Sero diagnosis of Legionella infection in community acquired pneumonia.

Background & objectives: Legionella pneumophila has been increasingly recognized as an emerging pathogen responsible for community acquired pneumonia (CAP) worldwide. In India, the actual burden is not known. The present study was thus undertaken to see the presence of Legionella infection in patients with community acquired pneumonia admitted in a tertiary care centre in north India. Methods: Both children and adults (n=113) with symptoms of pneumonia were included in the study. Clinical samples (blood, urine, nasopharyngeal aspirates, bronchoalveolar lavage, sputum, etc.) were collected and subjected to culture and other tests. Enzyme linked immunosorbent assay (ELISA) was done by commercial kits for all the three classes of immunoglobulins (IgG, IgM & IgA). Urinary antigen was also detected using commercial kits. Culture was performed on 51 respiratory tract fluid samples. Serum samples of 44 healthy controls were also screened for the presence of anti-legionella antibodies (IgG, IgM & IgA). Results: Thirty one of the 113 cases (27.43%) were serologically positive. Anti-legionella IgG, IgM and IgA antibodies were positive in 7.96, 15.92 and 11.50 per cent patients respectively. In controls, seropositivity was 9.09 (4/44). IgA was positive in 3 and IgM, IgG combined in one. Antigenuria detection by Microwell ELISA kit showed 17.69 per cent positivity. Four antigenuria positive patients were also serologically positive; of these two patients were positive for IgM, hence considered as confirmed cases of Legionella infection. None of the sample was culture positive. Interpretation & conclusions: Combination of serology and antigenuria detection may be a valuable tool for the diagnosis of Legionella infection in absence of culture positivity. In order to evaluate the actual burden of Legionella in community acquired pneumonia, further studies with larger samples need to be done.

[Isolation of legionella pneumophila from environment and water system samples and evaluation of immuno-protective efficiency of its whole killed cell in mice model]

Legionella pneumophila is a cause of pneumonia in human beings. The purpose of this study was to separate L.pneumophila from stagnant and waste water, city squares, coolers and faucets and evaluation of the immunoprotective efficiency of its whole killed cells in mice model. Water samples were prepared, concentrated and then cultured on selective [GVPC] media. After identification of L.pneomophila the biomass of the organism was fixed with 0.5% formalin in sterile saline at 37§C for 24 hours in order to prepare whole killed cells. Four groups of female BALB/c mices [each group consisted of 15 mices] were selected. Two groups of mice were immunized by three intraperitoneal administrations of prepared antigen in a dose of 4x108 CFU from whole killed cells at two week intervals and control groups received only sterile saline injections. Two weeks after the last injection, one group of immunized mice and one of the control groups were challenged with the lethal dose of virulent strain of L.pneuophila and also the two other groups of mice were challenged six weeks after the last immunization. From 120 water samples 27 samples were contaminated with L.pneumophila. Challenge results showed that the immune efficiency of whole killed cell was 93.33% after two weeks of the last immunization, and 86.66% after six weeks of the last immunization. This study showed that, stagnant water had the highest rate of contamination with L.pneumophila and the whole killed cell of L.pneumophila is a proper candidate for L.pneumophila vaccine studies

Legionella lipoprotein activates toll-like receptor 2 and induces cytokine production and expression of costimulatory molecules in peritoneal macrophages

Legionella bacterium, an intracellular pathogen of mononuclear phagocytes, causes acute fatal pneumonia, especially in patients with impaired cellular immune responses. Until recently, however, the toll-like receptor (TLR) engagement of bacterial proteins derived from Legionella is uncertain. We previously showed that a 19-kDa highly conserved peptidoglycan-associated lipoprotein (PAL) of Legionella pneumophila induced the PAL-specific B cell and T cell responses in mice. In this study, we observed that the rPAL antigen of L. pneumophila, as an effector molecule, activated murine macrophages via TLR2 and produced proinflammatory cytokines such as IL-6 and TNF-alpha. In both BALB/c and TLR4-deficient C3H/HeJ mice, pretreatment of macrophages with anti-TLR2 mAb showed severely impaired cytokine production in response to the rPAL. In addition, in vitro the rPAL treatment increased the cell surface expression of CD40, CD80, CD86 and MHC I/II molecules. We further showed that the synthetic CpG-oligodeoxynucleotides (CpG ODN) coadministered with the rPAL enhanced IL-12 and IL-6 production and expression of CD40, CD80 and MHC II compared to the rPAL treatment alone. In conclusions, these results indicate that Legionella PAL might activate macrophages via a TLR2-dependent mechanism which thus induce cytokine production and expression of costimulatory and MHC molecules.

Titration of specific antibodies to mycoplasma pneuomoniae, Chlamydia pneumoniae and Legionella pneumophila among Iranian pilgrims' sera during Hajj season in 2004

Respiratory tract infection is the most common diseases among Iranian pilgrims during Hajj season. To understand the possibility of bacterial involvement in such infections, we screened the pilgrims' sera to determine the titer of antibodies against Mycoplasma pneuomoniae [MP], Chlamydia pneumoniae [CP] and Legionella pneumophila [LP]. Serum samples from 128 pilgrims were collected, before the trip and one month after returning home. Antibodies to MP, CP, LP were assayed using Immunoflourecent and ELISA methods. IgM antibody titre to CP did not elevated, but IgG antibody titer was increased in 34.58% [n=48] and 15.82% [n=22] of cases, indicating of recent infection. The specific antibodies to MP and LP were not increased. In pilgrims infected with an atypical respiratory pathogen, C. pneumoniae should be considered as an important causative. The true prevalence of this pathogen should be investigated since it relies on the sensitivity and specificity of currently available diagnostic methods