Microbiology Learning Module Cardiovascular Infections Laboratory Module INTRODUTION Cardiovascular system consists of the heart, blood vessels and blood. As blood circulates throughout the body, it comes into contact with many tissues and organs. As such, the bloodstream serves as an excellent vehicle for the spread of pathogenic organisms; facilitating the infection of multiple organ systems. Bloodstream infections can manifest clinically into many forms. For example, persistent bacteremia is suggestive of intravascular infections such as endocarditis or catheter-related infections. These types of infections are life-threatening, and the appropriate diagnosis and management of such infections are critical to patient survival. PATIENT SCENARIO Anne 33 year old women who presents to ED with fever, chills, fatigue, SOB, and chest pain Anne examined by ED who observes injection marks on both arms Anne abusing heroin for past 3 years, and last injected drug 3 days ago Patient stats - BP 100/70 - HR 110bpm - Temperature 39 - RR 33 - Oxygen saturation 90% on room air Examination of Anne’s chest reveals a dullness to percussion in the left lung base and crackles and wheezes in the same area Loud heart murmur also heard; Anne tells ED physician first time hearing about murmur - Murmurs are abnormal heart sounds produced as a result of turbulent blood flow; heart murmurs most commonly result from 1) narrowing or leaking of valves, or 2) presence of abnormal passages through which blood flows in or near the heart; murmurs not usually associated with normal cardiac physiology and warrant further investigation Based on findings from physical exam, chest x-ray, and echocardiogram and 3 sets of blood cultures are ordered BLOOD SPEICIMEN COLLECITON When collecting blood specimens for culture in suspected cases of serious cardiovascular infection, 3 sets – each with one aerobic (green top) and one anaerobic (orange top) bottle should always be ordered - Multi-sampling strategy confers several advantages; drawing 3 sets of blood from separate venipuncture sits, spaced over 30-60mins: o 1) Increases total volume of blood cultured, improving blood culture sensitivity, and o 2) Generates separate samples that Help discriminate contaminants from pathogens when blood culture grow Improve blood stream infection detection in cases where intermittent bacteremia is expected When collecting blood specimens for culture in adult clients, 8-10mL is recommended volume for each bottle; to ensure optimal sensitivity BACK TO PATIENT SCENARIO Anne’s chest x-ray reveals several small areas of infiltrate in the lower lobe of her left lung, and preliminary data from the medical microbiology laboratory indicate Anne’s blood work positive for Staphylococcus aureus STAPHYLOCOCCUS AUREUS A gram-positive bacteria that appears microscopically as grape-like clusters. Staphylococci are ubiquitous, and can survive extreme conditions of drying, heat, and low-oxygen and high-salt environments S. aureus has many surface proteins that allow the organisms to bind to tissues and foreign bodies coated with fibronectin, fibrinogen, and collagen, thereby allowing the bacterium to adhere to sutures, catheters, prosthetic valves and other devices such as dirty needles S. aureus can colonize the skin and mucous membranes of healthy adults and children. Rates of nasal and cutaneous colonization with S. aureus is especially high in populations linked to frequent needle use (eg. DM, hemodialysis, illicit drug use, allergy shots). As such, these populations are at increased risk of vascular-access bacteremia. QUESTION At this stage in patient scenario, learning the following - Anne fever and low BP - Anne experiencing chest pain and increased HR - Anne has SOB and increased RR - Anne chest x-ray reveals lower lobe infiltrates in left lung - Anne has new heart murmur - 3/3 of Anne’s blood cultures are positive for S. aureus - Anne intravenous drug user Question – Based on information, what diagnosis is suspected? a) Infective endocarditis b) Community acquired pneumonia c) Cellulitis Answer – Infective endocarditis - IV drug users are at increased risk of bacteremia, which can lead to infective endocarditis. Annes new and loud heart murmur is consistent with heart valve damage. Right-sided endocarditis most often occurs when an aggressive species of skin bacteria, such as S. aureus, enters the bloodstream and damages a native valve. As the lesion on the valve grows, small clumps of bacteria called septic emboli can break off and enter bloodstream – spreading infection to other organs, such as kidneys, lungs, and brain. It is likely that the pneumonia seen on Anne’s chest x-ray is a result of septic emboli travelling from heart to lungs, causing a secondary infection - Not CAP – while infiltrates observed in Anne’s lungs suggest that she has pneumonia, it is unlikely that the lungs represent the primary site of infection. It is not uncommon for - individuals with pneumonia to become bacteremia, however S. aureus rarely causes community acquire pneumonia. As such, it is unlikely that Anne’s pneumonia is community acquired. Rather, it is likely a complication of an infection elsewhere in the body. Anne’s new heart murmur and IV drug use suggests cardiovascular infection Not cellulitis – while cellulitis is associated with bacteremia, and can be caused by S. aureus, it is unlikely that Anne has this type of infection. If Anne did have cellulitis, expect to find a hot, red, and swollen lesion on Anne’s body during physical exam. However, IV drug users are at an increased risk of skin and soft tissue infections. If Anne chooses to continue to abuse drugs, important for Anne to use new needle when injecting, and clean skin properly before injection INFECTIVE ENDOCARDITIS Infective endocarditis develops when bacteria from the bloodstream attach to rough surfaces of the endocardium and multiply. Thick vegetations composed of platelets and fibrin protect bacteria from host defenses, and impair valve function. Visualization of the vegetation on echocardiogram is an important diagnostic tool, and is a major criteria for infective endocarditis. - Echocardiogram – In general, transthoracic echocardiography (TTE) is the first diagnostic test for patients with suspected infective endocarditis. The sensitivity of this diagnostic test is ~75%. Consequently, the absence of vegetation on TTE does not eliminate a diagnosis of infective endocarditis. Transesophageal echocardiography (TEE) has a higher sensitivity (~90%) compared to transthoracic echocardiography and should be considered if TTE results are negative despite high clinical suspicion of infective endocarditis. Injection of particular matter found in illicit drugs such as heroin causes endothelial damage to the tricuspid valve, increasing the risk for right-sided infective endocarditis in this population. BACK TO PATIENT SCENARIO Based on Anne’s history, signs and symptoms, recent laboratory results, and echocardiogram, physician makes a diagnosis of acute infective (bacterial) endocarditis. Anne admitted to hospital and started on vancomycin (30mg/kg per 24hr IV in two divided doses for 6 weeks). Following day, laboratory reports that methicillin-resistant Staphylococcus aureus (MRSA) has been isolated from Anne’s blood cultures. Infection prevention and control is notified, and Anne placed on contact precautions - MRSA – strain of S. aureus that is resistant to all beta-lactam antibiotics. This resistance is caused by an alteration in the penicillin binding protein that is carried on the mecA gene. Because of this mutation, this organism is resistant to all the beta-lactam classes of antibiotics, such as penicillin’s, penicillinase-resistant penicillin’s (eg. cloxacillin) and cephalosporins. Consequently, MRSA can only be treated with a few agents (vancomycin, trimethoprim-sulfamethoxazole, linezolid and a few others). Patients can be colonized with this organism (eg. can live harmlessly on the skin, or in the nose or anus) or it can cause significant infection, as in Anne’s case. MRSA – INFECTION PREVENTION AND CONTROL In the hospital acute care setting, S. aureus can easily spread form one client to another, unless proper precautions are used. Infection prevention and control measures for MRSA involve implementation of contact precautions in addition to routine practices: - Single room preferred, door can remain open - Use of gown and gloves when entering patient room - Dedication of patient equipment wherever possible Some facilities also use procedure masks to reduce transmission. Procedure masks prevent patients and health care workers from touching their noses, thereby reducing risk of MRSA colonizing the nasal mucosa. However, not a consistent practice. Also important in cases where the client is under both contact and droplet precautions (eg. in cases where sputum specimens are positive for MRSA and client is coughing. Important to remember that MRSA not transmitted by airborne route. Contact precautions are necessary when treating clients with MRSA because 1) MRSA highly resistant to antibiotics 2) There are limited treatment options for MRSA 3) MRSA most often transmitted from one patient to another via healthcare workers 4) MRSA infections can be fatal 5) MRSA infections can be prevented by using proper infection control methods to reduce transmission INSIDE THE MEDICAL MICROBIOLOGY LABORATORY SPECIMEN COLLECTION AND LABORATORY DIAGNOSIS CARDIOVACULAR INFECTIONS BLOOD SPECIMENS Blood is a key specimen for the diagnosis of acute bacterial endocarditis. Blood specimens are collected as a set - One aerobic bottle (green) - One anaerobic bottle (orange) Bottles have a rubber stopper covering the opening. A needle can be inserted through this rubber to inoculate blood into the bottle. Each bottle contains: 1) A nutrient broth to promote the growth of bacteria and 2) Binding agents that remove antibiotics from the blood specimen The ratio of blood volume to broth volume within the bottle is important to allow adequate dilution of the specimen so that microbial growth isn’t inhibited if the specimen is collected while the patient is on antibiotic therapy. Bottom of each bottle contains a semi-permeable membrane which allows CO2 to diffuse from the specimen, causing a colour change at the bottom of the bottle. CO2 production is an indicator of bacterial growth. If the bottom of the bottle changes colour, an instrument in the lab detects tis change and alerts technician that bacteria is growing in the blood specimen. The workup of the specimen (such as Gram stain, blood culture, etc.) can then proceed. BLOOD SPECIMENS – GRAM STAIN Gram stain results are immediately reported out to clinical areas, The sample is then placed onto agar plates to grow the bacteria – allows bacteria to be identifies. Gram stain results from all three of Anne’s blood specimens show Gram positive cocci in clusters, suggestive of S. aureus. BLOOD SPECIMENS – CULTURE On sheep’s blood agar, S. aureus grows in round, golden-yellow-coloured colonies. Betahemolysis is also often observed. On chocolate agar, the characteristic golden colour of S. aureus is also apparent. S.aureus is also catalase positive. - - S. aureus is able to covert hydrogen peroxide produced by monocytes in the blood into harmless water and molecular oxygen via the production catalase. S. aureus can be easily distinguished from other types of Gram-positive organisms such as Staphylococci, Enterococci, and Streptococci (which do not produce this virulence factor) using the catalase test. Catalase test is performed by placing a drop of hydrogen peroxide on a microscope slide. Using an applicator stick, a sample of the bacterial colony is smeared into the hydrogen peroxide drop. If bubbles or froth form, the organisms is catalase-positive; if no froth forms, organism is catalase-negative. QUESTION Given that blood is normally a sterile specimen, could bacteria cultured from a blood specimen be considered a contaminant of the sample, and not a true pathogen? - Correct: Yes it could be a contaminant – from earlier, recall that the skin has a variety of microorganisms growing on surface (normal skin flora). If blood is not collected using aseptic technique, bacteria from the skin can get into the blood culture bottles. Glowing typical skin bacteria (such as Staphylococcus epidermidis a common contaminant) from only one bottle (or one set of cultures) suggests that skin contaminants were introduced to the specimen during collection. For this reason, at least 2 sets of blood specimens are always ordered. In cases of infective endocarditis, in which the client may be intermittently bacteremic, 3 sets are required to ensure culture sensitivity - Incorrect: No it is a true pathogen. Incorrect. During blood collection, contaminants from the skin (i.e.. normal flora) and other sources can enter the specimen. These microorganisms are not responsible for the infection, and should be considered contaminants of the sample. Differentiation between clinically significant positive blood cultures and a contaminant is based on the number of positive blood culture samples. The term ‘clinically significant positive blood cultures’ refers to any blood culture in which the signs and symptoms of infection observed in the client can be attributed to the microorganism(s) identifies by blood culture. For microorganisms associated with normal skin flora, at least 2 positive blood culture yielding the same microorganism are usually required to consider the result clinically significant. In general, the likelihood that a finding represents true bacteremia, as opposed to contamination, increases with the number of positive bottles reported. BLOOD SPECIMEN COLLECTION – CONTAMINANTS How are contaminants most commonly introduced during blood specimen collection? 1) When the disinfectant is not allowed to dry completely 2) When the blood culture bottle septum is not disinfected 3) When the site of puncture is palpated with non-sterile finger after cleaning 4) When a blood specimen is placed on a non-sterile surface Skin preparation Disinfect venipuncture site - Use chlorhexidine - Wipe in circular motion, from centre to periphery - Prepping agent should remain in contact with the skin for 30sec and allowed to dry Blood culture bottle preparation - Rubber septum is not protected by the cap on the bottle - Disinfecting the septum reduces contamination o Disinfect septum of the blood culture bottle with an alcohol wipe o Rub for 30sec and let air dry for 1min BACK TO PATIENT SCENARIO Once on antibiotic therapy, Anne shows some improvement, but continues to have fevers Repeated blood cultures remain positive for S. aureus, despite initiation of appropriate antibiotic therapy 2 days earlier. This finding does not suggest treatment failure. In Anne’s case, persistent positive blood cultures are due to the 1) intra-cardiac source of the infection, 2) virulence of the pathogen, 3) severity of Anne’s infection (infective endocarditis + pneumonia and septic emboli). The duration of therapy of serious S. aureus infections depends on the site and severity of infection, but is typically 6 weeks for cases of S. aureus endocarditis such as Anne’s. Two sets of blood cultures should be drawn every 48 – 72 hours until Anne’s infection has cleared (eg. blood is sterile). This level of monitoring is important to ensure successful resolution of the infection. Again, due to the severity of Anne’s infection, parenteral antibiotic therapy is recommended for the entire course of treatment. Individuals with a history of IV drug use should be referred to a drug treatment program, as this population is at increased risk of repeat cases of infective endocarditis.
