Specimen Collection and Laboratory Diagnosis

Special Specimen: Malaria

-Endemic in many tropical and sub-tropical countries -In Canada, malaria occurs in travellers and immigrants from endemic countries -Life-threatening with significant morbidity and mortality -Specimens: 1) Blood by venipuncture 2) Blood by capillary puncture - higher sensitivity when take blood from fingerprick capillary sample Clinical information: - Country of travel, symptoms Diagnostic tests: - Rapid antigen detection test - Thick and thin smear

Microorganisms in Positive Blood Test Cultures

-E. Coli -Coagulase negative staph → found on the skin -S. aureus → This can be a very significant pathogen with morbidity and mortality -Enterococcus -There can be a whole range of other pathogens including Klebsiella, Enterobacter, Pseudomonas, and Candida spp.

Fungal Diagnostic Methods

--Similar to the S. pneumonia, we use different media. Aspergillus nidulans is a mold. We identify the fungus based on microscopic examination of culture. -Mould detection requires an incubation time of up to 3 - 6 weeks • Identification is mainly based on microscopy, colonial features, growth at different T and some reagents

Blastomyces dermatididis

-It appears as neon blue

Sterile Fluids

-Pericardial fluid -Pleural fluid -Peritoneal fluid -Solid organ aspirates: spleen, liver, brain

Fungal Specimens

-Specimen requirements similar to bacteria except for: -Swabs are not recommended because fungi can also get stuck in the swabs, unless we're dealing with yeast -One of the most common causes of fungal infections is a mycosis, resulting in yellow and thickened nails. -One would scrape things off an infected nail or infected skin, put it in black paper and send it off to the lab.

Specimen Collection

-Testing must be kept to a minimum -All testing must be approved by Microbiologist/Lab Director -Use PPE as directed by Infection Control/Ministry Guidelines -PPE must be available -PPE donning and doffing - training required and regular refresher -Buddy system to ensure no breaches in PPE -Do not collect using glass containers -Disinfection of specimen, container, biohazard bag -Placement of specimen into leak-proof container with absorbent -Chain of custody - specimen should never be left unattended, all personnel with contact with the patient or specimen must be documented -Need for training - use of MOCK exercises to evaluate process

What are the clinical consequences

-The blood culture belongs to another patient and that patient requires treatment for the infection -The patient does not receive treatment and has complications and impact on morbidity and mortality -The blood culture ordered on the wrong patient may result in treatment that is not required 1) Potential side effects 2) Increased length of stay 3) Delay of surgery 4) Cost

What happens in the lab?

-The lab will receive the specimen, decide if it is acceptable and do the testing.

Listeria Monocytogenes

-This organism when it causes infection is a gram positive. It is a more of a rod but a small rod. It is a GP bacilli. It looks very different in culture than a S. pneumonia. -It moves in a typically umbrella like pattern

Examples of Retrievable Specimens

-Urine Sample

Stool Specimen Errors

-A common error would delay diagnosis and treatment as sample needs to be obtained using the correct container. -It would be increased workload for the nurses, lab and the cost of the delay in diagnosis would also matter -For C. difficile testing, stool will be rejected if not liquid

TB Specimen

-A whole different category of bacteria when we look at mycobacterium tuberculosis -For sputum, this is one of those bugs, we want to take repeated specimens. If you take a first morning specimen 3 days in a row, with each successive specimen, this will increase our likelihood of detecting and growing the TB bug -Do not rinse with tap water because this can result in contamination from environmental mycobacteria • Bronchoscopy is also good • Spinal fluid is also good • For BC, we would need a special tube • We might also want to consider a tissue or an aspirate -Swabs are not good because they have a small volume, but also because, for some types of organisms like TB which has a very very thick cell wall, it gets stuck very readily in the swab because it is lipophilic. -For pediatric patients, we may use a gastric lavage (usually first thing in the morning) reflecting the swallowing of sputum or secretions overnight -Requires a special container and neutralization of acid

Inoculation of Media

-After we process the specimen and make that slide, we actually inoculate it to different media. o We may inoculate a portion of the centrifuged sediment. o The media be enriched, selective or differential. -All of our media involve these kind of containers; we have 15 - 20 different types of media. We chose the media that targets the site so that we can isolate the organisms we need. -The media can either be enriched (blood or other vitamins) to help bacteria grow. We can add certain chemicals to make bacteria show up. This is called differential. -We might put antibiotics in to prevent certain bacteria from growing so that we can isolate different types. -For a child, age makes a big difference, so Group B strep and E. Coli would be more likely to cause bacterial meningitis. It would also matter if the patient was immunocompromised. -Other pathogens that could be found in SF include: o S. pneumonia o N. meningitis o H. influenza o Listeria monocytogenes

Collection of Stool Specimens

-Avoid sending rectal swabs unless they are for infants -When making the collection, do not collect directly from the toilet water. We would either fit a pan or saran wrap over the toilet. -Use a plastic collection device that fits into the toilet, or a sterile bedpan -Avoid laxatives or mixing urine or water with the stool -There is a recommended ratio of stool to preservative in the container, and this is indicated on the container. -Stool for bacterial culture containers and stool for parasitology SAF fixture containers have a red line. We do not want to go over it because it has a special liquid in there that is attempting to preserve the organism. -The organisms that we are interested in would die out if we did not have the liquid and couldn't get it to the lab fast enough -We want to preserve the bacteria so that it can grow. -We don't want to grow the parasite but we don't want it to get distorted. The liquid in here preserves it so that when looking under a microscope, we can see the eggs or organism clearly, thus making the diagnosis easier. This is the SAF fixture. -For a lot of the molecular testing, we only need a sterile container. Examples include virus detection by PCR or C. difficile, where we just need to detect the molecular presence. -Green bottle: bacterial culture -Yellow bottle: Paristology SAF fixture -Orange bottle: C. Difficile, virus detection by PCR

Blood Culture Instruments

-Blood culture bottles are placed in the instrument ASAP -The instrument incubates the bottle at 35°C, mixing the bottles -A continuous detection system looks for an increase in CO2 or decrease in oxygen -As soon as growth is detected the instrument alarms BC bottles are incubated 5 days -A positive blood culture bottle is then removed, a gram stain is performed and fluid is inoculated onto media plates -We place each of the bottles in its own specific spot and this instrument will incubate the bottles at 35 degrees and continually look to see if there is evidence of growth every 5 minutes -As soon as it detects growth, it alarms and technologist takes it out and makes that Gstain. o It detects growth through an increase in CO2 or decrease in O2. o Once this occurs, the instrument alarms.

Blood Cultures

-Blood cultures are one of the most critical specimens for diagnosis. We are trying to isolate either bacteria or yeast that are in the bloodstream. Any patient that has either of those are very sick. -When we have a patient with a positive blood culture, it can be a life threatening event due to one of several syndromes from endocarditis (infection of the heart valves), to pneumonia, urosepsis (infection started in the UT and then spilled over into the blood), meningitis, abscess and catheter related. -This is a critical specimen because timely and appropriate antibiotics are key to optimizing patient treatment. -It is one of those microbiology specimens that provides information regarding life threatening events. -Once the lab produces those results, action must be taken fairly quickly. If not on antibiotics, the patient is started on them. If started on antibiotics, the antibiotics must be reassessed to see if they were appropriate. -This is considered a microbiology specimen that can generate a critical result.

List the bronchoscopy specimens

-Bronchial wash -Broncho-alveolar lavage

Examples of Irretrievable Specimens

-Cardiac arrest or operating room gases -Blood gases by arterial puncture & scalp blood gases -Autopsy, surgical pathology & cytopathology specimens -Sterile body fluid, aspirate or tissue (e.g. Spinal fluid) -Blood cultures before antibiotics started

Intravascular Catheter Tips

-Central line, swan gans line: They have a longer plastic system so they go into the longer and bigger veins compared to a peripheral IV. This allows them to stay in a bit longer. -Especially in the ICU, this can be a big route of infection. When a central line is removed because they think it is the source of infection, they will actually send in the tip to the lab. The nurse or physician would be sending this it. Important to use sterile technique and cut it at 5 cm. -These details are to optimize so that we can detect what the organism is.

Disadvantages of Molecular Methods

-Contamination can occur in the laboratory -If the target site of the organism mutates, the assay will not detect organism = false negative -Susceptibility testing cannot be performed -Can be extremely sensitive so picks up "colonization" not necessarily disease Eg. C. difficile -Result can remain positive for a long period of time - can reflect dead organism -There are some organisms we cannot culture. We can only detect it using an MM. -If it is a new organism and we don't know how to detect it, MM can be useful. E.g. SARS -MM can be quantitative and this can give us a good idea as to whether they have disease and are responding to treatment. -In the lab setting, it can also amplify the contaminants which can be misleading -If the target site of the organism mutates, the assay will not work -Susceptibility testing will not work.

Consequences of Blood Culture Contamination

-Contamination of blood cultures has a negative impact on patient care -Consequences of false positives: -Is this is a real pathogenic problem that we need to look into and treat? o Longer hospital stay o Greater cost for antibiotics and investigations -If we report out coagulase negative staphylococcus, it would be gram positive cocci in clusters. This would probably generate that the patient be put on IV antibiotics and increase their length of stay. It may prevent the physician from thinking of another diagnosis for the symptoms -We need to minimize contamination -Contamination increases cost considerably in the hospital -The front line staff are collecting the blood cultures so we need to be equally focused on it as the lab is.

Stool Specimens

-Depending on the class of organism we are looking at, we may need very different specimens and containers. The following organisms are quite different and therefore need different containers and different transport media in order to preserve them. -Campylobacter spp. is an example of bacterial culture and we need a specific specimen collection container for that. Most common cause of community acquired infectious diarrhea -C. difficile -Viral detection: norovirus, rotavirus, enterovirus -Parasites: eggs and larvae -Common specimen where if put in the wrong container, the lab cannot test. We would need to re-collect.

Storage Recommendations

-Do not store blood cultures or spinal fluids for bacterial culture at 2 - 8 ºC. o These are critical specimens and we want to grow the organism. If we refrigerate them, some might die and some may be quite stunted. Will take a long time for the lab to be able to grow them. -Spinal fluids are critical specimens and they should get to the lab in an hour and blood cultures should always be stored at room temperature, not in a fridge.

Laboratory Requisition

-How does the lab know what we have ordered and what the test is? -Legally, there has to be a lab requisition. This can be a paper form. -When submitting a sample, there must be a paper requisition, which uniquely identifies the patient. • There are several requirements of a lab requisition: o Unique identification of the patient o Gender, age and DOB o Specimen type, anatomic site and clinical information o Test ordered o Date and time of collection o Who requested the test? (which physician was responsible?) • Must be uniquely identified • Copy to physician -There is unique identification of the specimen, patient and on the requisition. -On the requisition, the lab asks for other information. For microbiology, we need some clinical information and the anatomical site. If we see "wound" but not what kind of wound, the testing might not be set up to examine the appropriate and important scenarios. -For example, if somebody came in with a dog bite, we need to account for the flora in the dog's mouth would require additional testing media. -For example, a wound or abscess on the jaw may cause us to look for actinomyces that require further incubation. -Give us information where the swab was taken from and if there is ever any other important clinical information. -We need to know the test that was ordered. -When was the time of collection? For some specimens, they are time sensitive. -A blood culture must be received and put into the lab's instruments within 24 hours or else the instrument doesn't work as well as it could. -For spinal fluid, we want to ensure that gets to the lab right away. If we do not have the collection time, we don't know how appropriately it was transported. Spinal fluid should be in the lab within 1 hour of collection. -Who requested the test?

Wound Cultures

-If the abscess is closed, it must be aspirated -If we have a pustule or vesicle, look for intact pustules. Disinfect the skin with alcohol because it is normally contaminated. Then collect the cells at the base after unroofing the vesicle. -Collect fluid and cells at the base by rotating the swab vigorously -Certain patients may have chronic wounds, ulcers or fistulas. These are not great areas to swab because they are open to the environment all the time. They often don't reflect what the cause of infection is. If you do have to, disinfect and get a good sampling off the base (a fresh part of the base) and the exudate and the wall of the abscess or advancing margin of the lesion -Never send the dry crust because this is not useful. -Lab does not routinely do anaerobic culture unless fluid, tissue or swab -Open ulcers, wounds, fistula: -Open areas are usually contaminated -Disinfect area before sampling -Sample the base of the lesion and the wall of the abscess/advancing margin of the lesion + exudate -Dry crust is not useful -Volume allows the laboratory to maximize detection of aerobic organisms -Anaerobic organisms are best detected in tissue or fluid

Lower Respiratory Specimen: Endotracheal Aspirate

-If you are in the ICU or CCU setting, when patients are on ventilators, we take an aspirate of the breathing tube that is in there currently -Performed in patients who are intubated -Catheter passed through endotracheal tube and aspirated -Can be contaminated as it passes the tube

Immunoassays

-Immunoassays using antibodies directed to the analyte to detect a pathogen Methods: -Enzyme immunoassay (EIA) -Most common method Uses an enzyme label to detect that the patient's sample reacted with the antibody or antigen Eg/ HIV antibody detection, C. difficile toxin detection POC GAS kits in offices -Immunofluorescence NPS swab for virus detection: Cells from the patient placed on a glass slide in wells Fluorescent labelled antibody to the virus is added If the virus is present, fluorescence is detected using a fluorescent microscope

Lower Respiratory Specimen: Broncho-Alveolar Lavage

-In this, the bronchoscopy goes further. They feed that tube all the way through the particular lobes. -Fluid is instilled into the smaller airways and aspirated -This is better specimen quality as it reflects smaller airways in a particular lobe of the lung -The right middle lobe and the left lower lobe are candidates. They will wedge it in there, put in a bit of fluid and suction the secretions right from that particular lobe. This is a really good specimen and it reflects exactly what is going on in that area. -The bacteria can be quantitated by the lab and this provides better diagnostic information

Advantages of Molecular Methods

-Increased sensitivity compared to culture based method Eg/ herpes simplex virus in the spinal fluid C. trachomatis in genital or urine specimens Norovirus detection in stool samples -Can detect organisms where specimen may be delayed in transit eg/ N. gonorrhoeae difficult to culture if increased time to transport to the laboratory -Detect organisms that cannot be cultured eg/ Whipple's disease -Detect new organisms more rapidly then traditional methods eg/ SARS virus -Provide quantitative result

Occurence Management Report

-Is an important tool in any quality management system to look and see how well we are doing. The occurrence reports are the responsibility of the physician, nurse and lab tech to file when they realize an error has occurred. It gives us a good overview of what happens in real life. -The largest amount of errors involves incorrect patient registrations. The specimen and the requisition may come but the wrong patient may be picked, or the information may be provided but not entered into the system. -Next are the unlabeled specimens. Specimens may be unlabeled because somebody has forgotten it. We need to know who the patient is in order to work with the specimen. It is a consistent error even though it could be easy to decrease. -We could have poor specimen quality. -The specimen could be lost or broken. -We could have the incorrect tests -We have made inroads to improve incorrect specimen containers. This used to be one of the higher errors. -The patient report may be sent to the wrong location, which is a post analytical error. -We want to decrease errors to get appropriate results.

Maximize ability to isolate pathogen

-Maximize ability to isolate pathogen -Must collect specimens with the appropriate container and swab. -Transport media preserves the organism of interest until testing -The types of swab vary depending on the pathogen -Volume of specimen -Timing -When working with containers or swabs, there is actually either a gel or a liquid. This is pretty important because this is transport media. They are designed such that if we collect something and put it back in, that transport media will actually preserve that organism until it gets to the lab. -Volume of specimen: We want to optimize the volume → not too much and not too little.

Specimen Acceptance and Rejection

-Microbiology specific rejections may happen because...We need good quality of the specimen o We would reject formed stool for C. difficile o We need to receive our specimens on time o We can't have saliva in sputum samples -We would detect this by determining the ratio of squamous epithelial cells to pus cells. More than 10 squamous epithelial cells suggests oropharyngeal contamination. o We don't want our swab or blood culture media beyond expiry dates -Common reasons for rejection include the facts that we may reject it if... o It is the wrong container or a wrong swab o If it is leaking o If it is incorrectly labeled or misidentified o If we reject the specimen, the provider must redo it. If we get it right the first time, the patient get results back sooner.

Transport

-Might need to label it as STAT and calling a porter to deliver it to the lab -If you have collected a urine, need the courier to pick up the specimen in an appropriate period of time -All specimens should be transported to the lab within 2 hours -Spinal fluids and STATs should be transported within 1 hour -If you don't transport it and it sits for a day or two, if it is a urine sample, would likely be rejected. If it was processed, would likely be very contaminated. If the patient did have a pathogen, it may not be detected. -With N. gonorrhea, it can die out within 6 hours. Need to ensure appropriate and timely delivery to the lab. -A delay of more than 24 hours is not recommended for bacterial culture as the organism may die o Group A streptococcus from throat swabs o N. gonorrhea from cervical culture

Urine Cultures

-Most common, but BC are the most significant cultures we get. -This is probably our largest volume specimen. With urine cultures, they are often collected when the patient is asymptomatic. We do not want patients collecting when they have no symptoms. -When a patient has a UTI, they may experience local symptoms like dysuria (painful urination), hematuria, and urgency. If the infection starts ascending up (kidneys and blood), we will get fever, rigors and back pain. These are the standard symptoms for UTIs or ural sepsis. -Asymptomatic patients should have urine cultures taken if they are pregnant (through routine screening) and if they are undergoing invasive urologic procedures. We want to ensure that our site is sterile if we are going to be doing surgery -This is one of the most frequently collected specimens when there is no clinical indication to collect. • This results in an inappropriate use of antibiotics • The development of antibiotic resistance • Increased risk of C. difficile related disease -If a patient has cloudy urine, this does not indicate that there is a UTI. If there has been a cognitive change (e.g. a nursing home) it may be hard to communicate what the symptoms are. -The subsequent consequences are that if bacteria are found but the patient is asymptomatic, they will be put on antibiotics when they shouldn't be. This can be a risk factor for C. difficile.

Microorganisms invoelde in diarrhea

-Most hospitals have criteria for when they will do stool testing for the various pathogens. If somebody has been hospitalized for that period of time and develops new onset diarrhea, the most common organism is C. difficile. -This is likely due to nosocomial transmission. -Campylobacter spp. Is a very common bacteria. If you were sick and out in the community, this is the most common organism that causes infectious diarrhea and it can be obtained through a variety of different foods -Norovirus is that virus that can be very easily passed on; not the most common and we can see nursing home outbreaks -Giardia lambia is actually a parasite and therefore this would not be common to acquire in a hospital.

Urine Culture Instructions: Foley Catheter Specimens

-Never obtain the sample from the catheter bag as it is likely highly contaminated -That bag can be with a patient for days -We always use aseptic technique and we sample from a port. -We never want the tip of the Foley catheter -We sample from the sampling port using aseptic technique -If you are in a remote setting, we might put urine into special transport containers to preserve it for 24 hours

Safety for Needles and Sharps

-Never resheath (unless an approved aid used) -Never bend, break or cut -Dispose of in a puncture-resistant disposal container made of rigid plastic, metal or stiff cardboard -Container must have a lid -Do NOT overstuff -Mark container with biohazard sign -In Ontario: safety engineered needles or needle-less systems are required by law -Laboratory and hospital accreditation inspections will have observers watch practices

The most common breaks in technique

-Not allowing disinfectant to dry completely -Not disinfecting the BC bottle septum -Palpating the site of puncture after cleaning with non sterile finger (should be gloved or washed hand) -Placing the blood specimen on a non-sterile surface

Specimens

-Not expected to know everything by memory. Should know where the resources are. o Can consult the Hospital Specimen Collection Guide o Or the Specimen Collection Guide for private labs o Can also speak to a lab professional -If you don't get the right collection and the right container, you will not get the right test.

MALDI-TOF

-Novel method for rapid identification of microorganisms. -Laser pulses hit a sample of the grown bacteria or yeast, small ionized molecules are released and quantified based on mass, resulting in a pattern unique to each organism -Results are available in 20 minutes compared to routine methods that can take up to 24-48 hours for identification -Mass spec → we take the isolate that grows, zap it with a laser, look for the pattern based on the isolation time and then we identify it -This is a new technology that we have validated

Types of Swabs

-Nowadays we have many different types of swabs. They can differ in terms of tip composition and transport media -They are designed to collect, maintain organisms and then release the specimen when processed in the laboratory -Swabs differ in the type of: o Handle o Tip composition o Transport media that it is placed in -Each lab must evaluate the swab they use to make sure it will grow the expected organisms and can survive on the swab even if there is a delay of 24 hours in receipt to the lab -We can have a basic swab with a cotton tip and charcoal transport media, however the organism gets bound to the cotton tip and we cannot release it. -We have switched over to flocked swabs. The tip looks more like bristles than a completely solid surface and the transport media involves liquids. The advantage of the flocked swabs is that the organism gets released from the bristles once we put the tip back into the liquid. -With the cotton tip, the organism gets bound into the tip and we cannot release it. -When we are taking specimens for viruses vs bacteria because they have a different swab system and a different fluid to ensure viruses are preserved -Molecular swabs for detection of Chlamydia trachomatis and N. gonorrhoeae -Nasopharngeal swabs for viral studies

Molecular Methods

-Nucleic acid is extracted from the specimen or isolate -Can be applied to bacteria, viruses, parasitology and fungi -Different methods are used to amplify the RNA or DNA in the specimen: -Target amplification: PCR, Strand displacement, Transcription related amplification Probe and signal amplification -We are going down to the nucleic acid from the specimen or isolate. Can be used for almost anything but used a lot in viruses. -We are basically amplifying the virus' RNA or DNA. -We have a target that we want to amplify. The method will target that virus, amplify it and we will use a probe or different signal to detect that it is present.

Patient Care

-Safety, effectiveness, efficiency, equitability, patient-centeredness, and timeliness -These are important factors to reduce errors in healthcare and improve patient care -We want to create a safe environment both for the person collecting things and the patient. -We want to order tests that are effective to make the diagnosis. -We want to collect the specimen optimally to make that diagnosis. -We want to make things patient-centered and communicate with the patient; if we communicate with the patient and verify who they are, a lot of misidentification errors may not occur -We also want timeliness. We want results as quick as we can get them.

How to decrease blood culture contamination: Skin Preparation

-Skin prep is essential. We have to disinfect but we also need to use the right disinfectant. -We presently use a mixture of chlorhexidine and alcohol. We can use tincture of iodine but this one is not used as frequently. -One of the things we do have to take into consideration, depending on the type of disinfectant we use, we have to let it sit on the patient for a period of time. -Chlorhexidine and alcohol takes 30 seconds and proviodine requires 2 minutes -Chlorhexidine is effective and acts rapidly; hence it is the preferred disinfectant. -Tincture of iodine is also 30s but not used as much. -LET IT AIR DRY.

Specimen Priority

-Some specimens are very important and we want the results to be delivered STAT. -These specimens may be life threatening and also those specimens where the lab can give you the results quickly if the results get to us o If something cannot be delivered for a few days, there is no point in a STAT test o STAT is for significant specimen where rapid results are essential for appropriate management and where results could be life threatening -Lab can provide results rapidly -Good examples: o Spinal fluid. In the province of Ontario, the recommendation is that from the time of collection of SF to receipt in the lab, it should be one hour. We look at this very carefully. o If you are thinking you might have necrotizing fasciitis, the information the lab can give you can directly influence the treatment the patient should be on and if they need surgery, and the results can be quick. This can be life threatening o Malaria. Malaria is one of the syndromes that would be underrecognized. If you do suspect anybody who has travelled and they have fever, you should rule out malaria. Can do a direct smear and get results quickly.

Storage

-Specimens need to be stored at the appropriate temperature in the clinic or ward -Specimens are stored either at room temperature or in the refridgerator (2-8º C) -Storage at 2-8º C prevents growth of contaminants -Some specimens require room temperature as cold temperature reduce the growth and viability of the organism eg/ anaerobes -NEVER store blood cultures or spinal fluids for bacterial culture at 2-8º C

Direct Examiniation

-Spinal fluid is treated as a STAT specimen. We get the Gram stain out to you in 1 hour. The centrifuging is part of the processing, and we perform a gram stain. -With spinal fluid, the sample must be received by the lab within 1 hour of collection and Gram-stain and morphology are returned within 1 hour vs the normal 24. -The organism was described as GP because of the stain. We have GP diplococci. This is most likely Strep. pneumonia.

Spinal fluids and sterile fluids

-Sterile fluids involve fluids where we do not expect any bacteria or viruses. This would include pericardial fluid, pleural fluid, peritoneal fluid (intraabdominal) or if we take a needle and stick it into the spleen, liver or brain, we should not be expecting to grow anything. -These are sterile organs. If we did surgery on them, they should be Clean (Class I) -We can do solid organ aspirates -These are very important specimens. They are collected into sterile containers. -The physician does the collecting -Spinal fluid is the test that we would do for meningitis. We would have 4 tubes come to us and they are labeled as 1 - 4. That order of collection is very important. When we have a lumbar collection, the first tube that we collect is most likely to be contaminated because we are putting in a needle and it goes through skin. This is not the tube that we want in microbiology. We usually require tube 3 for micro and tube 1 and 4 can go for cell count where we are looking to see how many WBCs and RBCs are there. Tube 2 can go for chemistry. o Tube 1 and 4* → cell count. o Tube 2 → chemistry (glucose and protein) o Tube 3 → microbiology. We need large volumes (10 mL) if fungal or TB cultures are requested -When we label it, we must coincide the label and the test with the appropriate tube.

Blood Culture Volume

-The blood culture volume is the most important factor for the lab to detect a pathogen. -For adults the recommendation for blood cultures in the bottles shown was 8 - 10 mL. We don't routinely get those in the hospital. It is difficult for us to get 8 - 10. -The yield is proportional to the amount of blood. -The amount of blood put in is the portion of us isolating an organism of interest. We don't want to put too much blood in things because there are things in the BC bottles that neutralize antibiotics and if we put in too much, the ratio is off, so it will not neutralize. -The ratio of the volume of blood to the volume of the broth in the BC bottle is important to allow adequate dilution of the blood to prevent inhibition of growth if the patient is on antibiotics -Don't put too little or too much. -Before we actually take the blood culture, mark where the fluid is in the BC bottle and where you want it to be. There are very different ways of collecting BC. The bottle itself isn't going to collect the 8 - 10 mL by drawing it in. We have to look to make sure it is within the 8 - 10 mL. -We want to optimize our positivity rate of significant pathogens. -Adult volumes are 8 - 10 mL per bottle, but pediatric volumes vary by weight. Do not overfill or underfill. -Mark the meniscus before filling and mark the fill volume.

Patient Safety

-The error rate in healthcare can be quite significant. In the US, an error rate up to 69% has been determined. The error rate in the lab is fairly low. Most errors occur in PATP. -What are these errors? -The wrong tests may be ordered. We might want to diagnose one organism, we might order the wrong test or put it into the system incorrectly. -The patient or the specimen may be misidentified. This one of the most important errors that occurs. The patient may be misidentified or the specimen may have the wrong patient's name on it. -Once we go and collect the specimen, if it is not of good quality, it will impact the quality of lab testing - an insignificant result may be generated or the pathogen may be missed. We might even generate results that are misleading. -The specimen may be put in the wrong container or... -We may have issues with storage and transport.

Lower Respiratory Specimen: Sputum

-The first morning sputum is the best -If you have pneumonia and have been lying down, you have a lot more secretions that represent lung secretions that you can cough up. We don't just want the patient coughing up the saliva in their mouths. This will not help us detect the bacteria in the lungs, and it will be contaminated with the normal flora of the mouth -Patient should rinse their mouth, remove dentures and cough up sputum

How do we know what to collect?

-The lab will provide us with instructions. They provide information on how to collect things, what kind of container, what might any special requirements be (volume, timing, patient information), handling of specimen and how it should arrive o Order entry (in hospitals - lab info system) o How to collect o Appropriate container o Special requirements: volume, timing and patient information o Handling of specimen o Storage and transport o How quickly the specimen should arrive at the lab o Priority of the specimen: routine vs STAT o Turn around time for results -A laboratory test information guide can provide this information. It will become our guide as to how to collect specimens. It basically tells us exactly what kind of tube, how many samples we should take and when, what the volume is, and how you should send it to the lab. -These guides help everybody know what is the appropriate procedure of specimen collection -If you are collecting multiple specimens, there is actually an order of draw. This is based on what is in each of the different tubes. Because there can be different chemicals or reagents, we don't want to contaminate between the tubes. There is a defined order of draw. Blood cultures are usually taken first.

Viral Specimens

-The most common viruses include Influenza A and B. It is important to collect specimens to see what is occurring in the community -We also have parainfluenza, respiratory syncytial virus (RSV), adenovirus and metapneumovirus -With the swabs for viruses, we are trying to get the tip all the way to the back. Most of the viral swabs, in contrast to the ones seen before, are quite long and flexible (compared to bacterial swabs). These swabs can bend. -We have to take the swab and put it all the way through almost until the patient is coughing, and collect from right at the very back. The discomfort is momentary. -We either want a nasopharyngeal swab or a nasopharyngeal suction -We can also take an aspirate where we put in a little bit of fluid and suction it back. This is very good for babies. -There are 2 different ways for collecting for respiratory viruses

Urine Culture: Specimen Types

-There are invasive or non-invasive urine cultures: -Invasive cultures include... o Those collected by cystoscopy. These are cystoscopic. o Ureteral. The ureter goes from the bladder to the kidneys so this would only be collected in the OR. o Percutaneous nephrostomy. If they have kidney stones or a blockage, they might go to radiology where they will put in a drain directly into the pelvis of the kidney o Supra pubic aspirate -The lab needs to know if it is invasive or noninvasive because we treat them differently. We have a threshold for determining how many bacteria are present. If there is less bacteria in the invasive specimens, it is still significant because these have lower thresholds -Non-invasive cultures include... o Clean catch midstream specimen. It must be midstream o Catheter: • Indwelling catheter • In and out catheter o Ileal conduit if they have had bladder cancer and repair o Bagged specimens in pediatrics. These are really not good specimens. -The lab must always know the specimen type. -Do not collect a specimen if the patient has no symptoms

Lower Respiratory Specimen: Bronchial Wash

-They take a long black tube with a light and a microscope, will feed it all the way through. -We have two main bronchi in the lungs. -For the bronchial wash, we put it through so that it is in one of the main bronchi, put fluid through and bring it back. -This one is a good specimen but can be contaminated by a lot of things.

Specimen Label

-This is not a small step. The label is essential. The label has... o The patient's name o The hospital accession number o Date of Birth o Which physician collected it? o Collection date and time • As the nurse, often asked to fill in the date and time and who did the collection. o What the test is and any additional information? o Where is the source? o What is the clinical information? o Very important information in the PATP.

Patient Identification

-This is one of the most important aspects of specimen collection Why? 50% of deaths from transfusion related errors were related to identification errors. Transfusion medicine has beefed up its requirements for identification. -There are adverse outcomes related to errors in identification. -Multiple organizations have prioritized identification. -Laboratories undergo accreditation. As part of those processes, we want to look to see how well staff are using patient identification procedures. Want to ensure the lab and hospitals have procedures that are being followed. This is also part of hospital accreditation. -There may be teams of accreditation coming through and observing practices. We want to ensure that procedures are being followed. -One accreditation organization is the Joint Commission International. It prioritizes the accuracy of patient identification. -Benchmark: When we are collecting this information and trying to see how well we do, we also want to see what the expectation is. -Our benchmark is that less than 0.4% of specimens should be misidentified. These benchmarks allow us to see if we are meeting national requirements. -The patient and the specimen must be uniquely identified. We cannot just really on one unique identifier such as name. We need 2. We need the full name and another unique identifier. -Never go by bed location to identify the patient. We need to go by full name and communicate with them, even if they have a bracelet on, unless they are unconscious and the bracelet is all we have. The patient will tell us if the bracelet doesn't match but we need to ask. -It is the responsibility of every health care professional to confirm the patient's ID before any intervention. -Examples of unique identifiers include: • Full name • Hospital accession number • OHIP number • Birthdate -Compare the information to the requisition and specimen label -When we are making that collection, we must have single-patient processing. If we start collecting from 5 different patients and then process them all together, we might make an error. -Single-patient processing: Collect from Patient X, get the collection, label it and bring it to the ward clerk to enter in. We should be processing specimens from a single patient at a time. -Bedside labeling: • Label tubes and containers after collection but before leaving the bedside • We might not remember which containers go with which patient

Misidentification or Labelling Error

-This is still a big problem. -Retrievable: o Specimens that can be collected with minimal harm to the patient (e.g.a urine sample) -Irretrievable: o Might not be able to be retrieved or was taken invasively o If your patient had suspected meningitis, we used a lumbar puncture to collect CSF and we started antibiotics. We can never recover the original specimen. o E.g. If they took a liver biopsy in the OR, we can never get that again. o If we have a misidentification or labeling error on an irretrievable specimen, we will come back to you and verify that it is the right specimen for the right patient and then continue to analysis. The workup will proceed. o SF is irretrievable; it is taken invasively and is very difficult to recollect. Liver biopsies are very hard to recollect. Blood cultures before a patient was on antibiotics are irretrievable when a patient is. o Urine from a Foley catheter is retrievable at any time. - Notify clinical area correct labelling error & sign form accepting responsibility for verification of specimen identification (individual who obtained specimen)

Blood culture contaminiation

-This tells us how well we are collecting blood culture. We expect less than 3% of all our blood cultures to be contaminated. -Sometimes, we do a great job and our contamination rates are below the 3%. We then know that we have appropriately disinfected the skin and septum and collected appropriately. Sometimes, we have times where we go above the 3%. Then we need to know why things have changed or how things can be improved? -If we isolate normal flora in 1 out of the 2 sets, we call this contamination. This is how we calculate it in the lab. -There are a few other species such as bacillus etc that we consider common

Urine Culture Instructions

-Urine cultures can be easily contaminated because they are going through the urethral and perineal areas. -They are prone to contamination from bowel, vaginal and urethral flora -Men should clean the glans and women should be cleaning the periurethral and perineum with soap and water then rinsing. -They should be informed to void the first part of the urine into the toilet and then collect the midstream part, to ensure that we are not collecting any urethral contaminants. The urethra is part of the mucosal surface and we do have normal organisms there. -Very important for men -Pediatric urine bags: o High false positive results which may just reflect contamination o If negative, may be able to rule out infection

Transport Conditions

-Urine should go in ice packs. If transported in courier and it takes 3 - 4 hours to get to us and we don't have an ice pack, it will be grossly contaminated. Some bugs that are not the pathogen will start growing and reproducing and some of the other bugs would grow up. -We need ice to prevent overgrowth and contamination. -Transport bags must be able to maintain the appropriate T despite large seasonal differences in T -Room temperature vs 2-8°C vs dry ice -Specimens that require storage at 2-8°C require ice packs -Transport bags must be able to maintain the appropriate temperature despite large seasonal differences in temperature

Safety for Specimens

-Use leak proof containers -Transport all specimens in a sealable, leak-proof plastic bag -Paper requisition or labels, should be a in a separate compartment in the bag -Do not transport syringes with a needle to the lab prevent needle-stick for yourself, the porter, the lab worker -Syringes can be capped with a rubber cap or transfer contents to a sterile container -Proof plastic bag so that while they are being transported and when they arrive, there is a secondary means of protection -Never transport a needle to the lab. Syringes can be sent but not with a needle. They must be capped with a rubber cap or with contents transferred to the sterile container -Protect the patient, yourself, the lab tech and the porter

Meditech Order Entry

-Used in HHSC instead of paper requisition -Physician's order has to be translated into the computer system. It has a lot of the same information. o Unique identification of the patient o Location o Date of birth o Who the physician is o What test is ordered? o What is the source? Is urine midstream or from a catheter? o List of antibiotics -Things are generally moving to an electronic system.

Genital Specimens

-Vaginal swab: Vaginal discharge is a very common syndrome. It can be caused by yeast, bacterial vaginosis (RV) and trichomonas. Because these are the three most common, this is what the lab will look at. The vaginal swab is sent and we will do testing for these three areas. -If we delay it by over an hour, we have decreased sensitivity for trichomonas -Vaginal-rectal swab for Group B Streptococcus in pregnant women: Now done at around 35 - 37 weeks gestation. It is important because if we can detect the Strep. at that stage and can give them antibiotics at the time of delivery, we can decrease the same infection in the neonate. -These strategies have been commonly adopted throughout N. America and we have been able to reduce the incidence of Group B Strep. If she is a carrier, doesn't even have to be infected, she can pass it on the baby at the time of delivery -We need a vag-rectal. If we don't swab the right place, we will not isolate the organism we are interested in. If we do a cervical swab, at the time of PAP, this area is not that good for picking up Group B Strep which is more commonly colonized in the lower areas. -STD's detected through cervical swab (female), urethral swab (male) and urine (1st void): Allows for molecular detection of Chlamydia and N. gonorrhea. We have seen an increase in N. gonorrhea in university aged students and others. o One of the most common assays we use now is molecular. o In females, the site of infection is the cervix. o Sometimes we can take other specimens. For males, we can take a urine. It can reflect the same sensitivity as a urethral swab at picking up these specimens. For females, the urine collection can be quite good but the cervical swab is probably still preferred. -When do we go to a routine culture swab? N. gonorrhea is a bacteria and we can culture it. We can take a regular swab. Sometimes it is really important to take a swab for culture and not just molecular work because with N. gonnorhea, we find that there is an increased resistance to some of the more common empiric antibiotics that are started. The recommendation is that we want to take a swab to grow it and do susceptibility testing. -There are other situations too where we should do a culture, such as for sexual assault or pediatrics. The advantage of doing the molecular testing is that if we try and grow N. gonorrhea, we really need that swab pretty quickly into a lab because it is a fastidious organism because it can die out. o We might do a vaginal if pre-pubertal or an extra-genital swab -A molecular test does not require the organism to be alive - we are just testing for its presence. In some circumstances, we would take both. If we have a high risk patient, a swab for culture is preferred.

Fungal Infections

-Very similar to bacterial infections. We have direct examination of the specimen. We use different types of stains than with bacteria - but we want to find them in the direct exam. Calcofluor is a detergent used with a fluorescent microscope to detect fungi. -We may have a yeast, a mould or a dimorphic fungi

Viral Detection

-We can culture viruses just like we can bacteria. We can also directly detect if they are present. In order to see that they are there, we sometimes use immunofluorescence markers. -We can use electron microscopy -We can use a blood sample to see if they have antibodies. -We can use molecular methods to directly detect viruses (more and more common). Most molecular methods can amplify the organism. We can detect smaller amounts of the organism using molecular methods. A lot of the viral methods are going over to molecular.

Electron Microscopy

-We can use methods from molecular microscopy and pick up a lower amount in the specimen to test for viruses -Rotavirus and enterovirus have a very typical feature

Blood Culture Collection

-We have different bottles for aerobic and anaerobic bacteria -Anaerobic bacteria do not like oxygen. This bottle is designed to isolate anaerobes. -We have a pediatric bottle because the volume for a little child may be a lot less than for adults. o Aerobic: Green (think trees) o Pediatric: Yellow o Anaerobic: Orange -For routine blood collections, we recommend two sets (2 aerobic and 2 anaerobic). There are a certain volume that is essential for each bottle. In some situations we collect more than two sets, such as with endocarditis. -If a clinician is suspecting something that is a little bit unusual, such as a fungal culture, TB or a mycobacterium, we need to use the appropriate bottle. -For different types of pathogens, we need to have the appropriate blood culture collection system. -We have routine blood culture collection, which is 2 sets of 2. We want 8 - 10 mL in each bottle. -With endocarditis, we need 3 sets of 2. 8 - 10 mL in each bottle. -With fungi, we only want aerobic containers. 8 - 10 mL. -For other fungi and mycobacterium, we need 2 full tubes of a special stoppered bottle.

How to decrease blood culture contamination: Phlebotomy Team

-We know that when the lab or a dedicated phlebotomy team, either lab techs or nurses, take a culture, this usually results in lower contamination rates. -We routinely see higher contamination rates in some of the very busy settings such as the ER. In the ER, we have multiple different nurses taking cultures and managing patients. -Having a dedicated phlebotomy team does decrease contamination rates -In the pediatric ER, they found that if we put a peripheral IV and drew from it, it decreased contamination. We would not use this unless we needed to

How to decrease blood culture contamination: bottle preparation?

-We not only have to prepare the skin to decrease contamination, but we also need to prepare the bottle. This step may be skipped. -When we flip open the blood culture bottle, it has a little cap. Underneath we have the septum where we put our needle. If we don't disinfect that septum, we might have spores from different bacteria that are on them, and we do not want to inoculate them into the BC bottle. -We routinely see that there is an increase in Bacillus spp in contamination during the summer and when there is construction because the spores are very hardy and then end up underneath the little flip lid. -This rubber septum is not protected from the cap on the bottle -Rub for 30 seconds and air dry for 1 minute

Incubation of Media

-We take that media and put in incubators (at 35º) and we also vary the atmosphere because some bacteria are anaerobic, some like 5% CO2 and some like straight air or are microaerophilic -We vary the atmosphere so that we can isolate different organisms -We have to incubate usually for 18 - 24 hours and for some specimens, 5 - 7 days. o Spinal fluid takes about 5 days to culture -When we have to culture things, it takes at least a day for the organism to grow.

Vitek2 - Identification System

-We take the organism once it has grown, place it on a card and place it in here. In 6 - 18 hours, it identifies it -Rapid method involving biochemical and other reactions to identify a wide range of organisms -Identification usually 6-18 hours -Identifies bacteria and yeast

Appropriate Specimen

-We want to ensure we collect the right specimen -We want to collect the specimen in a way that maximizes our ability to identify the pathogen in question o E.g. if we are thinking of a viral pathogen, we need to use a viral swab and media. If we are thinking of a fungal pathogen, we need to use something differently. Specimens have to be collected in the appropriate container or swab. -Appropriate specimen depends on the site of infection as well as the type of pathogen (is it a bacteria, a virus or a fungus?)

Minimize contamination with normal flora

-We want to minimize contamination with normal flora. Our skin and mucosal surfaces have normal flora or normal microbiota. We see coagulase negative Staph, viridans streptococci, propionibacterium... -Coagulase negative Staphyloccocus is found on the skin. -In the mouth, nose, eyes, genitals and GI tract, we have a lot of bacteria that we expect. If we are taking a wound swab, we don't want to contaminate this with normal bacteria as this can lead to misleading results. We want to ensure disinfection so that we can eliminate the contamination with normal flora, especially for skin surfaces. -Female genital tract has lactobacillus, streptococci and anaerobic organisms -Need disinfection to decrease contamination with normal flora -Sites will have commensal flora: -Skin: coagulase negative staphylococcus, viridans streptococci, propionibacterium -Mouth full of normal flora: streptococci, anaerobic organisms -Female genital tract: lactobacillus, streptococci, anaerobes

Viral Culture

-We would take an actual cell line, put the specimen in and wait for 14 days, looking for pathogenic effects -With adenovirus, the cells are showing rounding. -Not common -For negative control cell lines, nothing appears - no lines or circles

How to decrease blood culture contamination: Percutaneous virus via vascular catheter?

-When taking a blood culture, we can either take it from a peripheral vein, or a patient may have catheters. If we take it from the catheter, we can have false positive cultures and this makes sense. -If a patient has a central line for a long period of time, organisms will start colonizing the inside of the lumen. If we only take it from there, we might only be finding out what is colonizing the lumen. -It is always good to take it percutaneously or peripherally -HOWEVER...Catheters put the patient at risk for blood stream infections. If we take it from the catheter, this may help us decide that the line is the source of the infection. It may also be good if the patient is a very difficult draw. -Only draw from vascular catheters if the catheter may be a source of infection or with a difficult draw

Surveillance Swabs

-When taking a surveillance swab, there are provincial guidelines on when to do it. Routinely if the patient is admitted to hospital and they have risk factors, they will be swabbed for MRSA and VRE. This is fairly standard. -If you have been hospitalized in the past 12 months -If you have been known to been exposed or been previously colonized -When we do surveillance, we are not looking to see if the patient is infected, just if they have that MDR organism. The type of specimen we take is quite different... -MRSA: Swab the nares and the rectal and perineal area -VRE: Swab rectal area (needs to be fecally stained) -ESBL/CRE: Screening of rectal swabs -Surveillance where we are looking for colonization versus when we are looking for an actual infection.

Specimen Processing

-When we have to centrifuge specimens, we centrifuge to concentrate the pellet in the bottom such that if we inoculate that into media, we can more readily grow things. We routinely centrifuge blood because we take the serum. We process things in a biological safety cabinet, which protects the technologist and the specimen. This is like a mini OR. There is laminar flow. The air flows in a certain direction so that we don't contaminate the specimen when we open it up. The air is actually drawn in so that there is no way that the technologist will have the aerosols spray out toward them. The BSEs are protecting the technologist. i. The sample is protected as is the tech in a BSE. -Automated microbiology processing instruments: Now we have this instrument where we can just put the specimen in. It will take the specimen, inoculate the plates, and eventually, using this track system, it will be able to determine which incubators it needs to go into. Their system is called a WASP.

Collecting Volumes

-Whenever possible, collect pus or tissue over a swab o We want the actual specimen vs a "swab" of the specimen -We want to get a container full of fluid from abscesses. We want to get as much fluid as possible. A routine swab can only probably absorb around 0.15 mL. If the physician did an aspirate and took the fluid, we could get 1.5 - 5 mL o Relieving for the patient as well -The more fluid we get, the better it is. We can grow different plates to isolate different organisms through inoculation if we have more of the sample -Collect pus or tissue over a swab whenever possible. -Swabs are the worst types of samples. • Pick up extraneous microbes • Extremely small specimen volume • Hard to release the microbes • Inoculum is not uniform across agar plates • Should vortex prior to inoculating media

Pre-analytical Testing Phase

-• The whole testing phase starts when a provider evaluates the patient. From there, they determine whether there is an infection or not. They want to select the right tests to determine if an infection is present or rule it out • From this we generate a test order. There are only certain people that can legally order tests (doctors, some NPs and chiropractors (limited tests)) and this is regulated on a provincial level • The pre-analytical test phase is very important. This goes from the test order all the way until it hits the lab and we do the analysis. This is important because if we look at where the errors occur, laboratory errors are fairly low. Most errors occur in PATP. We want to improve quality of care and decrease error, so we are focusing on PATP. • The preanalytical test phase consists of: o Patient evaluation o Test order o Requisition (Paper or electronic) o Specimen collection o Transport -As part of the total testing phase, after the lab has generated a report, we have the Post analytical testing phase where we actually communicate the results of the report, the provider looks and interprets the results and decides on management or treatment. This phase also has a higher rate of error than the analytical phase.

Specimen Collection

1) General Principles 2) Types of Specimens 3) How to collect specimens 4) Correct containers/tubes 5) Order of collection 6) Special timing of collection 7) Special requirements: volume, mixing

List all the steps involved in diagnostic testing

1) Specimen processing 2) Direct examination 3) Culture 4) Organism Identification 5) Susceptibility Testing

What are the four steps in labelling the specimens?

1) Take labels to the patient -Labels can go into all patient locations, including isolation rooms -Errors occur when unlabeled specimens are carried to another location for labelling 2) Take a moment to check patient identifiers -Always check 2 unique patient identifiers -This practice only takes a few seconds and improves patient safety, while saving time by avoiding errors 3) Write time of collection and your initials on the label -This practice is required for all specimens 4) Label immediately after collection -Avoids possibility of mix-ups

Common Microbiology Samples to be Stored or Shipped at Room Temperature

1. Spinal Fluids** 2. Sterile Fluids / Aspirates except for sterile fluids for fungal culture 3. Tissue / Biopsies / Bone Marrow** 4. Blood Cultures 5. Corneal / Eye Scrapings

Common Microbiology Samples to be Stored or Shipped at 2-8C

1. Urine 2. Respiratory (Sputum, Bronch Wash, BAL) 3. Faeces (except for Parasitology) 4. Virology Samples in UTM (Universal Transport Medium) 5. Blood for Viral Serology or Viral PCR 6. Spinal Fluid for Viral PCR

Bacterial Identification

Methods: -Sometimes we can look at it on the media and have an idea (colonial morphology on media) -We can do different biochemical reactions and assess the response (e.g. nitrate testing) and testing biochemical properties -Sometimes we can use automated instruments to test a whole series of chemicals to give us an answer -We could use antigen or antibody detection methods -Molecular methods: PCR, sequencing -MALDI-TOF -When we looked at the patient's culture, we see a highly mucoid organism. It is literally dripping. Sometimes organisms that are very pathogenic are mucoid. We do a series of test to know that it is S. pneumonia and report that

Specimen Labelling: Education

What are the problems with specimen labeling? Came up with "the usual suspects" o Unlabelled Libby: No label → second most common error after incorrect patient registrations o Wrong William: On the tube it says one name, and on the label it says another. This is a mismatch. We don't want to put the wrong patient on the container which is why we use single patient processing o Labels may be illegible o The labels may be placed such that the barcode is hidden o For everything to be acceptable... the label is under the cap, the barcode is vertical, the specimen quality was good, there is no mismatch in identification

Multi-drug resistant organisms

o MRSA: Methicillin resistant Staph. aureus • S. Aureus is a common organism that is quite pathogenic but it can be found on our skin. MRSA prevents us from using amoxicillin or any of the beta-lactams which are the standard treatment o VRE: Vancomycin resistant Enterococcus o ESBL: Extended spectrum B-lactamase producing organisms • We are starting to see this with the GN. We can't use the whole category of beta-lactam antibiotics. We see this with E. Coli and Klebsiella o CPE: Carbapenemase producing organisms. Carbapenams are things like Mirapenam and Imapenam; these are broad spectrum antibiotics. If we can't use these (this is a new problem that has been occurring), we cannot really treat the patient. (GNB)

Common reasons for microbiology rejection

• Common reasons for rejection include the facts that we may reject it if... o It is the wrong container or a wrong swab o If it is leaking o If it is incorrectly labeled or misidentified

Microbiology Specimen Rejection

• Microbiology specific rejections may happen because...We need good quality of the specimen o We would reject formed stool for C. difficile o We need to receive our specimens on time o We can't have saliva in sputum samples • We would detect this by determining the ratio of squamous epithelial cells to pus cells. More than 10 squamous epithelial cells suggests oropharyngeal contamination. o We don't want our swab or blood culture media beyond expiry dates