Clinical
The advances in laboratory diagnostic techniques and the completeness of epidemiologic investigations have led to the understanding that Legionella pneumophila can cause not only severe Legionnaires' disease, but also Pontiac fever, which resolves spontaneously with symptoms similar to those of the common cold. In addition to causing severe Legionnaires' disease, Legionella pneumophila can also cause Pontiac fever, which is similar to common cold symptoms; Legionnaires' disease is an important pathogen of community and hospital-acquired pneumonia; however, because its clinical manifestations are often atypical, and the medications used for treatment are different from those used for common community pneumonia, its complications and fatalities are no less serious than those of other pneumococcal organisms; therefore, early and correct diagnosis and rapid use of effective anti-microbial agents are important for the clinical management of Legionnaires' disease. Therefore, early and correct diagnosis and rapid use of effective antimicrobial agents are important in the clinical management of Legionnaires' disease. The Centers for Disease Control (CDC) receives approximately 8,000 to 18,000 reports of Legionnaires' disease each year, but many cases remain unreported or undiagnosed, and the incidence of the disease is much higher than these reports. About 1-5% of public ****site infections in adults are caused by L. pneumophila, and most are sporadic. The general population is susceptible to infection, but it is rare in persons under 20 years of age, and there have been several epidemics of hospital-acquired infections. Infection with Legionnaires' disease is more common in the elderly, smokers, diabetics, patients with chronic lung disease, kidney disease or malignant tumors, immunocompromised patients, steroid users, and organ transplant recipients. Infected patients usually present with high fever, chills, and a dry cough or a small amount of sputum, while others present with muscle aches, headache, abdominal pain, poor appetite, diarrhea, and even central neurological changes and abnormalities of liver and kidney function. Chest X-rays are indistinguishable from other pneumonia infections, and the diagnosis must be supported by specific laboratory cultures and detection of antigens and antibodies. The male-to-female ratio is about 25:1, and the mortality rate of Legionnaires' disease is about 5-30%. Currently, the diagnosis of Legionnaires' disease in China mostly relies on sputum or urine antigen testing or serologic changes in antibody potency, but rarely on culture of clinical specimens. Although sputum or urine antigen test can be used for rapid diagnosis, it is limited to a few serotypes, and serologic diagnosis requires the comparison of the second potency in the acute and recovery phases in order to establish the diagnosis, which is not conducive to the clinical treatment of patients. In Taiwan, the Indirect immunofluorescent test (IFA) has been used to determine the antibody potency of L. pneumophila in the sera of normal male youths as a reference for the diagnosis of this disease [13], and the first case of Legionnaires' disease in Taiwan was confirmed in 1983 [14], followed by other cases in succession. The first case of Legionnaires' disease in Taiwan was confirmed in 1983 [14], and other cases have been detected one after another, including pediatric cases [13-15].
Microbiology
The genus Legionella belongs to the family Legionellaceae, which contains more than 43 different species of bacteria, of which about 20 are associated with human disease; and more than 65 serogroups. Veterans are small, polymorphic Gram-negative bacilli with specific nutritional requirements for growth, and they are water-resident bacteria that optimally reproduce in warm environments at 32-45°C; the temperature range is 0-63°C, pH: 5.0-8.5, and the dissolved oxygen concentration in water is 0.2-15 ppm[15] . L. pneumophila is the most important and common of these; more than 90% of Legionnaires' disease is caused by L. pneumophila serotype 1. There are 15 serotypes of L. pneumophila, with types 1, 4 and 6 being the most common. Other strains of L. micdadei (Pittsburgh pneumonia agent), L. bozemanii, L. dumoffii, L. cincinatiensis, L. feeleii, L. longbecchae and L. orkridgensis are known to cause human infections. L. veterans is commonly found in natural and man-made water and soil environments, but it can also be found and isolated in trace amounts in hospital and domestic water supply systems.
The genus Veterans is very sensitive to the nutritional requirements for growth and does not grow in the BAP and chocolate media routinely used for bacterial cultures. In 1976, microbiologists from the U.S. Centers for Disease Control and Prevention (CDC) investigated an outbreak of a serious group of infections at the VA Annual Meeting and developed a new strain of the genus Veterans with the addition of the amino acids L-cysteine, ferrous pyrophosphate, and alpha-alpha-alpha-alpha-alpha-alpha-alpha-alpha. In 1976, when the microbiologists of the U.S. Department of Disease Control investigated the severe infections of the Veterans' Association, they developed a special basic medium, buffer-charcoal yeast extract agar (BCYE), with the addition of L-cysteine amino acid, ferrous acid, pyrophosphate, and alpha ketoglutarate, and ACES buffer, which has the ability to maintain the pH of the medium at a narrower range of 6.9±0.05. Because of the slow growth rate of Veterans' bacilli, it takes 3 to 5 days to observe the growth of colonies, and the colonies are easily covered by other fast-growing bacilli in upper respiratory tract samples, such as sputum and tracheal tube extracts, which contain normal bacilli and are not easy to observe. Antimicrobial agents are added to the culture medium to inhibit the contaminating organisms in the specimen. Therefore, isolation of pathogenic bacteria from clinical specimens requires the use of both BCYE and two selective media, PAC and PAV, which contain antimicrobial agents such as polymyxin B, anisomycin, and vancomycin, and bromocersol purple, respectively, BCYE-PAV medium with polymyxin B, anisomycin, and vancomycin and bromocersol purple and bromothymol bule dyes, respectively, gave a light green color to L. pneumophila colonies and a blue color to L. micdadei colonies; BCYE-PAC medium requires the addition of antimicrobials such as polymyxin B, anisomycin, and cefamandole. Veteran's bacillus cultures must be collected before antibiotics are started; they must be stored at 4°C for delivery because of contamination with upper respiratory flora. Conventional Gram staining is not diagnostic for Legionella infections. Because of the negative, very faint red coloration, the sputum may show many multinucleated white blood cells on Gram staining and no significant bacteria are stained. 0.05% basic funchin can be used as a substitute for the staining agent, which will still fail to differentiate between Gram-negative organisms. Sputum samples should also be treated with 0.2M HCL-KCL at pH 2.2 for 4 minutes or heated at 50°C for 30 minutes and then cooled immediately to minimize overgrowth of contaminating organisms in the samples, which may affect the interpretation. It should also be noted that the antimicrobials in the medium may affect the growth of some L. veterans, especially cefamandole, which is inhibitory to the growth of L. micdadei, but this bacterium can be grown in PAV. After incubating the agar plates at 70-80% humidity in a 35℃ (carbon dioxide is not required) incubator, the colonies were observed by dissecting microscope on the 3rd day; initially, the Veteran's bacilli colonies appeared to be protruding, rounded, and very pale blue with a slightly cut glassy center and pale iridescent coloring around the center; colonies incubated for a longer period of time would gradually turn white and waxy. Colonies incubated for a longer period of time will gradually turn white and waxy, and when picked with an inoculating loop, the whole colony will be picked up; if no suspected colonies are found after 10 days of incubation, the culture report will be negative [15].
Epidemiology
The humid environment of hospitals with a wide variety of medical equipment and the extensive use of aqueous solutions may provide a reservoir for aquatic microorganisms. Under appropriate environmental conditions (e.g., temperature and nutrient sources), these aquatic microorganisms can multiply rapidly and even stabilize or develop resistance and infectivity to the surrounding environment. Aquatic microorganisms responsible for patient infections during the course of medical care include glucose nonfermenter gram-negative bacteria (GNFGNB), non-tuberculosis mycobacteria (NTM), and Legionella species (LDL). Legionella spp. Transmission of aquatic microorganisms in the hospital environment can occur through (1) direct contact, e.g., hydrotherapy; (2) drinking contaminated water; (3) indirect contact, e.g., contact with water-contaminated medical equipment; (4) inhalation of contaminated water mist; (5) choking on contaminated water; and (6) inhalation of contaminated water. ) contaminated water. In hospitals, non-fermentative Gram-negative bacteria and non-tuberculous mycobacterial infections are often associated with glucose via the first to third routes of transmission, while Mycobacterium avium subspecies is often transmitted by inhalation or choking on contaminated water mist, resulting in respiratory infections in patients.
Cooling towers for air conditioning, evaporative condensers, hot water supply systems, whirlpools, dehumidifiers, fountains, rosettes, and tap water faucets are all high water environments that are favorable for the reproduction of this organism. Community- and hospital-acquired Legionnaires' disease is a serious infection that affects multiple organs of the body and is caused by exposure to water contaminated with Legionella spp. in the environment. Although the clinical manifestations of this disease are mainly respiratory infections, the source of the infection is related to the quality of the water in the environment. Many foreign studies have shown that most of the outbreaks of Veterans' Disease infections in hospitals occur when patients are exposed to showerheads, faucets, respiratory therapies, room-air humidifiers, etc. This increases the risk of the bacteria being exposed to artificial water. Factors that can increase colonization and amplification of this organism in man-made water environments include: (1) water temperatures of 25°-42°C (77-107.6°F), (2) stagnant water currents, (3) the production of scale and sediment such as biofilm, and (4) water that may support free-living, intracellular growth of L. Legionella species. The presence of free-living aquatic amoebae, which can colonize single-cell protozoa in the natural environment and human bronchial macrophages, may support intracellular growth of V. Legionella.
Environmental sampling
The method of prevention and control of Legionnaires' disease infection is recommended to use the Allegheny County Department of Health, Philadelphia, Pennsylvania, recommended guideline published for the first time in the United States in 1993 [16], according to which the method of sampling for the environmental survey of hospitals is to collect the appropriate amount of samples based on the number of beds of the hospitals, and to collect 10 points of samples in wards of less than 500 beds. For hospitals with less than 500 beds, 10 points of ward samples are collected, and for hospitals with more than 500 beds, 2 points of ward samples are collected for every 100 beds. The guidelines have three main recommendations: 1. identification of the problem - culturing of water supply systems for Legionella, 2. diagnosis of cases - providing clinicians with readily available laboratory diagnostic methods, and 3. disinfection of the source of infection - evaluating methods to reduce exposure to Legionella spp. in hospital water supply systems. Samples for environmental investigations include cooling water from the air conditioning system, water from the water supply system, water from the hot water system, limescale swabs from faucets or shower heads at the end of the water supply system, and water samples. After the samples were delivered to the laboratory in a low-temperature preservation mode, the swabs of limescale from faucets or shower heads were treated with 0.2M HCL-KCL acid solution at pH 2.2 for 3 minutes; the water samples were filtered through a 0.22-90 submicron membrane to concentrate the bacteria in the water samples, and then inoculated on the BCYE agar plate-DGVP (containing glycine, vancomycin, vinblastine, and vancomycin), which was specially designed for the environmental samples with selective and discriminatory properties. Then, they were inoculated into BCYE agar plate-DGVP (containing glycine, vancomycin, polymyxin B, bromothymol blue and bromocresol purple dyes) or CCVC for selective and discriminatory environmental samples, and cultured in an incubator with a humidity of 70-80% and an incubator at 35°C. After three days, we began to observe the colonies' patterns with a dissecting microscope, and then serotyped the bacteria with the method of direct fluorescent staining. All isolates were stored in a refrigerator at -70℃ for further antiserum and molecular biology typing studies. In order to establish the basic epidemiological data on the contamination of the hospital environment by this bacterium, and to serve as an indicator for disinfection of the hospital environment.
The suspected colonies were inoculated on BAP and BCYE agar plates respectively, and incubated in a humid 35℃ incubator every other day for the nutritional requirement test of L-cysteine; the colonies that grew only on BCYE agar plates but not on BP plates were picked; the biochemical reaction of L. veterinarum was not obvious, and the identification and typing methods were based on the combination of a single antiserum with fluorescence. Since the biochemical reaction of L. pneumophila was not obvious, the identification and typing methods were based on the immunofluorescent L. pneumophila (serogroup 1-14) antibody of the single antiserum of conjugated fluorescent L. pneumophila (serogroup 1-14) by the Direct Immunofluorescent Test (DFA) to measure its antigenic type. BCYE agar plates were placed under a long-wave 365-nm ultraviolet light to observe the spontaneous fluorescence of the colonies; L. bozemanii, L. dumoffii, L. cherrii, L. gormanii, L. tucsonensis, and L. anisa, which are associated with the disease, produced blue-white fluorescence; L. rubrilucens, L. tucsonensis, and L. anisa produced blue-white fluorescence; and L. rubrilucens, L. tucsonensis, L. tucsonensis, and L. anisa produced blue-white fluorescence. L. rubrilucens, L. erythra produce red fluorescence; this will help to limit the selection of single antisera for immunofluorescence typing and identification, thus saving time and reagent consumption.
Clinical Laboratory Diagnosis
Pneumocystis carinii is a naturally occurring waterborne organism that causes highly fatal pneumonia infections in hospitals and in the community; many cases go undiagnosed due to the inability to culture the organism in most hospital laboratories and because of unfamiliarity with the disease among clinicians. Since sputum samples are easily contaminated by the normal flora of the upper respiratory tract, it is impossible to isolate such a selective bacterium. It is necessary to supplement with other serologic tests such as urinary antigen determination and serum antibody determination of the patients to increase the diagnosis rate of Veterans' Disease. Respiratory secretions, serum, and urine are collected at the same time in patients diagnosed with pneumonia (once in the acute stage and once in the recovery stage after 3 weeks). Respiratory secretions are immediately collected for bacterial culture, and serum is screened for IgG+IgM qualitatively by enzyme immunoassay (ELISA) and then quantitatively by indirect immunofluorescence assay (IFA), while urine is examined by the rapid immunochromatographic assay of Binax NOW Legionella Urinary Antigen Test (immunochromatographic membrane assay). The presence of L. pneumophila serotype 1 antigen in urine samples was determined by the rapid Binax NOW Legionella Urinary Antigen Test (immunochromatographic membrane assay; ICT) within 30 minutes. The use of direct fluorescent immunoassay (DFA) to detect antigens in sputum from patients with Legionnaires' disease is associated with false-positive results due to the presence of respiratory flora (e.g., Bacteroides fragilis, Pseudomonas spp., Stenotrophomonas maltophilia) and a small number of pathogenic organisms in the test. The use of indirect fluorescent immunoassay has been progressively discontinued because of the small amount of pathogenic bacteria in the samples and the false-negative results[15] . The use of indirect fluorescent immunoassay (IFA) to detect antibody potency in the sera of patients with Legionnaires' disease is subjective because of the interpretation of the results of fluorescent staining, and according to the criteria of the National Institute of Disease Control (NIDC) for positive interpretation ≥ 1:256 potency, the correctness of diagnosis is poor, except for the four-fold increase in the antibody in the recovery phase compared to the acute phase, which is diagnostic, and the diagnosis of the serological diagnosis requires a comparison of the second potency between the acute phase and recovery phase. The use of serologic diagnosis requires a comparison between the acute phase and the recovery phase to establish a diagnosis, which is not helpful for the clinical treatment of patients; currently, ELISA is gradually being used abroad to determine the IgG+IgM potency instead. At the same time, all patients receive chest X-ray, biochemistry and hematology examinations.
The combined use of traditional culture techniques combined with the application of indirect fluorescent immunoassay and enzyme immunoassay to detect antibody potency in serum, and the application of the latest enzyme immunoassay to detect antigens in the patient's urine, to assist in the rapid clinical diagnosis of Veteran's Disease infection. The test specimens will include sputum, blood, and urine. According to the study plan, sputum will be cultured for traditional bacteria and L. pneumophila, and antigens will be detected by direct fluorescence immunoassay; serum will be collected from blood in the acute and recovery phases of the infection, and antibodies will be detected by indirect fluorescence immunoassay and enzyme immunoassay; and urine will be collected from the patients in the acute and recovery phases of the infection, and antibodies will be detected by enzyme immunoassay. At the same time, urine was collected from patients during the acute and recovery phases of the infection to detect the presence of LP1 antigen by enzyme immunoassay.
Conclusion
When a patient acquires any of the bacteria listed above, he or she usually ends up with symptoms of infection. The use of contaminated tap water for cleaning medical instruments and equipment that come into direct contact with patients in the health care process exposes patients to the risk of infection. Patients who are colonized by bacteria can also be a source of transmission of infection, especially if the medical equipment used by the patient is in a humid environment, such as a respirator. Bacteria that are widespread in aquatic environments, such as Legionella, Pseudomonas, Pseudomonas, Burkholderia onionis, and Fusobacterium are common glucose-neutral, non-fermentative Gram-negative bacteria found in hospital aquatic environments, and are absolutely relevant to the acquisition of infections from clinical and patient care exposures; they have been characterized by having a very low nutrient requirement (e.g., they can survive in distilled water), and they tolerate a wide range of physical environments (e.g., they can survive in distilled water). They are characterized by very low nutrient requirements (e.g., can survive in distilled water) and can tolerate a wide range of physical environments (e.g., temperature variations), and are therefore the cause of pneumonia, urinary tract infections, and bloodstream infections associated with the use of medical devices. These bacteria can spread through the hands of healthcare workers and infect patients, resulting in serious nosocomial infections. Therefore, all medical units in hospitals, especially intensive care units and burn units, must avoid the spread of these bacteria in water; healthcare workers must have good hygiene habits, absolutely comply with the requirement of hand-washing after taking care of patients, and wear gloves and appropriate isolation clothing to protect patients from infection; and at the same time, we must exclude sources of contamination in the hospital environment to avoid contamination of medical equipment and instruments and indirectly infecting patients. It is also important to eliminate sources of possible contamination in the hospital environment to avoid indirect infection of patients from contaminated medical equipment and instruments. Most of the hospitalized patients are people with poor immune and defense functions, but contact with water is unavoidable, so it is urgent to provide good quality and safe water for use in hospitals. In order to maintain good water quality, regular cleaning and disinfection of reservoirs and maintenance of piping are routine maintenance tasks.
According to the recommendations of the Allegheny County Department of Health in Philadelphia, Pennsylvania, USA, when the results of a survey of hospital environments show that the rate of positive cultures for Legionella bacteria at the end of the water supply system reaches 30% or more, it indicates that the environmental contamination of the environment is serious and that hospitalized susceptible patients may be infected, and that appropriate environmental disinfection methods must be adopted to avoid the emergence of outbreaks of infections in hospitals. Appropriate environmental disinfection methods must be adopted to avoid the emergence of outbreaks of hospital-acquired infections; after the implementation of this standard in this city, the rate of nosocomial infections of Legionella bacteria was found to have decreased from 31% to 8.4% with remarkable results [12]. Because of the special environment of hospitals, the disinfection of the water supply system is necessary and continuous, with the following disinfection methods: temporary heating-flushing (60°C water temperature at the terminal outlet, 30 minutes of flow), temporary (20-50 ppm) or continuous chlorination (0.5-1 ppm), continuous ultraviolet light, ozone (1-2 ppm), chlorine dioxide (3-5 ppm). dioxide 3-5 ppm) or monochloramines chemicals and copper-silver ion generating (copper ion 0.2-0.8 ppm, silver ion 0.02-0.08 ppm) systems [17]. Although the temporary heating-flushing disinfection method is simple and easy to operate, it is only applicable to the time-sensitive emergency alternative when the appropriate disinfection equipment cannot be used immediately, and it requires a lot of manpower support for disinfection with the possibility of patients being scalded, and the water supply system will be contaminated again soon without long-term effects. Although temporary or continuous chlorination is still the most widely used method and was the first to be applied to disinfect against Legionella bacteria, the feasibility of this method has been reassessed due to the need to monitor chlorine residual or effective chlorine, the carcinogenicity of chlorine compounds and piping breakage, and the laboratory evidence of chlorine resistance in Legionella bacteria. Ozone Disinfection In hot water supply system, ions will be destroyed and no disinfection effect can be achieved, and it will also cause damage to the piping, so this method is not suitable for hospitals. Ultraviolet radiation disinfection is easy to install and maintain, and is mostly used for localized disinfection in special units with low water consumption or for the start-up of new equipment, and is unable to remove remote contamination. Effective disinfection for Legionella spp. present in hospital water supplies requires the use of microbiological methods that effectively deliver disinfectant by water flow to eliminate remote outlets, including total water supply disinfection methods such as chlorination and copper-silver ion generation systems. When Legionella spp. is hiding in biofilms of scale or sediment in storage tanks or lines, it cannot be completely eliminated even with effective total water system disinfection methods. Since the hospital water supply system is a major potential source of infection, piping should be designed to avoid dead ends to avoid water stagnation, and regular disinfection and cleaning of storage towers is also necessary. Therefore, disinfection of the water supply system must be combined with effective disinfection methods and the avoidance of biofilm production from scale or sediment. After repair work on the water supply system, it should be avoided that a sudden increase in pressure through the piping will flush out the strains of bacteria in the biofilm that have been trapped in the walls of the piping, resulting in a sudden increase in the amount of bacteria in the piping that can cause infections. The excavation of the construction project should also avoid the dust, which may cause the bacteria in the soil to spread in the air and be inhaled by the patients. Copper-silver ionization systems are the current state-of-the-art method for treating waterborne microorganisms in hospital environments. After hospitals have adopted appropriate environmental disinfection methods to combat Legionella bacteria in the water, environmental culture tests should be conducted several times to determine the effectiveness of the disinfection and to assess whether it is possible to reduce the number of patients infected with this bacterium by droplets or choking.
Currently, the buildings of medical units are all closed buildings, the air-conditioning system is used extremely frequently, and the water supply system is densely packed, the migration of Legionella bacteria in the water supply system has been proved to cause serious outbreaks of infections in hospitals; in order to avoid the prevalence of Legionnaires' disease in hospitals, it is extremely important to carry out basic epidemiological investigations and regular disinfection and periodic testing in medical units. In order to prevent the epidemic of Legionnaires' disease in hospitals, it is extremely important to conduct basic epidemiological investigations and regular sterilization and testing in medical units. The mortality rate of patients infected with pneumonia due to Legionnaires' disease is very high if the disease is not properly diagnosed and rapidly treated with appropriate anti-microbial agents. The collection of specimens from suspected infected patients is needed to assist in rapid and accurate diagnosis, to reduce the number of days of hospitalization, to reduce the use of inappropriate antimicrobial agents, and to improve the quality of care.
Epidemiologic information on hospital-acquired L. pneumophila infections should be established immediately in all healthcare facilities; appropriate environmental disinfection methods should be used for sterilization. Traditional bacterial culture techniques are used to investigate the distribution of L. pneumophila in the hospital environment to provide clinicians with epidemiologic information and indicators of hospital environmental hygiene when diagnosing nosocomial pneumonia in hospitals. When the investigation shows that the hospital environment is indeed contaminated with this bacterium, the isolated strains should be typed by antisera and molecular biotechnology-pulse electrophoresis in order to check the possible reservoirs of infection in the environment when nosocomial infectious strains are generated. The authors believe that when the culture results show that the environment has been free of this bacterium, it should be planned for routine testing once a year, in order to establish the epidemiology of environmental contamination by this bacterium and to ensure the safety of the lives of hospitalized patients.