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Approximately 10-40 of all nosocomial infections are pulmonary, leading to grave complication. Elderly, debilitated, or critically ill patients are at high risk. Respiratory care equipments including ventilators, humidifiers, nebulizers and procedures have been identified as potential vehicle for major nosocomial infections if colonized by fungi or bacteria. Aim-To determine the rate of colonization by bacteria and fungi in oxygen humidifier chambers of portable cylinders and central lines at our hospital. The Hudson's chambers of nebulizers were also studied for the same. Methods-Swabs from equipment were obtained using sterile swabs on Tuesday, as these chambers are usually cleaned on every Saturday. Spot samples were taken from ICUs, wards, casualty and OPD on a single day. Air samples were also obtained on the same day to determine if the fungal spore load in the inhaled room air was normal or high. We performed disinfection with 70% ethanol after cleaning of these devices. Results-53/70 (75.71%) samples showed fungal growth; out of these, 23/33 (69.70%) were from ICU, 24/30(80%) were from wards and 6/7 (85.71%) were from OPDs. 23/30 (76.66%) swabs from central line humidifiers, 18/23(78.26%) swabs from O2 cylinder humidifier and 8/17 (47.5%) swabs from nebulizers grew bacteria. Of the total 61(87.14%) bacterial isolates, 42(68.85%) were gram negative bacteria and 19(31.14%) were gram positive cocci. Out of 42 Gram negative bacteria 17 were multi-drug resistant i.e. ESBL producers. Pseudomonas spp. (6) Acinetobacter spp.(4), Klebseilla pneumoniae (4), E.coli (2), Stenotrophomonas maltophila (1). Our finding (before disinfection) showed colonization rate for fungi was 75% and for bacteria it was 87%. After 70% ethanol disinfection and strict compliance of hand hygiene colonization rates significantly reduced. Fungal colonization rate reduced and only 15% fungus grew after disinfection while only 12% bacterial colonization rate were determined. Conclusion-The study indicates a potential in-hospital source of allergens and infection. The oxygen and nebulizer chambers need to be cleaned more frequently with disinfectants to control likely nosocomial infections.
Key wards- Microbial colonization, respiratory devices, Aspergillus fumigates, Pseudomonas spp. Acinetobacter spp.
Introduction- Respiratory infections are the commonest among the nosocomial infections. Nosocomial pneumonia is the second most common nosocomial infection worldwide and the most common infection in intensive care unit (ICU). In the United States, The Center for Disease Control and prevention (CDC) estimated roughly 1.7 million hospital-associated infections, from all type of microorganisms, including bacteria, fungi, viruses contributing 99,000 deaths per year [1,2]. The National Nosocomial Infections Surveillance (NNIS) system report, data summary from January 1992 through June 2004, issued in October 2004 reported a steady increase in the rate of nosocomial fungal infections, from 3.8 to 4.9 per 1,000 discharges .
Advances in medical and surgical therapy over the past two decades have changed the type of patients cared in the hospitals. Care in special units use invasive monitoring devices; parentral nutrition, broad- spectrum antimicrobial agents and assisted ventilation. These have helped to successfully treat patients suffering from previously known to be devastating or fatal diseases [3,4]. This severely ill, immunocompromized, hospitalized patient population is highly susceptible to nosocomial infections caused by variety of bacteria and fungi. The resulting illness is often severe, rapidly progressive and difficult to diagnose or treat. Approximately 40% of all nosocomial infections are pulmonary. Respiratory care equipments including ventilators, humidifiers, and nebulizers have been identified as potential vehicles for major nosocomial infections if these are colonized by fungi or bacteria. Nosocomial pathogens may also be acquired by the hands of hospital personnel, contaminated intravenous lines or fluids [5,6].Potential reservoirs of pathogens causing nosocomial puneumonia include- oropharynx, trachea, stomach, respiratory therapy equipment, paranasal sinuses, sanctuary(above tracheal tube cuff and below true vocal cords).
Contaminated respiratory care equipment may lead to nosocomial infection by two routes. Firstly, respiratory care equipment may serve as a reservoir for microorganism especially gram-negative bacilli. The fluid containing devices such as nebulizers and humidifiers may become heavily contaminated by bacteria and fungi capable of multiplying in water. Pathogens may then spread to the patient by aerosolization in the room. Secondly, contaminated equipment may lead to direct instillation or delivery of microorganisms to the airways, if the equipment is directly linked to a ventilator system or if contaminated medication is distillated aerosolized.[8-10].Many equipments such as oxygen mask, nebulizer chambers may be transferred from patient to patient several times daily but seldom cleaned daily. In our institute, these equipments are routinely cleaned once a week i.e. on Saturdays with soap and tap water. We thought that these should be considered as potential sources of bacterial colonization and subsequent transmission.
Systematic studies and reports evidently recognized that contaminated inhalation therapy equipments are the potential cause for nosocomial pneumonia but such studies have been relatively few. Bacteriological sampling plays significant function in establishing the success of programs intended to diminish the infection hazard related with inhalation therapy.
The substantial clinical and financial impact of nosocomial pneumonia makes this an important subject matter for hospital epidemiologists and microbiologists. Pneumonia is associated with the greatest mortality with nosocomial infections and with considerably increased costs of care. Preventive measures may reduce the incidence of nosocomial pneumonia by preventing transmission of highly pathogenic microorganisms to patient by reducing colonization of reservoir site. Infection control activities should emphasize the establishment of appropriate preventive guidelines and policies and the continuing education of health care workers to maintain optimal compliance with preventive practices [14, 15, 16].
Another important aspect is an increasingly common finding of fungal infections in hospital settings. Although fungi are generally thought to be less pathogenic than bacteria to humans, such prevalence is found to be increasing in ill patients, especially ICUs. The usual explanation given is that fungi take the lead in infecting an immunocopmromized patient who is well covered with antibiotics. However, we thought that there may be another potential reason for such infections i.e. the fluid containing reusable respiratory care equipments such as nebulizers and oxygen humidifiers. A thorough knowledge and understanding about colonization of respiratory devices by bacteria and fungi, is thus needed for clinicians and microbiologists to provide better patient care. Continued epidemiologic and laboratory research is required to better characterize these pathogens and thus to improve diagnosis and therapeutic strategies in the future. Hence, we aimed to qualitatively evaluate the microbial colonization rate in oxygen humidifier chambers (of portable cylinders and central lines) and Hudson's chambers of nebulizers; being used in various wards and ICUs of our hospital. We also performed a study to determine the efficacy of 70% ethanol wipe as decontamination of respiratory devices.
Methods- Study period- Jan 2011 to April 2011. The study was approved by institutional review board.
Sample collection methods-Total of 70 swabs samples were obtained from inner surface of oxygen humidifiers and Hudson's chambers of nebulizers using sterile swabs on a Tuesday as these chambers are usually cleaned on every Saturday at our institute. Spot samples were taken from ICUs (33), wards (30), casualty and OPD (7) on a single day. We performed disinfection with 70% ethanol to all above mentioned equipment and after disinfection, we have collected swabs samples from inner surface of equipments and followed same protocol to determine rate of colonization. Air samples were also obtained on the same day to determine if the fungal spore load in the inhaled room air was normal or high. Quality control-10 swabs were collected from new, unused oxygen humidifiers and Hudson's chambers of nebulizers as control and swabs were seeded on Sabouraud's dextrose agar (SDA) slants with antibiotics and SDA slants without antibiotic and blood agar to check quality control.
In order to maintain the blinding, the samples from various sites were coded. The pulmonologist investigators collected the samples and thus were un-blind. The microbiology investigators were blinded to the information about the site to which the swabs belonged to.
Bacteriological examination- Swabs were inoculated in glucose broth and incubated aerobically at 37ËšC in incubator for 4 hrs. Incubated glucose broth was then sub cultured on Mac Conkeys agar and blood agar and incubated at 37ËšC overnight. Growth after overnight incubation were identified and confirmed by standard conventional methods.
Antibiotic susceptibility tests - The Kirby- Bauer method recommended by the CLSI guidelines (2005) was used for antimicrobial susceptibility testing [17-20].
Detection of Extended Spectrum Î²-Lactamases-Screening Test (CLSI, 2010)
Initially screening test for ESBL production was done as part of routine susceptibility testing. Two antibiotic discs, ceftazidime (30 Î¼g) and cefotaxime (30 Î¼g) were used for screening for ESBLs. Plates with Mueller- Hinton Agar (MHA) were prepared and inoculated with the test organism (turbidity corresponding to 0.5 McFarland's standard) to form a lawn culture. The above discs were applied on the surface of the agar. The plates were incubated at 37 ° C overnight and sensitive pattern and resistant pattern were recorded by reading the zone diameter of the test organism. If a zone diameter of â‰¤ 22mm for Ceftazidime and â‰¤27 mm for cefotaxime was recorded these strain were considered "Suspicious" for ESBL production [20-22].
Double Disk Approximation Test (DDAT)
Bacterial suspension equivalent to 0.5 McFarland standards turbidity for testing ESBL production test were prepared. A sterile swab was dipped into standardized inoculum and the soaked swab was rotated against the upper inside wall of the tube to express excess fluid. The entire surface of the MHA was swabbed to form a lawn culture and the inoculum was allowed to dry for a minute with lid in place. With sterile forceps, Ceftazidime disk was placed on the agar plate near the centre giving a centre to centre distance of 15 mm Ceftazidime/clavulonic acid (30Âµg/10Âµg). The plates were inverted and incubated at 37°C for 16-18 hours. Each plate was examined for enhancement of zone of inhibition for ceftazidime disk at the side facing Ceftazidime/clavulonic acid disk. If the strain was an ESBL producer, then the zone around ceftazidime disk was extended towards Ceftazidime/clavulonic acid disk. ATCC Escherichia coli -25922 were used as negative control and ATCC K. pneumoniae -700603 was used as positive control [23-25].
Mycological examination- Swabs were inoculated directly on Sabouraud's dextrose agar (SDA) with and without antibiotics i.e, Chloramphenicol (50 mg/ml) & Gentamicin (20 mg/ml) and incubated at 25Â°C & 37Â° C separately over a period of four weeks. Fungus identification was done based on the growth rate, colony morphology, reverse and obverse surface colour of SDA slant & microscopic aspects such as mycelium & conidia types. Dematiaceous molds were considered when colonies that can develop dark gray to black mycelium, particularly prominent when a black reverse of the colony was observed.Species identification was done by lactophenol cotton blue (LPCB) of culture positive fungi [26-29].
Results and observations-
Table 1 Distribution of swab sampling from various sites
Central line humidifiers
O2 cylinder humidifiers
Total of 70 swab samples were processed i.e. 33 from ICUs, 30 from wards and 7 from OPD. 53/70 (75.71%) samples showed fungal growth; out of these, 23 (69.70%) were from ICU, 24(80%) were from wards and 6 (85.71%) were from OPDs. 23/30 (76.66%) swabs from central line humidifiers, 18/23(78.26%) swabs from O2 cylinder humidifier and 8/17 (47.5%) swabs from nebulizers grew bacteria.
Table 2 Frequency of Various Fungal Isolates in Swab Samples out of a total of 53 positive samples
Name of the species
Frequency of isolation
TB & Chest OPD (5/18)
Female Surgery Ward(2/18)
Male Med Ward(1/18)
Male Surgery Ward (1/18)
Central line humidifier 10/18
O2 cylinder humidifier 6/18
Pediatric OPD (2/10)
Female Medicine Ward(1/10)
O2 Cylinder Humidifiers(4/10)
Female Medicine Ward(1/8)
Male TB Chest Ward (1/8)
O2 Cylinder Humidifiers(3/8)
Male TB Chest Ward (2/7)
Female Medicine Ward(1/7)
Female Surgery Ward(1/7)
O2 Cylinder Humidifiers(5/7)
Central Line Humidifiers(2/7)
Female medicine Ward (1/5)
Male TB Chest Ward(1/5)
Female Medicine W(1/3)
Pediatric OPD (1/3)
TB&Chest OPD (1/3)
NICU - neonatal intensive care unit, PICU - pediatric intensive care unit, MICU - medicine intensive care unit, SICU - surgical intensive care unit, O2 -oxygen, OPD - out door patients.
Of the 51(75.71%) total fungal isolates, Aspergillus fumigatus 18 (33.96%) were predominantly isolated followed by Aspergillus niger10(18.86%).
Table 3 Frequency of Various bacterial Isolates in Swab Samples out of a total of 61 positive samples
Name of bacteria
Frequency of isolation
Methicillin resistant Staphylococcus aureus (MRSA)
Methicilin sensitive staphylococcus aureus (MSSA)
Coagulase negative Staphylococcus aureus (CONS)
NICU-neonatal intensive care unit, PICU- pediatric intensive care unit, MICU- medicine intensive care unit, SICU- surgical intensive care unit, O2 -oxygen, OPD- out door patients.
Of the total 61(87.14%) bacterial isolates, 42(68.85%) were gram negative bacteria and 19(31.14%) were gram positive cocci. Out of 42 Gram negative bacteria 17 were multi-drug resistant i.e. ESBL producer. Pseudomonas spp. (6) Acinetobacter spp.(4), Klebseilla pneumoniae (4), E.coli (2), Stenotrpphomonas maltophila (1).
One third of nosocomial infections are considered preventable. Information regarding the potential risk of transmission due to colonization of respiratory equipment/devices is insufficient. Aspergillus spp. is ubiquitous, commonly occurring in soil, water, and decaying vegetation. Potential reservoirs in hospitals may include unfiltered air, ventilation systems, oxygen humidifiers, nebulizer chambers and tubing's, contaminated dust dislodged during hospital construction, carpeting, food and ornamental plants . Aspergillus fumigatus, A. flavus, A. terreus have become common cause of nosocomial infections. Contaminated air or ventilation systems have been associated repeatedly with outbreaks of nosocomial aspergillosis . Construction and demolition activity nearby hospital, renovation, cleanings and moist environment within the ventilation system or air filter have been commonly cited. During our study period, our hospital construction work was in progress and it is a probable reason for isolation of Aspergillus spp. in higher proportion.
Hyalohyphomycosis i.e. non dematiaceous molds have recently been recognized as emerging nosocomial pathogens . Our study finding showed colonization by Fusarium spp. in 8(15.09%) swabs samples mainly from humidifiers. The mechanism of infection may include inhalation into the lungs or upper airways or breaks in the skin or mucous membranes.
The patients hospitalized with exacerbations of obstructive airway disease such as asthma often require nebulization and oxygen therapy. If the oxygen humidifier chambers or the nebulizer chambers are colonized by fungi, the clinician may actually be directly delivering the fungal allergen to the patient's airways. Rather, we suspect that this may be the cause for an occasional delayed response to asthma therapy.
Finding fungi or yeast cells in the sputum of patients receiving corticosteroid or antibiotic therapy is not uncommon. A study showed that fungi may be found in sputa in 42.4% of patients receiving prior antibiotics and 64.2% of patients receiving prior inhaled steroids . Majority of nosocomial infections are caused by Candida spp. . In our study, Candida tropicalis were isolated from two central line nebulizers. There are several reports on growing prevalence of non-albicans Candida among hospitalized patients [36-37]. Cross-infection from staff to patient may be common, even in ICU. Rangel-Frausto et.al reported that hands of health care workers were reservoir for Candida spp. and 85% were non- albicans Candida . An outbreak of Candida tropicalis fungemia in neonatal intensive care unit (NICU) was reported in neonates who were receiving total parenteral nutrition and antimicrobial agents [37, 38]. Same study showed that Candida tropicalis was isolated from two NICU workers and not from environment. Washing hands as promptly and thoroughly as possible between patient contacts and after contact with blood, body fluids, secretions, excretions, equipment or articles contaminated by them is an important means of infection control and isolation safety measures.
The etiology of bacterial nosocomial pneumonia depends on duration of hospitalization before pneumonia develops. Early onset nosocomial pneumonia is the occurring during the first four or five days of the hospital stay. It is more commonly caused by community acquired pathogens such as Streptococcus pneumoniae, Methicillin-susceptible Staphylococcus aureus, H. influenza, Morexella catahalis. In contrast, late onset nosocomial pneumonia (which usually occurring after five to six days of hospitalization) is commonly caused by Pseudomonas aeruginosa, Acinetobacter spp., Methicilline resistant Staphylococcus aureus etc.[40-]. After 10 or more days in hospital, Enterobacteriaceae and P. aeruginosaare the most common pathogens responsible . Several studies have reported the etiology of nosocomial pneumonia in the long-term care settings [42-45]. Present study showed high colonization rate by Pseudomonas spp. and Acinetobacter spp. followed by E. coli and Klebseilla spp. Klebseilla pneumoniae had been associated with 2%-5% of nosocomial infections, particularly from lower respiratory and urinary tracts. Nosocomial outbreaks by gram negative bacteria were associated with their drug resistance to third-generation cephalosporins and aminoglycosides [1-4]. Present study showed total 17/61 (27.86%) extended spectrum Î²-lactamases (ESBL) species.
We performed disinfection with 70% ethanol after cleaning of these devices (oxygen humidifiers and nebulizer chambers with distilled water and soap. Health care workers were educated for hand hygiene with alcohol-based hand rubs before and after each patient contact. We collected swabs again from same wards and ICUs after 70% ethanol disinfection and determined colonization rate by same methodology. Our finding (before disinfection) showed colonization rate for fungi was 75% and for bacteria it was 87%. After 70% ethanol disinfection and strict compliance of hand hygiene colonization rates significantly reduced. Fungal colonization rate reduced and only 15% fungus grew after disinfection while only 12% bacterial colonization rate were determined.
Limitation of this presents study is that we have not taken in to consideration any contamination with viruses or Mycobacteria. This is because prevalence of nosocomial viral or Mycobacterial infection is very very low. We have assessed only 70% ethanol wipe disinfection was and have not compared the results with other high level disinfectant; the reason being the cost of the various disinfecting agents and the practicability of their use.
We consider it to be prudent to perform periodic surveillance for bacterial and fungal colonization in the respiratory equipment, particularly water-sealed devices. Proper cleaning and sterilization or high level disinfection of reusable equipment is essential to prevent infections associated with respiratory therapy such as oxygen therapy, nebulization etc. Devices or parts of devices need to be rinsed in water after they have been chemically disinfected. Sterile water has been recommended because tap or locally prepared distilled water may harbor microorganisms that can potentially cause pneumonia. The implementation of new and regular hygiene measures for the maintenance of such equipment is desirable.