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Revision Term One QA Systems Quality Assurance measures apply to the laboratory analytical work overall, which includes; •identifying the person having the overall responsibility for quality •having laboratory equipment calibrated to recognised standards >Using only QA.ed materials •Using reference materials +ve and -ve •Validating analyses •Internal proficiency testing. •Joining proficiency testing schemes with other laboratories doing similar tests >Using correct SOP for each method/activity •Using correct record sheets •Traceability of changes to SOPs and records % Fat 3.30 QC systems % fa t Quality control measures apply to each analytical test in the laboratory by use of: 3.25 3.20 3.15 3.10 14/01/02 24/01/02 03/02/02 13/02/02 23/02/02 05/03/02 15/03/02 25/03/02 04/04/02 14/04/02 date Control chart • reagent blanks; • verified standard • blind samples solutions; • check samples • replicate analyses; • and control charts (from both within • Positive and the lab and from negative controls outside); • All materials QC ed. 24/04/02 Keeping track of the samples • Sample registration gives each sample a unique lab number. Lab. No. F7-002 Sample integrity • The sample register records all the information about the sample. • Just like a sample’s passport, you should not confuse any sample with any other. • The history of the sample should be traceable throughout. Samples recorded on receipt Beta ( β ) hemolysis (blood agar around bacterial colonies is completely clear, indicating complete breakdown and consumption of hemoglobin) Alpha (α) hemolysis (blood agar around bacterial colonies looks greenish-brown, indicating partial breakdown and consumption of hemoglobin) Mixed culture from a throat swab; note several β-hemolytic colonies Optochin (Taxo P) sensitivity - Results with no zone of inhibition + results with a zone of inhibition P P Catalase Test - + Chapter 21: Diseases of Skin and Eyes 1. Staphylococcal Infections – Gram-positive cocci in irregular clusters – Coagulase negative strains make up to 90% of skin microbiota (S. epidermidis). Only pathogenic when skin is broken or through invasive entry. – Coagulase positive strains tend to be pathogenic. Almost all pathogenic S. aureus strains make coagulase. High correlation between ability to produce coagulase and production of damaging toxins: • Leukocidin: Destroys phagocytic white blood cells. • Exfoliative toxin: Responsible for scalded skin syndrome. • Enterotoxins: Affect gastrointestinal tract. – S. aureus is commonly found in nasal passages. 1. Staphylococcal Infections (Continued) Common staphylococcal diseases A. Folliculitis: Infection of hair follicles (pimples). B. Sty: Infected eyelash follicle. C. Boil (Abscess or Furuncle): More serious infection of hair follicle in which pus is surrounded by inflamed tissue. Usually painful and firm. D. Carbuncle: Aggregate of several infected follicles (boils). May cause fever, chills, malaise, and death if not treated. Forms a round, hard, deep area of inflammation, typically on neck and back. 1. Staphylococcal Infections (Continued) Common staphylococcal diseases E. Impetigo: Problem in hospital nurseries and day care centers. Thin walled vesicles on skin rupture and crust over. Caused by S. aureus, S. pyogenes, or both. • Highly contagious, spread through direct contact and fomites. • Occurs almost exclusively in children. • Rarely produces fever and easily treated with penicillin. Staphylococcal diseases (continued) 6. Scalded skin syndrome: Caused by toxemia from S. aureus strains with two different exfoliative toxins. • Large sheets of bright red skin peel off. • Usually observed in children under 2, but may occur in adults. • Bacteremia and septicemia may occur, and can lead to death within 36 hours. • Require vigorous antibiotic treatment. • Exfoliative toxins are highly antigenic, preventing recurrence. Staphylococcal diseases (continued) 7. Toxic shock syndrome (TSS): Fever, vomiting, and sunburnlike rash, followed by shock. Rash later peels. • Presently about 25 cases per year reported. • Most cases are associated with use of superabsorbent tampons. • Males with boils or other staphylococcal infections are at risk. • A few cases associated with use of contraceptive sponge. • 5-15% of women have S. aureus in vaginal microflora. • Only a small percentage of these strains In addition to skin infections, the staph bacteria can cause: • Bacteremia – a blood infection • Deep abscesses –an abscess that occurs • • • below the skin surface Endocarditis – an infection on the valves of the heart Food poisoning – vomiting or diarrhea caused by a staph toxin Lymphadenitis – an infection of a lymph gland, which causes it to be red, swollen and painful • Lymphangitis – an infection of the lymph • • • • • • channels that drain to lymph glands, causing red streaks in the skin Osteomyelitis – a bone infection Paronychia – an infection of the skin folds of the nails Scalded skin syndrome Septic arthritis – an infection of a joint, like a hip or a knee Styes – an infection of the glands on the eyelid Toxic shock syndrome MRSA • MRSA stands for Methicillin-Resistant Staphylococcus aureus • MRSA are Staph aureus bacteria that have become resistant to this antibiotic. 2. Streptococcal Infections – Gram-positive cocci in chains. – Cause many disease including meningitis, pneumonia, sore throat, otitis media, endocarditis childbirth fever, and dental caries. – Produce multiple toxins and virulence factors. • • • • Stretokinases: Dissolve blood clots. Proteases: Destroy proteins. Hyaluronidase: Breaks down connective tissue. Hemolysins: Lyse red blood cells. Alpha, beta, and gamma hemolysis. – Beta hemolytic streptococci are often associated with human disease. – Streptococcus pyogenes = Group A bhemolytic streptococci. – Infections are often localized, but can Chapter 21: Diseases of Skin and Eyes 1. Staphylococcal Infections – Gram-positive cocci in irregular clusters – Coagulase negative strains make up to 90% of skin microbiota (S. epidermidis). Only pathogenic when skin is broken or through invasive entry. – Coagulase positive strains tend to be pathogenic. Almost all pathogenic S. aureus strains make coagulase. High correlation between ability to produce coagulase and production of damaging toxins: • Leukocidin: Destroys phagocytic white blood cells. • Exfoliative toxin: Responsible for scalded skin syndrome. • Enterotoxins: Affect gastrointestinal tract. – S. aureus is commonly found in nasal passages. 1. Staphylococcal Infections (Continued) Common staphylococcal diseases A. Folliculitis: Infection of hair follicles (pimples). B. Sty: Infected eyelash follicle. C. Boil (Abscess or Furuncle): More serious infection of hair follicle in which pus is surrounded by inflamed tissue. Usually painful and firm. D. Carbuncle: Aggregate of several infected follicles (boils). May cause fever, chills, malaise, and death if not treated. Forms a round, hard, deep area of inflammation, typically on neck and back. Damages surrounding tissue, with extensive scarring. May need to lance and drain surgically. 1. Staphylococcal Infections (Continued) Common staphylococcal diseases E. Impetigo: Problem in hospital nurseries and day care centers. Thin walled vesicles on skin rupture and crust over. Caused by S. aureus, S. pyogenes, or both. • Highly contagious, spread through direct contact and fomites. • Occurs almost exclusively in children. • Rarely produces fever and easily treated with penicillin. Staphylococcal diseases (continued) 6. Scalded skin syndrome: Caused by toxemia from S. aureus strains with two different exfoliative toxins. • Large sheets of bright red skin peel off. • Usually observed in children under 2, but may occur in adults. • Bacteremia and septicemia may occur, and can lead to death within 36 hours. • Require vigorous antibiotic treatment. • Exfoliative toxins are highly antigenic, preventing recurrence. Staphylococcal diseases (continued) 7. Toxic shock syndrome (TSS): Fever, vomiting, and sunburnlike rash, followed by shock. Rash later peels. • Presently about 25 cases per year reported. • Most cases are associated with use of superabsorbent tampons. • Males with boils or other staphylococcal infections are at risk. • A few cases associated with use of contraceptive sponge. • 5-15% of women have S. aureus in vaginal microflora. • Only a small percentage of these strains In addition to skin infections, the staph bacteria can cause: • Bacteremia – a blood infection • Deep abscesses –an abscess that occurs • • • below the skin surface Endocarditis – an infection on the valves of the heart Food poisoning – vomiting or diarrhea caused by a staph toxin Lymphadenitis – an infection of a lymph gland, which causes it to be red, swollen and painful MRSA • MRSA stands for Methicillin-Resistant Staphylococcus aureus • MRSA are Staph aureus bacteria that have become resistant to this antibiotic. 2. Streptococcal Infections – Gram-positive cocci in chains. – Cause many disease including meningitis, pneumonia, sore throat, otitis media, endocarditis childbirth fever, and dental caries. – Produce multiple toxins and virulence factors. • • • • Stretokinases: Dissolve blood clots. Proteases: Destroy proteins. Hyaluronidase: Breaks down connective tissue. Hemolysins: Lyse red blood cells. Alpha, beta, and gamma hemolysis. – Beta hemolytic streptococci are often associated with human disease. – Streptococcus pyogenes = Group A bhemolytic streptococci. – Infections are often localized, but can Common streptococcal skin diseases – Erysipelas: From Greek erythos = red, and pella = skin. Also called St. Anthony’s fire. Common skin infection associated with S. pyogenes. • Spread through contact or insect bites. • Skin erupts into reddish patches with raised margins. • High fever is common. • Organisms can spread through lymphatics and cause septicemia, abscesses, pneumonia, endocarditis, arthritis, and even death if untreated. • Mortality was high before use of antibiotics. • Responds well to antibiotic (b-lactams) treatment. Common streptococcal skin diseases – Flesh eating bacterial infections: • Caused by invasive group A streptococci. • 15,000 cases per year in U.S. • Exotoxin A acts as superantigen causing damage by the immune system. • Attacks and destroys muscle (myositis), muscle covering (fasciitis), and solid tissue (cellulitis). • Can destroy several inches of tissue per hour. • Antibiotics are not effective because dead tissue has no circulation. • Requires amputation or surgical removal of tissue. • Mortality rate up to 40% – Impetigo: Refer to previous description. Flesh eating bacterial infections • Caused by invasive group A streptococci. • 15,000 cases per year in U.S. • Exotoxin A acts as superantigen causing damage by the immune system. • Attacks and destroys muscle (myositis), muscle covering (fasciitis), and solid tissue (cellulitis). • Can destroy several inches of tissue per hour. • Antibiotics are not effective because dead tissue has no circulation. • Requires amputation or surgical removal of tissue. • Mortality rate up to 40% Bacillus • Classification – – – – – All are large Gram-positive bacilli Are aerobic Form endospores Most are found in dust and soil Bacillus anthracis is the major pathogen in the group • Morphology and Cultural Characteristics (Bacillus anthracis) Bacillus – Grow well on ordinary lab media producing large granular colonies with a coarse texture. • Virulence factors – Capsule helps organism to resist phagocytosis but antibodies are not protective. – Exotoxin is very complex and is produced only when the bacteria is growing in animal tissues. • Toxin production is mediated by a temperature sensitive plasmid. • The toxin consists of three protein components (maximum toxicity occurs when all three components are present). Bacillus • Clinical significance – Anthrax which is the disease caused by B. anthracis is essentially a disease of animals who acquire the organism by ingestion or inhalation of spores. • The spores are extremely resistant to adverse chemicals and physical environments. • They may remain a source of infection in soil for 2-3 years. – Man acquires anthrax usually from contact with animal products; less commonly from working in an agricultural setting with infected animals. Bacillus – Man may acquire the organism through skin abrasions, by inhalation of spores, or by ingestion. The disease that develops depends upon the mode of transmission: • Pulmonary (Woolsorter‘s disease) Spores are inhaled and germinate in the lungs where they multiply and spread to cause a fatal septicemia or meningitis. – This is the most serious form of the disease. • Intestinal anthrax results from ingestion of spores. Bacillus • Antibiotic susceptibility and treatment – Penicillin or tetracycline – A short-term PA vaccine is available for industrial workers and others at high risk. • Other Bacillus species – Bacillus subtilis, and occasional other species may occasionally cause opportunistic infections. – Bacillus cereus is a major cause of enterotoxin food poisoning; • The toxin is protein in nature and can be destroyed by heating; • Food poisoning occurs after ingestion of pre-formed toxin; – Vomiting occurs 1-5 hours after ingestion Bacillus – B. cereus is also an opportunistic pathogen that has been cultured from cases of septicemia, endocarditis, meningitis, wound infections, pneumonia, and fulminant eye infections • In addition to the enterotoxin that bacteria may produce, a dermonecrotic and a lethal toxin, hemolysins, lecithinase, proteases, and nucleases may be involved in its pathogenesis • Clindamycin with or without gentamycin may be used for treatment of infections Clostridium Clostridium form endospores under adverse environmental conditions Spores are a survival mechanism Spores are characterized on the basis of position, size and shape Most Clostridium spp., including C. perfringens and C. botulinum, have ovoid subterminal (OST) spores C. tetani have round terminal (RT) spores Clostridium Clostridium form endospores under adverse environmental conditions Spores are a survival mechanism Spores are characterized on the basis of position, size and shape Most Clostridium spp., including C. perfringens and C. botulinum, have ovoid subterminal (OST) spores C. tetani have round terminal (RT) spores Clostridium perfringens Micro & Macroscopic C. perfringens NOTE: Large rectangular NOTE: Double zone of hemolysis gram-positive bacilli Inner beta-hemolysis = θ toxin Outer alpha-hemolysis = α toxin Clostridial Cellulitis Clostridium tetani Clostridium tetani Gram Stain NOTE: Round terminal spores give cells a “drumstick” or “tennis racket” appearance. Opisthotonos in Tetanus Patient Mechanism of Action of Tetanus Toxin Clostridium botulinum Mechanism of Action of Botulinum Toxin Rates of Isolation of C. botulinum and Botulinum Toxin Tuberculosis (TB, consumption) • M. tuberculosis • major human disease – healthy people • problems – association with AIDS – multiple drug-resistance M. avium- M. intracellulare complex (M. avium) • non-AIDS – infection almost never • AIDS – major bacterial opportunist • multiple drug-resistance M. bovis • spread from cattle • infected cattle are culled – positive skin test • rarely seen in US M. leprae • leprosy • major disease of third world • Leprosy rates in Australia are only about • • • one case per million, but are higher in Aboriginal Australians and immigrants from infected areas. Worldwide, leprosy is still endemic mostly in Africa and around India. There is also some leprosy in parts of the USA Laboratory diagnosis - tuberculosis • skin testing – delayed hypersensitivity – tuberculin – protein purified derivative, PPD • X-ray Laboratory diagnosis M. tuberculosis • acid fast bacteria – sputum Positive skin test tuberculosis • indicates exposure to organism • does not indicate active disease Laboratory diagnosis M. tuberculosis (culture) • grows very slowly – several weeks – non-pigmented colonies – niacin production *differentiates from other mycobacteria Mycobacterium leprae Leprosy (Hansen's Disease) • M. leprae – causative agent • chronic disease – disfigurement • rarely seen in the U.S. • common in third world • millions of cases • infects the skin Leprosy • tuberculoid • few organisms • active cell-mediated immunity • lepromatous • immunosuppression • few organisms Leprosy • lepromin – skin testing • acid-fast stains – skin biopsies • clinical picture Corynebacterium diphtheriae Corynebacterium diphtheriae • Gram positive • strict aerobe • pleomorphic (e.g. club-shaped) Diphtheria • infection – upper respiratory tract (pharynx) – pseudomembrane – chocking – bacteria do not spread systemically . Diphtheria toxin • B binds to host cell • A inhibits protein synthesis • ADP-ribose moiety (NADH) attaches – rare amino acid, diphthamide – elongation factor 2 inhibited C. diphtheriae should not be confused with: • diphtheroids – other corynebacteria – propionibacteria Corynebacterium spp. Listeria monocytogenes Listeriosis (invasive disease & non-invasive enteritis) • The organism – G+ve ovoid to • Widespread in environment rod-shaped bacterium Listeria monocytogenes • Characteristics - grows in wide range of temperatures (1 to 45o C) - survives freezing - aerobic & anaerobic conditions Listeria monocytogenes • The illness – invasive form - incubation – 30 days - flu’-like symptoms, diarrhoea, vomiting, meningitis, septicaemia, spontaneous abortion Listeria monocytogenes • The illness – invasive form, continued - infective dose – 100 to 1 000 cells - pregnant women, newborn babies, the elderly & AIDS patients - Rx – penicillin, ampicillin +/gentamicin Listeria monocytogenes • The illness – non-invasive - incubation – 18 hours - diarrhoea, fever, muscle pain, headache, abdominal cramps & vomiting Listeria monocytogenes • The illness – non-invasive - infective dose – > 100 thou. cells/gm - all individuals susceptible - Rx - penicillin, ampicillin +/gentamicin Listeria monocytogenes • Sources - human – person-to-person rare - animal – diseased animals shed in faeces, contamination of red meat; silage - food – ready-to-eat cooked food with long shelf-life - raw foods - environment – widespread in soil, water & sewage (Hospitals & occupational exposure) Listeria monocytogenes Year 2004 2002 2000 1998 1996 1994 1992 1990 1988 1986 1984 1982 40 35 30 25 20 15 10 5 0 1980 Number of cases Listeriosis cases in NZ from 1980 to 2004