Clinical Microbiology Lecture Slides PDF

Clinical Microbiology Clinical Microbiology Ain Shams University Faculty of Medicine Clinical Pathology Department Microbiology Unit  Diagnostic Errors Preanalytical Phase (Collection process) Analytical error (performance...

Clinical Microbiology Clinical Microbiology Ain Shams University Faculty of Medicine Clinical Pathology Department Microbiology Unit  Diagnostic Errors Preanalytical Phase (Collection process) Analytical error (performance of the test) Post analytical Phase(reporting, recording, or interpreting Results). Proper specimen collection in microbiology forms the back bone of the investigation procedures  Biological causes of errors Specimens that Colonization contain normal flora  Non-sterile  Sterile Colonization: is a process by which bacteria These These sites normally do which aresites notare open part oftothe normal body flora at that the external environment not contain any bacteria site are present without  Mid stream urine invading or bacteria so any causingfound anyif damage. What  isThroat swabs the difference between collected properly. colonization and infection?  Wound swabs  Urine( suprapubic) Infection : organisms gain access to a susceptible  Blood  Ear swabs hostand there is evidence of tissue  CSF invasion or Nasal swabs damage(local  High vaginalor systemic symptoms swab  Bile or signs). Organisms that cause infection when  Sputum samples one  Fluids: or peritoneal, Pleural, more of the host’s defense mechanisms are  Faeces disrupted synovial, or pericardial, malfunction are known as opportunistic amniotic pathogens(  Deep tissue samples? they are usually of low pathogenicity), and the infections they cause are referred to as opportunistic infections  Laboratory diagnosis of infectious diseases. I. Direct methods: A-Macroscopic examination B-Microscopic examination Wet film:. Stained Smear: 1.Gram’s stain: 2. Ziehl Neelsen stain (ZN): 3. Leishman stain: C. Culture and antimicrobial susceptibility: 1. Cultivation. 2. Identification of isolated organisms 3. Determination of antimicrobial susceptibility of pathogenic organisms. D. Antigen detection (on clinical specimen): E. Nucleic Acid Detection (Molecular Diagnosis): II. Indirect methods:  Laboratory diagnosis of infectious diseases Whether the cause is bacterial, viral, fungal or parasitic. This is carried either through direct or indirect methods. Direct methods( on clinical specimen): A- Macroscopic examination: Inspection of gross morphology of the specimen. B-Microscopic examination Wet film: A part of the specimen is taken on a slide and examined directly. This is done for the detection of pus cells, red blood cells, fungi and motile organisms (Trichomonas vaginalis and amoeba). Stained Smear: 1. Gram’s stain: This is useful in identifying the organisms as gram- positive e.g., Staphylococci, and Streptococci or gram-negative e.g., Neisseria, E. coli, and Klebsiella…etc. Importance of direct gram stain 1. Detect the presence of organism and d.d between org. acc. to gram reaction & morphology. 2. Diagnostic.  Meningitis: gram negative diplococci intra and extracellular  Throat swab : Vincent angina  Urethritis :gram negative diplococci intra and extracellular  Vaginitis : Candida or anaerobic vaginosis 3- Support diagnosis Suspected case of gas gangrene or tetanus. 4- Used to check quality of the specimen.  Sputum and wound swab.  Stained Smear: 1.Gram’s stain: 2. Ziehl Neelsen stain (ZN): This is used for the detection of acid fast bacilli (e.g. Mycobacteria) in clinical specimens. 3. Leishman stain: This is used to define the type of cells in a smear Differentiate viral from bacterial infection e.g meningitis , sputum , Differentiate between exudate and transudate e.g synovial, pleural effusion and peritoneal effusion. C. Culture and antimicrobial susceptibility. this includes: 1. Cultivation on conventional and / or selective culture media 2. Incubation for 24 hours to 48 hours or more aerobically.  Facultative anaerobes will grow in the presence or absence of O2.  Strictly aerobic organisms will grow only in the presence of O2.  Anaerobic organisms cannot grow except in the absence of O2 as it has a lethal effect on these organisms.  Microaerophilic bacteria (Campylobacter) can grow only in an atmosphere with reduced O2 and increased CO2. N.B: if anaerobic culture is required, it should be requested specifically. 3. Identification of isolated organisms 4. Determination of antimicrobial susceptibility of pathogenic organisms (requires another 24 hours). So, the minimum time required to release culture and sensitivity results is ≥ 48 hours. Growth of E. coli Antibiotic Susceptibility Ampicillin R Co-amoxiclav I Cephradine R Cefuroxime R Cefotaxime I Ceftazidime S Cefepime I Cefoxitin S Pip-tazobactam I Meropenem S Ciprofloxacin R Nitrofurantoin S Co-trimoxazole R Amikacin S Gentamicin R D. Antigen detection: This is a rapid method for identification of organisms in a specimen Through identification of its surface antigens Identification of the soluble antigen produced by the organism in the blood of the patient. E. Nucleic Acid Detection (Molecular Diagnosis): Molecular techniques used in the microbiology laboratory include nucleic acid probes, amplification systems e.g., Polymerase chain reaction (PCR) and others. These molecular techniques may be used mainly for 1. Rapid detection of microorganisms Organisms non-cultivable on artificial culture media (e.g. Chlamydia and viruses. Slow grower organisms :Mycobacterium tuberculosis. 2. Determination of genes responsible for antimicrobial resistance 3. Bacterial typing in epidemiological investigations.  Indirect methods( serum): Serum antibody detection for organisms non- cultivable on artificial culture media or difficult to be cultivated (e.g. Mycoplasma), also certain parasitic infections. 2. Paired serum samples should be obtained from the patient. The first sample is taken in acute stage to know 10-14 days the baseline titer; the second sample can be taken 10-14 days after the initial exposure to infection to detect  4 fold rise of antibodies  Antibiotics Definition: Antibiotics are chemicals produced by microorganisms that have the capacity to kill or inhibit the growth of other organisms.  Clinical Use of Antibiotics: Description Type of Therapy Antibiotics used to prevent Prophylactic-1 infection :Therapeutic-2 Organism is unknown but a-Empiric syndrome is known Culture results is available b-Definitive  Antibiotic combination Synergism: combination has a greater effect than the sum of the two individual drug effects Antagonism: combination has less activity than that of individual drug. Indifference: The combined action of both equals the action of the effective one only Antibiotic combination :- Indications :- Mixed or severe infections e.g. synergistic comb. of pencillin + gentamycin for a patient with E. faecalis endocarditis. Prevention or delay of development of drug resistance e.g. Antituberculous drugs. Empirical treatment for high risk patients with serious infection. 17 Failure of Antimicrobial chemotherapy:- Improper choice, dose, route, time and duration of therapy. Presence of natural barrier as prostatic barrier in chronic prostatitis. Antagonism upon use of certain combination of bactericidal and bacteriostatic antibiotics. Antibiotic therapy alone without surgical drainage of abscess. Development of antimicrobial resistance. 02/16/2025 D. Malaka Zakaria Amer 18  Emerging Antimicrobial Resistance: "ESKAPE” pathogens currently cause the majority of hospital acquired infections and effectively “escape” the effects of antibacterial drugs Enterococcus faecium, E Staphylococcus aureus, S Klebsiella pneumoniae K Acinetobacter baumanii A Pseudomonas aeruginosa P Enterobacter species. E Gram positive cocci: vancomycin-resistant Enterococci: enterococci (VRE). Vancomycin is the drug usually reserved as a last resort for treating life-threatening infections caused by gram positive organisms that are resistant to all β- lactam drugs. Staphylococcus aureus: Staphylococcus aureus, another common cause of nosocomial infections. Methicillin-resistant Staphylococcus aureus (MRSA) are resistant to methicillin as well as all other β-lactam drugs. Infections caused by these strains are generally treated with vancomycin. Gram negative bacilli: The main mechanisms of resistance are through production of β-lactmases mainly: Extended spectrum β-lactmases (ESBLs): They are enzymes that confer resistance to first, second and third generation cephalosporines and monobactam. This mechanism of resistance is common mainly among Enterobacteriaceae. Carbapenamases: Resistance to carbapenems is the most important mechanism of resistance among gram negative bacilli (E.coli, Klebsiella, Enterobacter as well as Pseudomonas aeruginosa and Acinetobacter). Carbapenamases confer resistant to most types of antibiotics, including carbapenems which are the last resort in treating life-threatening infections.  INFECTION CONTROL AND SAFETY MEASURES HEALTH CARE ASSOCIATED INFECTIONS (HAIs) (NOSOCOMIAL INFECTIONS / HOSPITAL ACQUIRED INFECTIONS) Health care associated infection: An infection which is acquired during hospitalization and which was not present or incubating at the time of admission. Infection which is acquired in the hospital and becomes evident after discharge is also considered HAI. Types: 1. Blood stream infection (BSI). 2. Surgical site infection (SSI). 3. Hospital acquired UTI. 4. Hospital acquired pneumonia. 5. Catheter related infections.  Sources: 1. Autogenous (i.e. self infection = from the own flora of the patient). 2. Cross infection via hands is the common source of nosocomial infection. Hands Contamination occurs upon contact with Microbial Flora patients or of the their Hand: environment 1. Resident Flora: Flora of low pathogenic potential inhibited or reduced 3. Environmental sourcesbyas skin air,antiseptic water, solutions (e.g. food, Diphtheroids, surgical Coagulase instruments, negative I.V. catheters, Staphylococci). urinary catheter, ventilators…etc. 2. Transient Flora: Organisms which colonizes the superficial layers of the skin , is more amenable to removal by routine hand hygiene. They are often acquired by medical staff during direct contact with patients or contaminated environmental surfaces and are the organisms most frequently associated with nosocomial infections (e.g. S. aureus, Gram- negative bacilli) Adverse Effects of Nosocomial Infections: 1. Prolong the period of hospitalization. 2. Increase the overall hospital morbidity and mortality. 3. Increase the health care cost. 4. Emergence of hospital resistant strains and spread of antibiotic resistance. Precautions to prevent transmission of infectious agents: These precautions are designed to protect patients, staff, and visitors from contact with infectious agents and to reduce transmission of microorganisms in healthcare settings. 1-Standard precautions: Standard precautions are a set of basic infection prevention practices that should be applied to every person every time to assure that transmission of disease to staff or patients does not occur. These practices include; 1. hand hygiene. 2. Use of personal protective equipment (e.g. gloves, gowns, masks) depending on the anticipated exposure. 3. Respiratory hygiene/cough etiquette. 4. Safe injection practices (intended to prevent transmission of infectious diseases between one patient and another, or between a patient and healthcare personnel during preparation and administration of parenteral medications). 5. Safe handling of potentially contaminated equipment or surfaces in the patient environment. 2-Transmission based isolation precautions:  Transmission Based Isolation 1-Contact isolation 2-Droplet isolation Gloves + Gown gown if there is substantial contact with patient Standard Mask Meningi equipment, Precautions SeparatesPertus environment and if patient had diarrhea room????sis A colonized patient Influen may be a source MDR of Isolation za infection inspite of Precautions O being not infected. So isolation of probable pathogen does not necessarily mean 3-Air-borne isolation Mycobacterium infection Mask(N95) tuberculosis, the Private room with negative pressure varicella-zoster virus, and N.B: TB patient leaving the room Should wear mask. measles.. Laboratory diagnosis of infectious diseases :I. Direct methods A-Macroscopic examination B-Microscopic examination. :Wet film :Stained Smear :Gram’s stain.1 :Ziehl Neelsen stain (ZN).2 3. Leishman stain: C. Culture and antimicrobial susceptibility: 1. Cultivation. 2. Identification of isolated organisms 3. Determination of antimicrobial susceptibility of pathogenic organisms.. Antigen detection (on clinical specimen): Nucleic Acid Detection (Molecular Diagnosis): Indirect methods:  Case A 30 years old man C/O fever (39 C), headache, irritability & neck stiffness. FastingMeningitis blood glucose was120mg/dL Characteristic CSF Changes in Meningitis.  CSF exam. showedBacterial the following: Viral (Aseptic) Bacteri Normal Tuberculous (Septic) o Protein : 150 mg/dl ( N: 20-40 Clearmg/dL). or Slightly al Clear or S. Appearance Clear Turbid o Glucose: 20 mg/dL (N: 60-70% bl. Turbid Glucose). Turbid White Cell 0 – 5/mm3 MeningLymphocytes  PML Lymphocytes o Cell count count: 500/cmm (N: 0-5/cmm). Mainly lymphocyte itisPNLs. + PNL Protein 20-40mg/dl  slightly or N  o What are theofcausative 60-70% organisms? Glucose  N  blood glucose o Other tests to be requested???? PML: polymorphonuclear leucocytes  Meningitis Causative Pathogens: Age of patient Organisms Neonates38 C or 90 beats/minute oRespiratory rate >20 breaths/minute oWhite cell count >12 000 cells/mm3, 10% immature white blood cells (band forms).  BLOOD STREAM INVASION Indications for blood culture : 1. Clinical features of sepsis 2. Suspicion of infective endocarditis 3. Pyrexia of unknown origin 4. Infection related to an intravascular device. 5. Typhoid fever, Brucellosis, early meningitis and pneumonia. 6. Probable bacteremia or fungemia occurring when pathogens enter the blood stream from abscesses, infected wounds of burns, or from area of localized disease as in pneumonia, meningitis, osteomyelitis, cholangitis, pyelonephritis, peritonitis, enterocolitis, and puerperal sepsis. Principles for blood culture sample collection: 1. Two or more blood culture sets should be collected over 24 hours period, before administering antibiotics. 2. If patient is critically ill or in whom the likelihood of continuous bacteremia is high as in case of infective endocarditis, it is sufficient and appropriate to obtain blood from 2 separate sites within minutes of each other. 3. For patients already on antibiotics, blood culture bottles with additives (such as charcoal or resins) that inactivate antibiotics are preferable. If blood culture results are negative and clinical condition of the patients allows, stop the intake of antibiotics and consider blood re-culturing after 48 hours. 4. It is necessary to use aseptic procedure for sample collection as blood culture contamination may lead to confusion in interpreting the significance of a “positive” blood culture result Diagnosis: 1-Specimen: blood sample collected under complete aseptic precautions and added to blood culture bottles. 2- Blood culture bottles are incubated for 7 days and they are examined continuously for any evidence of growth during incubation period. Any bottles showed growth will be subjected to routine culture and sensitivity and the results will be released once finished. Bottles that showed no growth will be released by end of incubation period as "no growth". Interpreting a “Positive” Blood Culture: 1- Virtually any organism, including normal flora, can cause bacteremia. 2- A positive culture result does not necessarily indicate bacteremia. Guidelines can assist in distinguishing probable pathogens from contaminants are as follows: Probable pathogens 1. Certain organisms should almost always be thought to represent true pathogens (bacteremia or fungemia)when isolated from a blood culture. These organisms included Staphylococcus aureus (S.aureus), Streptococcus pneumoniae (S.pneumoniae), E. coli and other Enterobacteriaceae, Pseudomonas aeruginosa, Streptococcus pyogenes, Streptococcus agalactiae, Listeria monocytogenes, Neisseria meningitidis, Neisseria gonorrhoeae, Haemophilus influenzae, Bacteroides fragilis, all Candida species, and Cryptococcus neoformans. 2. Isolation of commensal microbial flora??? (commonly coagulase negative Staphylococci) from blood of immunocompromised patients or those having prosthetic devices or central venous catheter and patient suspected to be bacteremic. 3. Growth of the same organism in repeated cultures obtained either at different times or from different anatomic sites.  Probable contaminants: 1. Growth from only one of several cultures  multiple organisms  Growth of known commensal flora 2. Growth occurred after 72 hours is usually assumed as contaminant if sufficient volumes of blood have been inoculated into the bottles. 3. The clinical presentation and/or course are not consistent with sepsis (physician- based, not laboratory-based). 4. The organism causing the infection at a primary site of infection is not the same as that isolated from the blood culture.  Case A 28-year-old woman is transferred from an outside hospital with a chief complaint of fever 39°C for the past 3 weeks with no defined cause. Pyrexia of unknown origin (PUO)  Case A 29-year-old farmer complained of intermittent fever, anorexia, profuse sweating, malaise, headache, and muscle pain especially of the neck and shoulder, and arthralgia for 3 d. The complaints, persisted on and off for the next one month and the conditions were getting worse. Abdominal palpations revealed PUO hepatomegaly and splenomegaly with pain. BRUCELLOSIS  BRUCELLOSIS Brucellosis is a common disease in many developing countries and it is one of the differential diagnoses of PUO. It has been known by various names, including Mediterranean fever, Malta fever, and undulant fever. Causative organisms: B. abortus (cattle), B. melitensis (sheep or goats), B. suis (pigs) and B. canis (dogs). Mode of transmission: Humans acquire brucellosis by occupational exposure among farmers, slaughterhouse workers, and veterinarians through: 1. Ingestion of undercooked meat or consumption of unpasteurized dairy products. 2. Bacteria can also enter wound in skin or mucous membranes, through contact with infected animals. 3. Inhalation of bacteria during handling of infected animal. Clinical presentation: Brucellosis is a systemic infection that can involve any organ of the body and clinically presented as asymptomatic infection or as acute, subacute or chronic infection. Relapse is considered an important feature of brucellosis. Laboratory diagnosis: A-Microbiological examination Direct Methods: Specimens for culture and sensitivity : Blood, bone marrow and other specimens as CSF, pleural and synovial fluids, and urine. It is essential that the clinical microbiology laboratory be notified whenever brucellosis is suspected to ensure that specimens are cultivated in an appropriate manner for optimum recovery of the organism. Direct stains not particularly useful for the diagnosis of brucellosis. Culture: Blood culture: It should be collected in the first three weeks and it is recommended to use bottles of the automated blood culture system. Conventional culture of other specimens but incubation should extend to 7 days. Fluid enrichment increase yield if collected from sterile site. Molecular diagnosis are reliable and specific means of detecting Brucella spp in clinical specimens but not available for routine use.. Indirect Methods (Serological diagnosis): a. Standard agglutination test [SAT]. This technique detects antibodies to B. abortus, B. melitensis, and B. suis, but not B. canis antibodies (there is no serological test available to detect antibodies to B. canis). SAT may give false negative results due to the relative excess of antibodies against antigens (prozone phenomenon) and due to non-agglutinated IgG in subacute and chronic cases. So, anti-human globulin test (indirect Coombs' test) should be performed after the SAT to detect non-agglutinating antibodies in subacute and 1:640chronic1:320 cases of brucellosis. 1:160 1:80 1:40 1:20 b. Enzyme-linked immunosorbent assays (ELISA) for IgG and IgM antibodies. B-Complete blood count: Neutropenia with leukocytosis. Treatment: To prevent relapse of infection, patients with brucellosis should undergo prolonged treatment (6 weeks) with antibiotics that can penetrate macrophages.  ABSCESSES AND WOUND INFECTIONS Abscesses are collections of pus in confined tissue spaces, Wound infections occur primarily from breaks in the skin as a result of complications associated with surgery, trauma, and bites, or from diseases that interrupt the mucosal or skin surface. Causative Organisms: Aerobic organisms Gram-positive cocci as Staphylococcus aureus, Coagulase-negative staphylococci, Enterococci, other streptococci and other gram-positive aerobes. Gram-negative bacilli as Enterobacteriacae , Pseudomonas aeruginosa. Anaerobes e.g. Clostridia, Bacteroides, and anaerobic cocci. Fungal: Candida species infections in patients who receive prolonged antibiotic therapy. Notes: Human bite wounds are particularly subject to anaerobic infections, in contrast, infected bites of domestic animals (dogs, cats) are almost always due to Pasteurella multocida. Lab. Diagnosis: Specimens: Pus may be aspirated in sterile syringe or deep wound swab. Specimens will be subjected to the followings: 1- Direct Film Wet film for motile ameba in liver abscess. Gram stain To detect causative organism. Can improve the accuracy of evaluating wound culture. The presence of PML is an indication of an inflammatory or infectious process, while the presence of epithelial cells indicates surface contamination of the specimen. 2- Culture both aerobic and anaerobic culture if specimen is suitable In abscesses, mixture of aerobic and anaerobic organisms could be the cause. The most commonly isolated aerobic organism is E. coli and staph. aureus, and the most commonly observed anaerobic organism is Bacteroides fragilis.  ANAEROBIC INFECTIONS Anaerobic microorganisms are classified as: I-Non-Spore Forming e.g. Bacteroides species, anaerobic cocci, Actinomyces and P. acnes. Bacteroides species are the most common cause of bronchiectasis and closed abscesses, anaerobic intraabdominal infections commonly following colon surgery. Although most anaerobic infections are polymicrobial, B. fragilis alone may be responsible for infection. II-Spore-Forming Organisms (Clostridia spp) Organism Disease C. perfringens Gas gangrene or clostridial myonecrosis is an acute and rapidly progressive invasive process producing marked changes in muscles. Anaerobic cellulitis is a gradual necrotizing process of the soft tissues. Gas is produced; but it does not involve muscles. N.B: Distinguishing between the two conditions is critical to avoid performing unnecessarily aggressive surgery in the former condition. Food poisoning. C. tetani Tetanus. C. botulinum Botulism: The organism produce neurotoxin→ dry food poisoning (without vomiting or diarrhea). C. difficile Antibiotic associated diarrhea is a non-bloody diarrhea occurring after broad spectrum antibiotics e.g. clindamycin. Suppression of normal flora by antibiotics allows proliferation of C. difficile with subsequent production of toxins and occurrence of pseudomembranous colitis. It can be treated by stopping the antibiotic intake with administration of oral vancomycin or metronidazole. Lab. Diagnosis: 1- Specimens: Tissue, fluid or pus in sterile anaerobic container. N.B: Throat, nasopharyngeal, sputum, gastric contents, stool, food remnants, material adjacent to skin or mucous membranes, voided urine, and vaginal or cervical specimens are not suitable for anaerobic culture. 2- Direct film stained with Gram stain: The presence of numerous, large, “boxcar”-shaped, gram- positive bacilli provides presumptive confirmation of the clinical diagnosis of Clostridium perfringes infection. The presence of gram positive bacilli that have terminal bulging spore with drumstick appearance, support the clinical diagnosis of tetanus. May be diagnostic of anaerobic streptococcal myositis. 3- Culture on conventional culture media under anaerobic conditions. 4- Toxin Detection for Clostridium difficile toxins (A& B) and Clostridium perfringens toxin in stool and Clostridium botulinum toxin in food remnants.