International Journal of Medical Laboratory

Introduction
Legionnaires’ disease (LD) is an acute pneumonic illness caused by gram-negative bacilli of the genus Legionella, the most common of which is Legionella pneumophila (L.pneumophila) [1]. L.pneumophila is a motile, gram-negative, rod-shaped facultative intracellular bacteria belonging to the genus Legionella. The genus Legionella consists of 48 species and 70 serogroups. There are currently at least 16 serogroups of L.pneumophila. Serogroup 1. L.pneumophila is the most common cause of Legionnaires’ disease; it causes 80% of all reported cases of legionellosis [2]. It distributes in the natural aquatic environment and spreads by inhalation of aerosolized biofilm droplets containing the bacteria and by invading the lungs, cause pneumonia. Pneumonia due to L.pneumophila is termed Legionnaires’ disease. The Legionnaires' disease is a severe, life threatening pneumonia [3, 4]. Legionella infections are responsible for 2-15% of community-acquired pneumonia. Morbidity and mortality depends on the underlying diseases of the patient, early treatment and whether the disease is sporadic, nosocomial or part of an outbreak. Outbreaks occur in community acquired and nosocomial settings. Due to the increased tolerance to chlorine, it can proliferate in thermal habitats, including air-conditioning towers, hot water systems, showerheads, taps, spas and respiratory ventilators [2]. There are some specific diagnostic tests for legionnaires’ disease caused by L.pneumophila including:
1-         Culture, which done in sputum or other lower respiratory tract secretions, blood and extra pulmonary tissues or fluids. Sensitivity and specificity of the culture of Sputum is 20-95% and 100%, respectively. May be positive up to several days after treatment; it requires special media and expertise [1].
2- Urine antigen testing: This specific test can be easily performed by those without special skills and is often positive when other tests are negative. The test is not perfect because it is most sensitive for the detection of the Pontiac subtype of L.pneumophila serogroup 1 (up to 90%). Sensitivity and specificity of urinary antigen is 60-95% and >99%. The highest sensitivity is for L.pneumophila serogroup 1, pontiac type; may remain positive for days to months 3-       Immunofluorescent microscopy: Sensitivity and specificity is 20-50% and 99%. The highest specificity is associated with monoclonal antibody; but it requires high level of technical expertise 4-            Serology: Sensitivity and specificity of antibody-paired serum is 20-70% and 95-99%. The highest specificity is for L. pneumophila serogroup 1. Serology is the method most commonly used for the diagnosis of Legionella infections. The sensitivity of serology is generally limited by the time required to develop a detectable antibody response during the course of the infection and by the proportion of infected patients who respond immunologically. Approximately 20-30% of patients do not develop significantly elevated antibody titres, even after prolonged observation. This limits the overall sensitivity of serology to 70-80%. Some patients (10%) even seroconvert as late as 6-9 weeks after onset of the disease. The specificity of seroconversion (Fourfold titre rise) using L.pneumophila SG 1 antigen in the immunofluorescence assay test has been reported to be approximately 99%.
5- Molecular amplification: Sensitivity and specificity of molecular amplification sputum, other lower respiratory tract secretions; urine is 70-95% and 90-95%, it is not well standardized [1].
The frequency of L.pneumophila is different in different societies, which can be derived from environmental, health, social or samples and diagnostic methods used to determine the frequency of this microorganism [5]. Garbino et al., in a Swiss study in 2002, reviewed 318 patients with community acquired pneumonia  in a prospective study. The prevalence of Legionella antigen in this study was 1.8% [6]. In the study of  Den Boer, L.pneumophila was responsible for 2-5% of acquired pneumonia cases in different regions [7]. Limited studies conducted in Iran (Tehran and Isfahan) have obtained this rate from 2.5 to 8.8% [8, 9].
In spite of dramatic improvement in infectious medicine, but the prominence of pneumonia as a clinical entity remains. The clinical challenge of community-acquired pneumonia (CAP) includes the increasing number of microbial agents that can cause infectious diseases, the difficulty in making etiologic diagnosis due to lack of all new laboratory diagnostic method, and the fact that no single antimicrobial regimen can cover all the possible causes. Because a specific etiologic diagnosis is often not possible at the beginning of the treatment, the empirical therapy is most appropriate. The increasing prevalence of antibiotic resistance among many of the most common pathogens has made this challenge more difficult. Understanding of microbial etiology aids to reasonable therapy [1].
The information about the prevalence of this bacterium in the patients with pneumonia in Kashan is not available, and that the study of urine antigens is a simple method, while also having high sensitivity and high resolution. This study was designed to determine the frequency of legionell pneuomonia and identification of L.pneumophila among the patients with CAP referred to Kashan Shahid Beheshti Hospital by using urinary antigen test.
Materials and Methods
Study population and setting
This descriptive cross sectional study was conducted in Kashan Beheshti Hospital, in 2015. Our sampling strategy was simple. Inclusion criteria were recorded pneumonia as the primary diagnosis in medical record according to the clinical symptoms and signs (fever, cough, chest pain, sputum, dyspnea) and infiltration in chest X ray in a hospitalized patient aged above 13 years and the presence of signed consent form. Exclusion criteria were as follows: age under 13, and normal chest X ray. Participants were given information about the objectives of the study and informed consent was obtained. 10 ml of urine sample was collected and sent to laboratory where the urine was used to be tested by enzyme-linked immunosorbent assay (ELISA) method. The Legionella urinary antigen EIA (Binax, Inverness Medical: Scarborough, Maine) with sensitivity of 70-90% and specificity approaching 100% for L.pneumophila serogroup 1 was used. A questionnaire consisting the demographic information, risk factors, clinical symptoms and physical findings was filled out through direct interview with the patients and physical examination.
Interpretation
Interpretation of results was done by referring to the enclosed visual read card for color comparisons.
Interpretation of results - visual (Manual)
Any sample well that had obvious and significant yellow color interpreted reactive and any sample well that does not have obvious and significant yellow color was non-reactive: A sample well must be obviously darker than the negative control well to be called a positive result.
Interpretation of results
ELISA Reader: Read all wells bichromaticallyat 450 nm and 620-650 nm.
Reactive: Absorbance reading of 0.15 OD units and above indicates the sample contains Legionella antigen.
Non-reactive: Absorbance reading less than 0.15 OD units indicates the sample does not contain detectable levels of Legionella antigen.
Limitation of procedure: Test results should be used as an aid in diagnosis and should not be interpreted as diagnostic by themselves. A negative result can occur from an antigen level lower than the detection limits of this assay. Multiple samples over time may be indicated for those patients that are suspected of being positive for Legionella.
Excretion of Legionella: Antigen in urine may vary depending on the individual patient and the stage of the disease. Some individuals have been shown to excrete antigen for an extended period of time, so a positive ELISA reaction may reflect a recent but not active infection. Early treatment with appropriate antibiotics may also decrease antigen excretion in some individuals. Antigen excretion may begin as early as 3 days after onset of symptoms and persist for up to a year afterwards.
Statistical analysis
Following the recording of all the data, they were analyzed using statistical SPSS version 16. Descriptive statistics, including mean, standard deviation in addition to frequency rate was calculated; Chi-square test was used to test the associations. The significance level was set to 0.05.
Results
In this study, 69(59%) from 117 pneumonia patients were males and 48(41%) were females. The most patients were above 60 years old (Mean age 61.66±1.7 min: 18 max: 92). The most common job was housekeeper 53.8%. Most of the patients were Iranian (99.9%) and residing in urban (71.8%) and literate (64.1%). The most cases (85.5%) were living and working (73.6%) in humid environments. There were 13(11.1%) smokers and 5 (4.3%) addicts (Table1).
Cardiovascular diseases were the most common underlying diseases. The most common chief complaint was productive cough (74.4%), dyspnea (64.1%), myalgia (46.9%), loss of consciousness (11.1%), and diarrhea (6%). Physical examaniation showed rale (52.1%), fever (40.2%), tachypnea (40.2%) and tachycardia (6.4%). There was infiltration in chest X ray of all the patients. There was increased aspartate aminotransferase (38.5%) and alanine aminotransferase (61.5%), leukocytosis 31.6%, increased erythrocyte sedimentation rate 49.9% and positive C-reactive protein 54.7%. There was no significant association between age, sex, job, level of education and humidity in residential site with detection of urinary antigen L.pneumophila, only there was found significant association between smoking and detection of urinary antigen L.pneumophila (Table 2). The study was approved by the Ethics Committee of the Research Deputy in Medical Sciences University of Kashan.
 
 
Table 1. Demographic characteristics of studied patients

 
Table 2. Relationship between clinical symptoms and laboratory finding with detection of urinary antigen L.pneumophilla

 
Discussion
The prevalence of L.pneumophila in our study was 9.4%. In the study carried out by Goudarzi et al., which done on 210 hospitalized children in Tehran (2011), the sputum of 12(5.7%) children with acute respiratory infections was positive for L.pneumophila [5]. In a study of Yazdani et al among the 96 bronchoscopic specimens, 4 strains of gram negative bacilli were isolated. Further specific direct flourcent antibody revealed that they were L.pneumophila [8]. Among 80 serum samples from CAP patients in Ahvaz, 12 cases (15%) were positive for L.pneumophila [10]. In a study done by Benito et al, seroconversion was observed in 54.8% of 97 studied patients [11]. In a study conducted by Garbino et al in Switzerland on 318 CAP patients, L.pneumophila was isolated in 4.4% of cases [6]. In a study of Viasus et al. among 3934 non-immunosuppressed hospitalized patients with CAP, 214 patients (5.4%) had L.pneumophila pneumonia [12]. In the research of Kanavaki et al. in Greek on 88 respiratory infection patients by examination on sputum, serum and urine, L.pneumophila was isolated in 2 (4.3%) in sputum and 6 (6.8%) in urine [13]. From 204 CAP patients in Thailand, only 3 (1.5%) had urinary antigen of L.pneumophila [14]. In a study performed by Dionne et al., 1154 tests were performed on 1007 patients. Seven patients had nine positive Legionella urinary antigen tests. Three of these patients had confirmed L.pneumophila pneumonia. Three others had probable or possible L.pneumophila pneumonia [15].
The prevalence in our study was more than other studies, which is probably due to the non-standardization of our heating and cooling systems.
In addition, it seems that the difference in the prevalence may be due to several factors, the most important of which are: a) The diversity of studied societies in terms of social conditions, climate and season of study time. b) Seroprevalence of legionella infection in general population, reflecting the rate of exposure to this organism, for example, in European countries, Spain has the highest prevalence and Austria has the lowest. c) Demography of the patients and the presence of people with underlying illnesses and risk factors such as smoking [16-18]. In our study, among legionella positive cases, 45.5% of cases were over 60 years old and the frequency of legionella prevalence was not significantly correlated with age. In the study carried out by Alavi and Mirkalantary et al., also, there was no significant difference between the positive and negative serology, in this respect, we are in agreement with this study [9, 10]. In our study, 54.5% of positive cases were male. The prevalence of legionella was not significantly correlated with the gender. In Alavi et al, positive antibody in males was more than females, but the statistical analysis did not show a significant difference and in this respect, our results were consistent with this study [10]. In our study, the prevalence of legionella was not significantly correlated with habitat and workplace humidity and underlying diseases; these results were consistent with study of Alavi et al. [10]. 54.5% of the positive cases were smokers and 9.1% were hookah users, and there was a significant relationship between legionella prevalence and tobacco use. In Alavi  et al study, all the patients with positive serology had a positive history of smoking [10]. Therefore, cigarettes are risk factors for the development of legionella pneumonai. Criteria to identify the patients at high risk for L.pneumophila pneumonia are as follows: male gender, cigarette smoking, chronic heart or lung disease, diabetes, end-stage renal failure, organ transplantation, immunosuppression, some forms of cancer, and age older than 50 years [19]. Despite there are some risk factors and clinical features and laboratory findings, which are helpful to suggest a diagnosis of L.pneumophila pneumonia, but exact clinical differentiation from other causes of pneumonia is not possible and the rate of correct diagnosis is about 3% and many cases are often not considered [15]. While L.pneumophila is increasingly recognized as a significant cause of CAP in many countries, it becomes an important public health problem worldwide. Since clinical signs and symptoms are not reliable to diagnose Legionnaires' disease, the use of diagnostic laboratory tests for Legionella is necessary [20]. According to the importance of disease caused by L.pneumophila, laboratory diagnosis of this organism has increased. The value and sensitivity of culture for L.pneumophila has decreased because of the need to the specific environments and specialists for working with it, inability to obtain sputum from half of the patients, inability of microorganism to survive in respiratory secretion for long time, inability of microorganism to grow in culture after starting the antimicrobial treatments [13]. Serology, polymerase chain reaction tests, urinary antigen test are other laboratory diagnostic tests for CAP. Urinary antigen testing has grown in popularity for several significant respiratory infections, particularly Legionella pneumophila, Streptococcus pneumoniae, and Histoplasma capsulatum [21]. Rapid urine antigen tests are very useful to determine CAP etiology in adults. A positive urinary antigen test for Legionella spp. allows an early switch from empiric to targeted treatment in hospitalized, community-acquired pneumonia patients [22]. In a research conducted by Kanavaki et al., sensitivity and specificity of urinary antigen g test was calculated 68-90% and 100%, respectively [13]. Guerrero et al. compared the Bartels enzyme immunoassay, Biotest enzyme immunoassay, and Binax NOW immunochromatographic test urinary antigen kits for the detection of L.pneumophila serogroup 1 using 178 frozen urine samples. When non-concentrated urine samples were used, the sensitivity levels of both enzyme immunoassays were significantly higher than the sensitivity level of the immunochromatographic test. After concentration of the urine samples, no significant differences in sensitivity were found among the three tests [23]. The advantages of L.pneumophila urinary antigen tests are prompt diagnosis due to rapid performing, high specificity, usually detectable at the time of presentation. Urinary antigen tests are quick and simple tests helping to provide an etiological diagnosis in community-acquired pneumonia. Legionella urinary antigen test is the most commonly method used for the diagnosis of legionellosis, but must be prescribed in a specific clinical context [24].
Conclusion
The rate of L.pneumophila pneumonia was 9.4%. Using urinary antigen test could help us to detect L.pneumophila simple and rapid. Urinary antigen test should be the first diagnostic method in our hospital because it is often easier to obtain urine in ill patients and the results could be available within hours and reliable to commence treatment.
Conflict of Interest
The authors have no conflict of interest.
Acknowledgements
The present study was the thesis of a medical student, Maryam Shamaee Zavareh, which was supported by the Research Deputy of Kashan university of medical sciences. The authors gratefully acknowledge the help of Dr. Mansoor Sayyah. We would like to thank our participants for their patience and ongoing participation.
 
 
References