phagocytic cells. The 44-kDa antigen was also expressed during growth with Acanthamoeba castelanii, MRC-5, and U937 cells but with different kinetics. Synthesis of this antigen was not dependent on protein synthesis of the host cell. Since the 44-kDa antigen could be precipitated by an antiserum produced against a recombinant Escherichia coli harboring a plasmid with an L. pneumophila insert which also codes for the mip gene, we believe that the corresponding gene is within the vicinity of the mip gene. We named this protein legionella intracellular growth antigen (LIGA), since it could be found exclusively in intracellularly grown L. pneumophila.

Characterisation of Legionella pneumophila antigen in urine of guinea pigs and humans with Legionnaires' disease

To study and develop the urinary antigen detection assay, urine was obtained from aerosol infected guinea pigs. The appearance of Legionella pneumophila antigen in guinea pig urine was then compared with that detected in urine from human Legionnaires' Disease (LD) cases. This study demonstrated that the antigen expressed in the experimental model of LD was of identical molecular weight, reacted almost identically in an ELISA based assay and in Western blots with the antigen found in human urine. The guinea pig urine can therefore be used as a reliable, consistent and controllable source of antigen for use in the urinary antigen assay.

Human and guinea pig immune responses to Legionella pneumophila protein antigens OmpS and Hsp60

We studied the immune responses of guinea pigs and humans to two Legionella pneumophila antigens. Guinea pigs surviving a lethal intraperitoneal challenge dose of virulent L. pneumophila exhibited strong cutaneous delayed-type hypersensitivity (DTH) reactions to purified OmpS (28-kDa major outer membrane protein) and Hsp60 (heat shock protein or common antigen), while weak DTH reactions were noted for extracellular protease (major secretory protein [MSP] [ProA]) and no reaction was observed with an ovalbumin (OA) control. Lymphocyte proliferation responses (LPRs) were measured for peripheral blood and spleen lymphocytes from guinea pigs surviving sublethal and lethal challenge doses of L. pneumophila. Lymphocytes from uninfected animals showed no proliferation to Hsp60 or OmpS, while lymphocytes from sublethally and lethally challenged animals exhibited strong proliferative responses to Hsp60 and OmpS. Guinea pigs vaccinated with purified OmpS exhibited low antibody titers and strong DTH and LPRs to OmpS, whereas lymphocytes from animals vaccinated with Hsp60 exhibited weak DTH responses and high antibody titers to Hsp60. All guinea pigs immunized with OmpS survived experimental challenge with L. pneumophila (two of two in a pilot study and seven of seven in trial 2) versus zero of seven OA-immunized controls (P = 0.006 by Fisher's exact test). In three vaccine trials in which animals were vaccinated with Hsp60, only 1 guinea pig of 15 survived lethal challenge. Peripheral blood lymphocytes (PBLs) from humans with legionellosis showed stronger LPRs to OmpS than PBLs from humans with no history of legionellosis (P = 0.0002 by Mann-Whitney test). PBLs of humans surviving legionellosis exhibited a lower but highly significant proliferative response to Hsp60 (P < 0.0001 compared with controls by Mann-Whitney test). These studies indicate that OmpS and Hsp60 are important antigens associated with the development of protective cellular immunity. However, as determined in vaccine trial studies in the guinea pig model for legionellosis, the species-specific antigen OmpS proved much more effective than the genus-common Hsp60 antigen.

Use of latex agglutination technique for detecting Legionella pneumophila (serogroup 1) antibodies.

Following the outbreak of Legionnaires' disease in Stafford in 1985, 500 serum samples were submitted to the indirect immunofluorescence antibody test and a latex agglutination. Latex agglutination using ultrasonically disrupted Legionella pneumophila antigens coupled to latex particles, proved a rapid, simple method for detecting circulating antibodies to L pneumophila in a one minute slide latex agglutination test. There was good correlation with the indirect immunofluorescence antibody test (IFAT), and the specificity and sensitivity with respect to a diagnostic result were 98.3% and 97.6%, respectively, using a series of well characterised sera. The latex agglutination test seems well suited as a screening test for presumptive cases of Legionnaires' disease; the latex reagent is easy to prepare and seems to remain stable at 4 degrees C for up to six months.

Induction of Interferon-Gamma and Tumor Necrosis Factor by Legionella pneumophila: Augmentation of Human Neutrophil Bactericidal Activity

We have previously reported that Legionella pneumophila antigens can induce interferon-gamma (IFN-gamma) and tumor necrosis factor (TNF) in vitro and in vivo in mice. Furthermore, treatment of murine polymorphonuclear leukocyte (PMN) cultures with these cytokines resulted in augmented killing of the bacteria in vitro. The purpose of the present study was to determine if these findings could be extended to human responses. Here we report that Legionella antigens induced IFN-gamma and TNF in nonimmune human leukocytes cultures, and that these cytokines were able to stimulate the bactericidal activity of isolated PMN against L. pneumophila in vitro. Furthermore, optimal production of IFN-gamma was found in cultures which were enriched for large granular lymphocytes (LGL). The phenotype of IFN-producing cells was determined to be CD11+, CD16+, CD2+, and negative for CD4, CD8, CD14, and Leu 7. Additionally, Legionella-infected monocytes were found to produce TNF in a dose-dependent response to the number of infecting bacteria, and the addition of recombinant IFN-gamma to infected monocytes resulted in augmented production of TNF in a synergistic manner. Finally, treatment of PMN with recombinant IFN-gamma and recombinant TNF augmented their bactericidal activity against Legionella in a dose-dependent response. Thus, cytokines which can be induced by L. pneumophila antigens are able to stimulate PMN function in vitro, suggesting that resistance to infection results from a complex interaction of cytokines and cell responses.

The early diagnosis of legionnaires' disease in a Legionella pneumophila aerosol-infected guinea pig model; comparison of a method developed for detecting Legionella pneumophila antigens in urine and the demonstration of circulating antibody by enzyme-linked immunosorbent assay

The appearance of Legionella pneumophila antigen in the urine of guinea-pig with experimental airborne legionnaires' disease was investigated and compared with that of emerging antibody by use of enzyme-linked immunosorbent assay. Models with high dose (acute) and low dose (chronic) infection were studied. Antigen was detected after 40 h in the high dose group but animals died (at 3 days) before an antibody response could be elicited. In the low dose group, antigen was detected 4 days after infection, well before serum antibody was detected (7-10 days). Small, but significant, amounts of antigen were detected up to 17 days after infection in surviving animals. Although detection of L. pneumophila antigen in urine has been proposed before, and achieved on an ad hoc basis, the technique is not in routine, general use. This is due mainly to difficulties of evaluation in relation to other methods of early diagnosis in the human situation where infectious dose, time of infection, host uniformity and availability of samples present difficulties. The use, in this study, of a highly relevant aerosol-infected guinea-pig model of legionnaires' disease has avoided these uncertainties and hopefully proved the value of this technique for general routine use. The antigen detection test was shown to be rapid, sensitive and reliable, and allowed diagnosis of legionnaires' disease earlier than was possible by demonstrating antibody. In addition, the detection of antigen in urine is a convenient and non-invasive procedure.

The simultaneous direct detection of Mycoplasma pneumoniae and Legionella pneumophila antigens in sputum specimens by a monoclonal antibody immunoblot assay

An immunoblot (Western) assay was developed employing a species-specific monoclonal antibody to a 43 kDa Mycoplasma pneumoniae membrane polypeptide and a species-specific monoclonal antibody to 29 kDa Legionella pneumophila outer membrane protein. This assay could simultaneously detect these two different antigens directly in sputum. The 43 kDa M. pneumoniae antigen was detected by this assay in each of three M. pneumoniae culture-confirmed sputum specimens. In addition, the 29 kDa L. pneumophila antigen was detected in three of three L. pneumophila culture-confirmed sputum specimens. Neither of these two specific antigens were detected in induced sputum specimens from ten normal individuals.

Induction of interleukin 1 by Legionella pneumophila antigens in mouse macrophage and human mononuclear leukocyte cultures

Exposure to Legionella pneumophila antigens has been reported to result in both an adjuvant effect and pathophysiological changes such as fever, headache, myalgia and arthralgias. Immunoenhancement and inflammatory changes have been associated with the production of interleukin 1, and we, therefore, sought an involvement of interleukin production in the alteration of biological responsiveness following exposure to Legionella pneumophila antigens. Killed Legionella pneumophila cells, incubated with mouse splenocytes, induced the formation of a soluble substance which enhanced splenocyte antibody production to heterologous antigen. The immunoenhancing substance was also produced by mouse peritoneal macrophages and supernatants from these cultures were demonstrated to also contain thymocyte co-mitogenic activity. Following gel filtration, this co-mitogenic activity eluted in the 15,000 molecular weight range suggesting an involvement of interleukin 1. Experiments with Legionella pneumophila cells, and cell extracts containing endotoxin, and purified endotoxin suggested that the interleukin 1 activity was induced by both endotoxin and non-endotoxin antigens. The Legionella pneumophila antigens were also found to be potent inducers of interleukin 1 activity in human peripheral blood mononuclear cell cultures. These results suggest that Legionella pneumophila antigens are potent inducers of interleukin 1 in both mouse and human cells. The induction of this monokine may partially acount for both the immunoenhancing property of this bacterial species and the associated pathophysiological changes following infection with this microorganism.

Kinetics and characterization of interferon production by murine spleen cells stimulated with Legionella pneumophila antigens

Formalin-killed Legionella pneumophila bacterial cells, as well as a purified cell wall preparation (designated F-1 antigen) containing lipopolysaccharide (LPS), stimulated production of interferons (IFNs) in mouse spleen cell cultures. L. pneumophila whole-cell vaccine induced an IFN that was pH 2 labile and neutralized by anti-IFN-gamma indicating that IFN-gamma was the dominant form present. F-1 antigen induced a mixture of IFNs, depending upon the age of the culture and cell types present. In freshly prepared whole-spleen cultures and in 2-h adherent cultures, F-1 induced predominantly IFN-alpha/beta. In whole-spleen cultures that were allowed to age for 24 to 48 h before stimulation, F-1 was seen to induce mostly IFN-gamma, with low levels of IFN-alpha/beta present. Since only IFN-alpha/beta was produced in T-cell-depleted populations (at 2 h or at 48 h), it is suggested that T cells are responsible for IFN-gamma production in aged cultures. Additionally, heat-treated F-1, Escherichia coli LPS, and heat-treated E. coli LPS all induced similar levels of IFN-gamma in whole-splenocyte or nonadherent cell cultures which were incubated 48 h before stimulation. This suggests that LPS present in F-1 is responsible for IFN-gamma production and that an activated cell population is required. These results show that L. pneumophila antigens can induce the production of various types of IFN in mouse spleen cell cultures through several mechanisms.