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Healthcare Associated Infections (HCAIs)
& Environmental Sample Collection
‫الرحيم‬ ‫الرحمن‬ ‫اهلل‬ ‫بسم‬
Sajjad Ahmad PhD Scholar Microbiology
Laboratory Coordinator NIH-CDC

2. Objectives:
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INTRODUCTION
• WHAT ARE HEALTH CARE ASSOCIATED INFECTIONS?
• WHY COLLECT SAMPLES?
• TYPES OF SAMPLES
• SITE SELECTION
• SAMPLE COLLECTION METHODS
• STERILIZATION AND DISINFECTION
• TRANSPORTATION AND STORAGE
• SAMPLE PROCESSING
• LABORATORY ANALYSIS
• INTERPRETATION OF RESULTS
• REPORTING
• QUALITY CONTROL
• CHALLENGES IN SAMPLE COLLECTION
• ETHICAL CONSIDERATIONS
• LEGAL CONSIDERATIONS
• TRAINING AND EDUCATION
• COLLABORATION AND COMMUNICATION
• FUTURE DIRECTIONS
• CONCLUSION

3. Introduction
• There is increasing evidence that the hospital surface environment contributes to the spread of
pathogens.
• Health care associated infections (HAIs) are a serious concern in hospitals, as they can lead to
prolonged hospital stays, increased healthcare costs, and even death.
• Healthcare-associated infections (HCAIs) lead to poor clinical outcomes and death. In high-income
countries HCAIs affect approximately 5-15% of patients, whereas in low-income countries prevalence
rates are in the region of 15-19%.
• In Europe, HCAIs are attributed to approximately 37,000 deaths per year and 25,000 people per year
die from antibiotic resistant HCAIs.
• The hospital surface environment is an important factor in infection risk as it may act as a reservoir
for nosocomial pathogens.
• On average, patients being 73% (28.8-87.5%) more likely to acquire HCAIs if a previous room occupant
was colonized or infected.

4. What are Health Care Associated Infections?
• Health care associated infections, also known as nosocomial
infections, are infections that patients acquire while receiving
medical treatment in a hospital or other health care facility.
• These infections can be caused by bacteria, viruses, fungi, or
other microorganisms, and can occur anywhere in the body,
including the bloodstream, lungs, urinary tract, and surgical
wounds.
• Health care associated infections are a major concern in
hospitals because they can lead to serious complications,
prolonged hospital stays, and even death.
• Patients who are already sick or have weakened immune
systems are particularly vulnerable to these infections.
• In addition, health care associated infections can be spread
from patient to patient, and from health care workers to
patients, if proper infection control measures are not followed.

5. Why Collect Samples?
• Collecting samples from hospitals is an essential
step in identifying and preventing health care
associated infections.
• By analyzing these samples, healthcare
professionals can determine the presence of
harmful microorganisms and take appropriate
measures to prevent their spread.
• In addition, collecting samples allows for the
identification of antibiotic-resistant strains of
bacteria, which can help inform treatment
decisions and prevent the development of further
resistance.
• Overall, the collection of samples plays a crucial
role in maintaining the safety and well-being of
patients in healthcare settings.

6. Types of Samples
There are several types of samples that can be collected from hospitals to detect health care associated infections.
One common type of sample is a swab, which is used to collect samples from surfaces in patient rooms, such as
bed rails, call buttons, and door handles.
Another type of sample is a specimen, which is collected directly from a patient's body, such as blood, urine, or
sputum. Environmental samples, such as air or water samples, can also be collected to identify potential sources of
infection.
In addition to these traditional sample types, new technologies are being developed to detect health care
associated infections. For example, some hospitals are using electronic monitoring systems to track hand hygiene
compliance among staff members.
Other hospitals are using genomic sequencing to identify specific strains of bacteria and viruses that may cause
infections. These new technologies have the potential to revolutionize the way we detect and prevent health care
associated infections in hospitals.

7. Step 1: Select the appropriate sampling method based on the type of environment being
sampled.
Step 2: Label the sample container with the date, time, location, and type of sample
collected. Use a waterproof marker to prevent smudging or fading.
Step 3: Collect the sample using the appropriate sampling equipment, such as swabs,
wipes, or air samplers. Follow established protocols for each type of sample.
Step 4: Seal the sample container tightly to prevent contamination during transport.
Step 5: Transport the sample to the laboratory as soon as possible. If there will be a delay
in transport, store the sample at the appropriate temperature and follow established
storage protocols.
General Guidelines/Protocols

8. Transportation and Storage
• Proper transportation and storage of samples collected from hospitals is crucial to ensure accurate
laboratory analysis.
• Samples should be transported as soon as possible after collection, ideally within 24 hours, to
prevent degradation of the sample and minimize the risk of contamination.
• During transportation, samples should be kept at a temperature between 2 and 8 degrees Celsius to
maintain stability.
• Once samples arrive at the laboratory, they should be stored in a secure, designated area with
appropriate temperature controls.
• Samples should be labeled clearly with patient information and the date and time of collection to
ensure proper tracking and prevent mix-ups.
• It is important to follow standard operating procedures for both transportation and storage to
maintain the integrity of the samples and ensure accurate results.

9. Basics in Culturing of Surgical Sites Infections
(SSI)-Wound Swabs
Why Wound Swabs are Important in Patient Care
• Wound Swabs are one of the Important Specimens sent to
Microbiology Departments for Bacteriological and Fungal isolation
and Anti-microbiologial Evaluation.
• However, many basic lacuna in Collection, Laboratory identification
and reporting makes the Deficit in Effective care of the Patients and
increases the potentials for Morbidity And mortality.
Events in Infected Wounds:

10. • Methods of obtaining a specimen from a wound include wound swabbing,
needle aspiration and wound tissue biopsy. Although wound swabbing is
the most practical and widely used, it is important to use a technique
that produces reliable samples for microbiological analysis.
Obtaining a Specimen
Wound Culture Protocols
•Soft skin and tissue infections with open or draining lesions
(Appearance of) insect/spider bite with necrotizing centre,
drainage & erythema.
•Abscess – ulcer
•Infected laceration
•Culture wound prior to initiation of antibiotics if signs or symptoms of infection are present. If Culture &
Sensitivity is obtained after antibiotics have been started, list the drug on the laboratory C& S request.

11. • The technique usually employed for transferring clinical samples from wounds to microbiology
laboratories.
• Uncertainty about when swabs should be taken, the correct collection procedure and the
appropriate protocols for submitting swabs for investigation have led to a situation where
clinicians regularly collect and process unsuitable sample.
• Swabs should be collected with Suspected Infection. Swabs should therefore be collected only
when clinical criteria point to a wound infection and before any antimicrobial interventions have
been initiated.
What are guideline to Follow
• When a swab is indicated, the patient should be given a concise explanation of the need for
microbiological investigation and what the procedure involves, for example, that swabs are mainly
used to recover species from the surface layers rather than from the deep tissues of a wound.
• Cleaning the contaminating materials is a priority Before a representative sample is collected, any
contaminating materials such as slough, necrotic tissue, dried exudate and dressing residue
should be removed by cleansing the wound with tap water, sterile saline or debridement.

15. Catheter-Associated UTIs (CAUTI)
• A catheter-associated urinary tract infection (CAUTI) is a urinary tract infection (UTI)
in which the positive culture was taken when an indwelling urinary catheter had
been in place for > 2 calendar days.
• Patients with indwelling bladder catheters are predisposed to bacteriuria and UTIs.
• Symptoms may be vague or may suggest sepsis.
• Diagnosis depends on the presence of symptoms.
• Testing includes urinalysis and culture after the catheter
has been removed and a new one inserted.
• The most effective preventive measures are avoiding
unnecessary catheterization and removing
catheters as soon as possible.

16. • Bacteria can enter the bladder during insertion of the catheter, through the catheter lumen, or from
around the outside of the catheter. A biofilm develops around the outside of the catheter and on the
uroepithelium.
• Bacteria enter this biofilm, which protects them from the mechanical flow of urine, host defenses, and
antibiotics, making bacterial elimination difficult.
• Even with thoroughly aseptic catheter insertion and care,
the chance of developing significant bacteriuria is
3 to 10% every day the catheter is indwelling.
Of patients who develop bacteriuria, 10 to 25%
develop symptoms of UTI. Fewer develop sepsis.
• Risk factors for UTI include duration of
catheterization, female sex, diabetes mellitus,
opening a closed system, and suboptimal aseptic
techniques.
• Indwelling bladder catheters can also predispose
to fungal UTI.

17. Urinalysis and urine culture for patients with symptoms or at high risk of sepsis. Testing is done only in
patients who might require treatment, including those who have symptoms and those at high risk of
developing, such as
•Patients with granulocytopenia
•Organ transplant patients taking immunosuppressants
•Pregnant women
•Patients undergoing urologic surgery
•Diagnostic testing includes urine analysis and urine culture.
•If bacteremia is suspected, blood cultures are done.
•Urine cultures should be done, preferably after replacing the
catheter (to avoid culturing colonizing bacteria), then by a direct
needlestick of the catheter, all done with aseptic technique,
so that contamination of the specimen is minimized.
•When a patient presents with symptoms, the lack of pyuria points
to a different diagnosis than CAUTI
Diagnosis of Catheter-Associated UTIs (CAUTI)

18. Catheter-Associated UTIs (CAUTI) Sample Collection
• Clean catch midstream urine samples (10–20 mL) is to be
collected in a sterile container.

19. • Collected samples are to be inoculated onto blood agar, MacConkey agar, and cysteine
lactose electrolyte deficient agar (CLED).
• The inoculated culture media were incubated in an aerobic atmosphere at 37°C for 24 h.
• After overnight incubation, the bacterial growth on the respective media was inspected
visually and graded for the presence of significant bacteriuria.
• A significant bacteriuria was considered, if pure culture at a concentration of 10
≥ 5
colony
forming unit (CFU)/mL.
• All isolates were further identified using colony morphology and biochemical tests.
• Antimicrobial sensitivity was determined by modified Kirby–Bauer disc diffusion method.
Lab Analysis & Result Interpretation:

20. • A central line-associated bloodstream infection (CLABSI) is a
laboratory-confirmed bloodstream infection not related to an
infection at another site that develops within 48 hours of
central line placement.
• Most cases are preventable with proper aseptic techniques,
surveillance, and management strategies.
A central line-associated bloodstream
infection (CLABSI)

21. CLABSI - Centers for Disease Control and Prevention (CDC) definition:
• CLABSI is a surveillance definition used by the CDC and defined as the recovery of a
pathogen from a blood culture (a single blood culture for an organism not commonly
present on the skin and two or more blood cultures for organism commonly present
on the skin) in a patient who had a central line at the time of infection or within 48
hours before the development of infection.
• The infection cannot be related to any other infection the patient might have and must
not have been present or incubating when the patient was admitted to the facility.
• Exit site infection: Signs of inflammation confined to an area (typically < 2 cm)
surrounding the catheter exit site and the presence of exudate that proves to be
culture positive.
• Tunnel infection: Inflammation extending beyond 2 cm from the exit site (along with
the track or cephalad towards the vein entry site or extending beyond the cuff),
typically associated with pain and tenderness along the subcutaneous track and
culture-positive exudate at the exit site that may not be seen unless expressed by
palpation.

22. Ventilator associated pneumonia (VAP) is the most common nosocomial
infection in patients receiving mechanical ventilation is defined
as pneumonia occurring more than 48 h after patients have been
intubated and received mechanical ventilation.
Its reported incidence depends on case mix, duration of mechanical
ventilation, and the diagnostic criteria used. It occurs in 9-27% of
mechanically ventilated patients, with about five cases per 1000
ventilator days.2 The condition is associated with increased ICU and
hospital stay and has an estimated attributable mortality of 9%.

23. • The most common techniques of sampling are the endotracheal aspirate
(ETA), which is considered a noninvasive method, the protected specimen
brush (PSB) and the bronchoalveolar lavage (BAL), both being invasive
methods of investigation.
• Diagnosing VAP requires a high clinical suspicion combined with bedside
examination, radiographic examination, and microbiologic analysis of
respiratory secretions.
• The multiplex PCR–based Unyvero pneumonia cartridge assay can
directly identify 20 bacteria and one fungus, amongst the most frequently
causing VAP, and 19 of their resistance markers in clinical specimens
(bronchoalveolar lavage or tracheal aspirate), with a turnaround time of
4–5 h.
• The most common pathogens are gram-negative bacilli and
Staphylococcus aureus; antibiotic-resistant organisms are an important
concern.
VAP Sample collection & Lab Investigations:

24. • Generally, hospital environments are only sampled in response to an outbreak. Routine sampling is
not usually indicated for healthcare environments.
• Environmental sampling collection in hospitals requires specialized equipment to ensure accurate
and reliable results. Some of the most commonly used equipment includes swabs, wipes, and air
samplers.
• In light of the changing awareness of the risk posed by the surface environment, more hospitals are
considering instigating routine monitoring of their environments, either to assess cleaning or as
part of a continuous risk assessment- microbiological protocols required.
• Microorganisms: Bacterial, viral, and fungal contaminants were included.
• In this guideline, targeted microbiologic sampling connotes a monitoring process that includes
• a. a written, defined, multidisciplinary protocol for sample collection and culturing
• b. analysis and interpretation of results using scientifically determined or anticipatory baseline
values for comparison; and
• c. expected actions based on the results obtained.
• Samples: Surface samples were included like floor, walls, beds, equipment, instruments, chairs,
stools, tabletops, water, and air.
• Random & Targeted sampling, geographical location or specialty were included.
Environmental Sampling

25. Site Selection
• Proper site selection is crucial when collecting samples from hospitals.
• The location of the sample collection can greatly impact the accuracy and usefulness of the
results. For example, if a sample is collected from an area that is frequently cleaned or
disinfected, it may not accurately represent the overall level of contamination in the hospital.
• Similarly, if a sample is collected from an area that is rarely used, it may not provide a good
indication of the risk of infection to patients and staff.
• To ensure proper site selection, it is important to consider factors such as the frequency of use,
the type of activity that occurs in the area, and the likelihood of contamination.
• Areas that are frequently touched by multiple people, such as door handles or handrails, are
often good candidates for sample collection.
• It is also important to consider the type of surface being sampled, as different surfaces may
harbor different types of bacteria or viruses.

26. Methodology
• There are several methods for collecting samples from hospitals to detect health care associated
infections.
• One method is surface swabbing, which involves using a sterile swab to collect a sample from a
surface in the hospital, such as a bed rail or doorknob.
• Another method is air sampling, which involves using special equipment to collect air samples from
different areas of the hospital.
• A third method is water sampling, which involves collecting samples of water from different sources
in the hospital, such as taps and showerheads.
• Each method has its own pros and cons. Surface swabbing is relatively easy and inexpensive, but it
may not be effective at detecting certain types of bacteria that are difficult to culture.
• Air sampling can provide information about the overall microbial load in the hospital, but it may not
identify specific pathogens.
• Water sampling can help identify potential sources of contamination, but it may not be useful for
detecting airborne pathogens.

27. • To properly use a swab, first moisten it with sterile water or saline solution. Then, gently rub the swab over the surface being
sampled, making sure to cover as much area as possible. After collecting the sample, place the swab in a sterile container and
label it with the appropriate information.
• Wipes are another common tool used for environmental sampling collection in hospitals. They are particularly useful for
collecting samples from large areas such as walls or floors. To use a wipe, simply moisten it with sterile water or saline solution
and wipe the surface being sampled. Like with swabs, be sure to cover as much area as possible. After collecting the sample,
place the wipe in a sterile container and label it with the appropriate information.
• To use an air sampler, first select the appropriate collection media for the type of particle you want to sample. Then, set up the
sampler according to the manufacturer's instructions and turn it on. After sampling is complete, remove the collection media
and place it in a sterile container labeled with the appropriate information.
• Proper maintenance of environmental sampling equipment is crucial to ensure accurate and reliable results. Before and after
each use, all equipment should be properly cleaned and sterilized. Additionally, all equipment should be regularly inspected
and calibrated to ensure it is functioning correctly.
• By using the right equipment and following proper procedures for maintenance and use, healthcare workers can ensure that
environmental sampling collection in hospitals is accurate and reliable, helping to maintain a safe and healthy environment for
patients and staff.
General Guidelines/Protocols

28. • There are several types of environmental sampling collection methods
used in hospitals, including air sampling, surface sampling, and water
sampling.
• Sample collection (pre-analytic), culture (Analytic), and results
interpretation
(Post Analytic analysis).
• Microbiologic sampling of air, water, and inanimate surfaces
(i.e., environmental sampling) is an expensive and
time-consuming process that is complicated by many
variables in protocol, analysis, and interpretation.
• Infection control, in conjunction with laboratorians, should assess the
health-care facility’s capability to conduct sampling and
determine when expert consultation and/or services are needed.
• Sampling devices: swab, sponge, petrifilm, dipslides, and contact
plate.
• There are both direct and indirect methods of sampling.
Direct methods, such as contact plates, are self-enclosed and
require no further processing.
• Indirect methods, such as swabs, require an extraction step

29. • Each type of environmental sampling collection method has its own benefits and limitations.
• For example, air sampling can provide information about the overall quality of the air within a hospital
environment, but it may not identify specific sources of contamination.
• Surface sampling can identify potential areas of high risk for infection transmission, but it may not
capture all types of microorganisms present on a surface.
• Water sampling can identify potential sources of waterborne pathogens, but it may not detect other
types of contaminants that could affect patient health.
• Understanding the benefits and limitations of each type of sampling method is important for ensuring
accurate and effective environmental monitoring within hospitals.

30. Contact Plates:
• Contact plates are convex agar plates that can be directly pressed on to a surface to take a quantitative sample. Contact plates can
be made with selective or non-selective agar, with or without a neutralizing agent, all of which lead to differences in recovery of the
target organism.
• The main advantage of contact plates is the production of semiquantitative data in the form of colony counts. Recovery of
organisms ranged between 23% and 56% depending on the plate and organism.
• Surface sampling involves collecting samples from various surfaces within the hospital environment, such as
doorknobs, countertops, or medical equipment. This can be done using swabs or wipes, which are then analyzed
for the presence of bacteria, viruses, or other pathogens.
Surface Sampling

31. Dipslides
• Dipslides are a direct contact method, like contact plates, held inside a plastic container which reduces
contamination risk and agar drying. Dipslides have a paddle formation with two separate sides, which can
contain two different selective or non-selective agars.
• The two sides can be used to take two samples with different media, or to take two separate samples
using the same media. Most commonly, dipslides will have one side with a selective agar and one side
with a non-selective agar.

32. Swabs are indirect sampling devices
made of various materials, including
cotton, rayon, polyester, calcium alginate,
or macrofoam, and they may be flocked
by design. Swabs can be manipulated
around difficult or uneven surfaces, such
as door handles, bed rails, and around
sinks and taps.
They are the most frequently used
sampling method. This is perhaps due to
their simplicity, affordability, and
availability in the hospital environment.
Flocked swabs have a nylon fibre coating
added in a flocking process. This coating
allows better sample adsorption through
capillary action.
Rayon- and polyester-tipped swabs are
manufactured similarly to cotton swabs,
though the bud material is different.
Brush-textured swabs are produced by
spraying nylon flock on to a plastic
spatula or swab bud. Handles are made
of plastic, wood, or metal.
Swabs:

33. Swab Contd….
• It was found that cotton swabs recovered significantly more colonies
than other swabs from a wet surface. These results emphasize the
need to understand the surfaces that will be sampled to optimize
swab choice.
• Across the literature, macrofoam swabs are generally found to be the
most effective type of swab.
• Variation in results is not only explained by difference in device,
target organism, and surface state, but by the difficulty in
standardizing sampling pressure, size of sampling area, angle of
swab, and pattern while sampling. This can cause variation in
recoveries.

34. • Sponges are an indirect sample device which can be
manipulated around uneven surfaces, can sample a
wider surface area with ease, and some pressure can
be exerted during sampling.
• As such, sponges are often reported to have better
recoveries than other methods and have been shown to
be significantly better for Clostridioides.

35. Petrifilms
Petrifilms are more often used in the food industry, though they
should not be overlooked for use in clinical environments. They
are fast, simple to use, and have a wide variety of
applications. Petrifilms can be inoculated with a swab or can be
used as a direct contact method for both surface sampling and
finger dabs.
Once the surface of the petrifilm paper has been wetted, the
paper is pressed against the surface for testing, the film closed,
and incubated. A plate count can be read directly from the
petrifilm. They are available impregnated with either selective or
non-selective media for colony counts or specific pathogen
detection.
Petrifilms have an advantage over contact plates as they are
flexible and can adapt to the topography of a surface. Petrifilms
were the best method for recovering MRSA from linoleum,
mattress, coated steel, and polypropylene.

38. • Different methods and additional processing steps and options to improve recovery are available.
• Swabs, sponges, and wipe methods can be enhanced by pre-wetting prior to surface sampling.
• Wetting solutions and diluents can either aid or hinder recovery, depending on the target organism.
• There are many wetting agents available, ranging from sterile saline, buffered peptone water, various strengths
of Ringer solution and letheen broth.
• It is also possible to use a wide variety of transport media and neutralizers. When choosing a neutralizer, it is
important to consider the potential presence of chemical residue on the surface.
• When selecting transport medium, time between sampling and processing must be determined in advance.
• Samples were generally processed immediately, within 4 h or stored in transport media at 4 C for no more than
24h.
• Wetting agents
Microbial recovery from surfaces was significantly improved by pre-moistening for all swab types. A dry cotton
swab gave 8.0% recovery and pre-moistening improved recovery to 41.7%.
• Wetting solutions with letheen broth and solutions with buffered peptone water significantly increased recovery
rates of S. aureus and Escherichia coli at room temperature.
• Phosphate-buffered saline was optimal for E. coli and Bacillus cereus, whereas phosphate-buffered saline with
Tween was better for Burkholderia thailandensis recovery.
• Cotton-tipped swabs in one-quarter-strength Ringer solution were best for E. coli recovery alone.
• The Cyto-brush textured swab in Copan rinse formula was best for S. aureus recovery.
Pre-analytical sampling choices: sample device
wetting, transport, and storage.

39. Transport Media and Neutralizers:
• Transport medium, such as anaerobic universal transport medium, aerobic Amies medium and
neutralizing buffer, is the solution used for sample storage before processing.
• Choice of transport medium is important, and the choice should vary depending on the target
organism, time taken to transport to the laboratory, and post-test storage conditions and storage time.
• Neutralizing broths help to keep microbial cells intact while also neutralizing any chemical cleaning
substances that may have been collected along with the microbiological sample.
• Some transport media allow inhibition of growth to enable more accurate estimation of counts.
Polyurethane swabs without transport medium gave the highest recoveries if tested within 2 h, and
viscose swabs with aerobic Amies transport medium were second best, giving 90.7% and 25.7%
recoveries, respectively.
• Viscose swabs with no transport medium had the lowest recoveries overall at just 8.4%. However, if
swabs were not processed within the first 24 h, addition of transport medium was critical to avoid cell
death or excessive growth, leading to inaccurate counts. It was shown that bacteria that adhere to dry
fibers can become desiccated, allowing only 3-5% recovery.

40. Sample processing-Analytical Analysis:
• If using an indirect sampling method, following sampling, direct plating on to agar, enrichment or
molecular processing are the available options. The choice of processing method is dependent on the
organisms being investigated, cost, and time available.
• Culture analytical processing options
Sample extraction. Swab, sponge, and wipe samples require extraction (i.e. removal of the target from
the swab) in order to undergo further processing. Extraction solutions include phosphate-buffered saline,
Butterfield’s buffer, Butterfield’s buffer and Tween, and maximum recovery diluent.
• After target organism, choice of extraction solution was found to have the next biggest impact on
extraction efficiency.
• Ensuring optimum extraction of the sample is important in the reduction of associated losses.
• Vortexing, agitation, or sonication of the swab or sponge are three methods that may increase recovery.
• An optimum time of 2 min vortexing was shown to be superior over 12 min of sonication, followed by
agitation to remove Bacillus anthracis spores from a swab.

41. Sample enrichment:
• Enrichment involves placing the sample directly into a broth and incubating,
providing time to grow in favorable conditions. It can be useful for slower-
growing organisms, cells that have become stressed, or to select the target
organism from a swab or non-selective sample.
Incubation conditions: Incubation times and temperatures varied in the
literature, ranging from 18 to 48 h, or non-specific 'overnight.

42. • Molecular methods are extremely valuable for analysing the microbiological contaminants of the
hospital surface environment.
• Whereas historically organisms were identified using culture methods, not all clinically relevant
organisms are culturable, such as norovirus, for which polymerase chain reaction (PCR) methods
based on nucleic acid detection must be used.
• Studies which investigated the presence of other viruses on surfaces also used PCR methods. As
such, molecular methods
using next generation sequencing, such as metagenomic approaches and 16S rDNA gene
sequencing, which support the
capture of total bacterial or organism diversity, should be considered in order to provide a true
picture of the contaminants in the hospital environment.
• To ensure that diversity is accurately assessed, consideration should begivento targets withinthe16S
rDNA gene. As with all detection methods, these can also be affected by primer design and inhibition
due to contaminants
such as cleaning agents and sample processing bias.
• For the majority of studies focusing on bacteria in this review, only traditional microbiological culture
methods were used (N ¼ 43).
Molecular Biology Processing

43. • Molecular methods were generally only used for comparisons of environmental and patient strains (N
¼ 6) or to further identify specific pathogens after performing phenotypic tests (N ¼ 7).
• Only two studies used high-throughput sequencing to investigate the entire collection of isolates
further identified using molecular methods to give a comprehensive reflection of the microbiome:
one of these looked at the hospital microbiome, the other examined the microbiome of surfaces on
the International Space Station.
• For studies focusing on viral contamination, molecular methods were the only way of assessing
presence, absence, and species identification.
• Another molecular identification method that has been adopted in many clinical laboratories is
matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry (MALDI-TOF).
• This method is able to identify a range of bacteria, mycobacteria and fungi by looking at their protein
fingerprint, based on the charge and size of the proteins. A number of the studies included in this
review used MALDI-TOF to confirm species identification after using selective media and phenotypic
tests.

45. • Air sampling involves collecting samples of air to test for the presence of airborne pathogens or
contaminants. This can be done using specialized equipment such as air samplers, which draw in a
certain volume of air and trap any particles or microorganisms present.
Air Sampling

47. • Biological contaminants occur in the air as aerosols and may include bacteria, fungi, viruses,
and pollens. Aerosols are characterized as solid or liquid particles suspended in air.
• Particles in a biological aerosol usually vary in size from <1 μm to 50 μm. These particles may
≥
consist of a single, Unattached organism or may occur in the form of clumps composed of a
number of bacteria. Clumps can also include dust and dried organic or inorganic material.
• Vegetative forms of bacterial cells and viruses may be present in the air in a lesser
number than bacterial spores or fungal spores. Factors that determine the survival of
microorganisms within a bioaerosol include:
a. the suspending medium,
b. temperature,
c. relative humidity,
d. oxygen sensitivity, and
e. exposure to UV or electromagnetic radiation.

48. • Pathogens that resist drying (e.g., Staphylococcus spp., Streptococcus spp., and fungal spores) can survive for long
periods and can be carried considerable distances via air and still remain viable. They may also settle on surfaces
and become airborne again as secondary aerosols during certain activities (e.g., sweeping and bed making).
• Microbiologic air sampling is used as needed to determine the numbers and types of microorganisms, or
particulates, in indoor air.
• Health-care professionals considering the use of air sampling should keep in mind that the results represent
indoor air quality at singular points in time, and these may be affected by a variety of factors, including:
a. indoor traffic,
b. visitors entering the facility,
c. temperature,
d. time of day or year,
e. relative humidity,
f. relative concentration of particles or organisms, and g) the performance of the air handling system
components.
• To be meaningful, air-sampling results must be compared with those obtained from other defined areas, conditions,
or time periods. Several preliminary concerns must be addressed when designing a microbiologic air sampling
strategy.

49. • Consider the possible characteristics and conditions of the aerosol, including size range of particles,
relative amount of inert material, concentration of microorganisms, and environmental factors.
• Determine the type of sampling instruments, sampling time, and duration of the sampling program.
• Determine the number of samples to be taken. Ensure that adequate equipment and supplies are available.
• Determine the method of assay that will ensure optimal recovery of microorganisms.
• Select a laboratory that will provide proper microbiologic support.
• Ensure that samples can be refrigerated if they cannot be assayed in the laboratory promptly.
• Bacteria, fungi, and particulates in air can be identified and quantified with the same methods and equipment.
• The basic methods include:
a. impingement in liquids,
b. impaction on solid surfaces,
c. sedimentation,
d. filtration,
e. centrifugation,
f. electrostatic precipitation, and
g. thermal precipitation.
Preliminary concerns for conducting air
sampling.

50. Methodology/Instruments used:
• Several instruments are available for sampling airborne bacteria and fungi.
• Some of the samplers are self-contained units requiring only a power supply and the appropriate collecting medium,
but most require additional auxiliary equipment (e.g., a vacuum pump and an airflow measuring device [i.e., a
flowmeter or anemometer]).
• Sedimentation or depositional methods use settle plates and therefore need no special
instruments or equipment.
• Selection of an instrument for air sampling requires a clear understanding of the type of
information desired and the determinations that must be made.
• Information may be needed regarding:
a. one organism or all organisms that may be present in the air,
b. the concentration of viable particles or of viable organisms,
c. the change in concentration with time, and
d. the size distribution of the collected particles.
• Before sampling begins, decisions should be made regarding whether the results
are to be qualitative or quantitative.
Comparing quantities of airborne microorganisms to those of outdoor air is also standard operating procedure.
• Infection control professionals, hospital epidemiologists, industrial hygienists, and laboratory supervisors, as part of a
multidisciplinary team, should discuss the potential need for microbial air sampling to determine if the capacity and
expertise to conduct such sampling exists within the facility and when it is appropriate to enlist the services of an
environmental microbiologist consultant.

55. Selecting an Air sampling device
The following factors must be considered when
choosing an air sampling instrument:
• Viability and type of the organism to be sampled
• Compatibility with the selected method of
analysis
• Sensitivity of particles to sampling
• Assumed concentrations and particle size
• Whether airborne clumps must be broken (i.e.,
total viable organism count vs. particle count)
• Volume of air to be sampled and length of time
sampler is to be continuously operated
• Background contamination
• Ambient conditions
• Sampler collection efficiency
• Effort and skill required to operate sampler
• Availability and cost of sampler, plus back-up
samplers in case of equipment malfunction

56. • The use of settle plates (i.e., the sedimentation or depositional method) is not recommended when sampling air for
fungal spores, because single spores can remain suspended in air indefinitely. Settle plates have been used mainly
to sample for particulates and bacteria either in research studies or during epidemiologic investigations.
• Results of sedimentation sampling are typically expressed as numbers of viable particles or viable bacteria per unit
area per the duration of sampling time (i.e., CFU/area/time); this method can not quantify the volume of air
sampled.
• One advantage of using a settle plate is its reliance on gravity to bring organisms and particles into contact with its
surface, thus enhancing the potential for optimal survival of collected organisms. This process, however, takes
several hours to complete and may be impractical for some situations.
• Air samplers are designed to meet differing measurement requirements.
• No one type of sampler and assay procedure can be used to collect and enumerate 100% of airborne organisms
obtained for biological analysis. Newer analytical techniques for assaying air samples include PCR methods and
advanced techniques.

57. Water Sampling
Water sampling involves collecting samples of water from various sources within the hospital, such as taps or showerheads. These
samples are tested for the presence of harmful bacteria or other contaminants i.e used detect waterborne pathogens of clinical
significance or to determine the quality of finished water in a facility’s distribution system.
Routine testing of the water in a health-care facility is usually not indicated, but sampling in support of outbreak investigations can
help determine appropriate infection-control measures.
Health-care facilities that conduct water sampling should have their samples assayed in a laboratory that uses established methods
and quality-assurance protocols.
Water specimens are not “static specimens” at ambient temperature; potential changes in both numbers and types of microbial
populations can occur during transport. Consequently, water samples should be sent to the testing laboratory cold (i.e., at
approximately 39.2°F [4°C]) and testing should be done as soon as practical after collection (preferably within 24 hours).
Because most water sampling in health-care facilities involves the testing of finished water from the facility’s distribution system, a
reducing agent (i.e., sodium thiosulfate [Na2S2O3]) needs to be added to neutralize residual chlorine or other halogen in the
collected sample.
If the water contains elevated levels of heavy metals, then a chelating agent should be added to the specimen.

58. • Volume: The minimum volume of water to be collected should be 100 mL is considered a suitable minimum volume.
• Sterile collection equipment should always be used.
• Sampling from a tap requires flushing of the water line before sample collection and hot and then cold water must
be run through the tap before collecting the sample.
• Microorganisms in finished or treated water often are physically damaged (“stressed”) to the point that growth is
limited when assayed under standard conditions. Such situations lead to false-negative readings and misleading
assessments of water quality.
• Appropriate neutralization of halogens and chelation of heavy metals are crucial to the recovery of these
organisms.
• The choice of recovery media and incubation conditions will also affect the
assay. Incubation temperatures should be closer to the ambient
temperature of the water rather than at 98.6°F (37°C), and
recovery media should be formulated to provide appropriate
concentrations of nutrients to support organisms exhibiting
less than rigorous growth. High-nutrient content
media (e.g., blood agar and tryptic soy agar [TSA])
may actually inhibit the growth of these damaged organisms.
• Reduced nutrient media (e.g., diluted peptone and R2A) are
preferable for recovery of these organisms

59. • Use of aerobic, heterotrophic plate counts allows both a qualitative and
quantitative measurement for water quality. If
bacterial counts in water are expected to be high in number (e.g., during
waterborne outbreak investigations), assaying small quantities using pour plates
or spread plates is appropriate.
• Membrane filtration is used when low-count specimens are expected, and larger
sampling volumes are required ( 100 mL). The sample is filtered through the
≥
membrane, and the filter is applied directly face-up onto the surface of the agar
plate and incubated.
• Control or comparison samples should be included in the experimental design.
• Any departure from a standard method should be fully documented and should be
considered when interpreting results and developing strategies.
• Assay methods specific for clinically significant waterborne pathogens
(e.g., Legionella spp., Aeromonas spp., Pseudomonas
spp., and Acinetobacter spp.) are more complicated and costly
compared with both methods used to detect coliforms and other
standard indicators of water quality.
Methodology

60. • Interpreting the results of laboratory analysis of samples collected from hospitals for health care
associated infections requires a thorough understanding of microbiology and infectious diseases.
• The presence of certain microorganisms in a sample does not necessarily indicate an infection, as
some microorganisms are part of the normal flora of the human body.
• Therefore, it is important to identify the specific microorganism and determine whether it is
pathogenic or not.
• The interpretation of results also involves considering the clinical context of the patient. For
example, a positive result for a particular microorganism may be significant in a patient with
symptoms of infection, but not in a patient who is asymptomatic.
• Additionally, the interpretation of results may be influenced by factors such as the sensitivity and
specificity of the laboratory test used and the prevalence of the microorganism in the hospital
population.
Interpretation of Results

61. • Reporting the results of sample analysis to hospital staff and public health officials is a
crucial step in preventing the spread of healthcare-associated infections (HAIs).
• By sharing this information, hospitals can identify potential outbreaks and take steps to
prevent further transmission.
• In addition to informing hospital staff, reporting also plays an important role in public
health surveillance.
• Public health officials use this information to track the incidence and prevalence of HAIs
in the community, identify trends, and develop strategies for prevention and control.
Reporting

62. • Quality control measures are essential in ensuring the accuracy and reliability of sample
collection and analysis for healthcare-associated infections in hospitals.
• These measures include proper training of personnel, use of appropriate equipment and
materials, adherence to standard operating procedures, and regular monitoring of results.
• One important aspect of quality control is the use of positive and negative controls, which
are samples known to either contain or not contain the target pathogen.
• These controls are used to validate the accuracy of the testing process and ensure that
results are not affected by external factors such as contamination or improper handling.
Quality Control

63. • Collecting samples from hospitals for health care associated infections can be a challenging process.
• One of the main challenges is ensuring that the sample is collected from the right location. This
requires careful planning and coordination between hospital staff and laboratory personnel.
• Another challenge is ensuring that the sample is collected in a sterile environment to prevent
contamination.
• In addition, collecting samples from certain patient populations can be difficult. For example,
patients who are immunocompromised may have a lower number of microorganisms present in
their samples, making it more difficult to detect an infection.
• Overcoming these challenges requires a combination of proper training, attention to detail, and
collaboration between hospital staff and laboratory personnel.
Challenges in Sample Collection

64. Ethical Considerations
• When it comes to collecting samples from hospitals for health
care associated infections, there are a number of ethical
considerations that must be taken into account.
• For example, patients have the right to privacy and
confidentiality, which means that any samples collected must be
done so in a way that respects these rights.
• Additionally, there is also the issue of informed consent. Patients
must be fully informed about what the sample collection
process involves, as well as the potential risks and benefits of
participating.
• This can be particularly challenging in cases where patients may
not fully understand the implications of their participation, such
as those with cognitive impairments or language barriers.

65. Legal Considerations
• When collecting samples from hospitals for health care associated
infections, it is important to consider the legal implications involved.
• Hospitals are subject to various laws and regulations that govern the
collection, handling, and storage of patient samples. Failure to comply with
these laws can result in serious consequences for both the hospital and the
individuals involved.
• One important legal consideration is patient privacy. The Health Insurance
Portability and Accountability Act (HIPAA) requires that patient information
be kept confidential and only disclosed to authorized individuals. This means
that any samples collected must be properly labeled and stored to protect
patient privacy.
• Additionally, hospitals must obtain informed consent from patients before
collecting any samples. Informed consent ensures that patients understand
the purpose of the sample collection and how their information will be used.

66. Training and Education
Proper training and
education are essential for
hospital staff involved in
collecting samples for health
care associated infections.
It is important that staff
understand the proper
techniques for sample
collection, including site
selection, sample
processing, and storage.
In addition, staff should be
trained on the importance of
sterilization and disinfection to
prevent contamination of
samples.
Furthermore, staff should be educated on
the importance of timely and accurate
reporting of sample results to hospital
management and public health officials. This
ensures that appropriate measures can be
taken to prevent the spread of health care
associated infections within the hospital and
the community at large.

67. • Collaboration and communication between hospital staff, public health
officials, and laboratory personnel are crucial to effectively collect and
analyze samples for healthcare-associated infections. Each group has a
unique perspective and skillset that is necessary for the success of the
process.
• Hospital staff provide valuable insight into patient care practices and can
identify areas of concern for infection prevention. Public health officials
have a broader view of community health and can provide guidance on
best practices and outbreak control.
• Laboratory personnel have expertise in sample processing and analysis,
which is essential for accurate results. Effective collaboration and
communication among these groups can lead to improved patient
outcomes and a safer healthcare environment.
Collaboration and Communication

68. Future Directions
In the future, sample collection and analysis for health care associated infections in
hospitals will become more streamlined and efficient.
Advances in technology will allow for faster and more accurate identification of pathogens,
leading to quicker treatment and prevention of infections.
Additionally, there will be a greater emphasis on data sharing and collaboration between
hospitals, public health officials, and laboratory personnel to better track and prevent
outbreaks.
Another important direction for the future is the development of new sampling techniques
and tools. For example, there may be new methods for collecting samples from hard-to-
reach areas or for detecting pathogens that are currently difficult to identify.
These advances will lead to more comprehensive and effective surveillance of health care
associated infections in hospitals.

69. Conclusion
• In conclusion, health care associated infections are a serious concern in hospitals
and proper sample collection and analysis is crucial for preventing their spread.
• We have discussed the importance of collecting samples from hospitals, the
different types of samples that can be collected, proper site selection, sample
collection methods, sterilization and disinfection, transportation and storage, sample
processing, laboratory analysis, interpretation of results, reporting, quality control,
challenges that may arise, ethical and legal considerations, training and education,
collaboration and communication, and future directions.
• It is important to emphasize that preventing health care associated infections
requires a team effort between hospital staff, public health officials, and laboratory
personnel.
• By working together and following proper procedures for sample collection and
analysis, we can reduce the incidence of these infections and improve patient
outcomes.

70. THANK YOU