MICR 111 Lesson 15: Anaerobic Bacteria PDF

MICR_111 NATHANIEL VERON M. CASAMINA, MSMT, RMT, ASCPI | 3MT03 3RD YEAR, 1ST SEMESTER BY: MANALO, A. & MILAR, K. LESSON #15: ANAEROBIC BACTERIA...

MICR_111 NATHANIEL VERON M. CASAMINA, MSMT, RMT, ASCPI | 3MT03 3RD YEAR, 1ST SEMESTER BY: MANALO, A. & MILAR, K. LESSON #15: ANAEROBIC BACTERIA OUTLINE 5. Growth on Anaerobic Culture Plate 6. Double Zone of Hemolysis incubated anaerobically I. Indicators of Anaerobic Bacteria II. Endogenous Anaerobes common in Human ENDOGENOUS ANAEROBES Infections COMMON IN HUMAN INFECTIONS III. Clostridium spp. IV. Clostiridium perfringens (Gas Gangrene Table No. 1 Endogenous Anaerobes Common in human infections Bacillus) ANAEROBE INFECTION A. Clinical Infections of C. perfringens Actinomyces spp. Actinomycosis 1. Gas Gangrene Clostridium difficile Antibiotic-associated diarrhea; Pseudomembranous 2. Food Poisoning Enterocolitis V. Clostiridium tetani (Tack Head Bacillus) Bacteroides fragilis, Fusobacterium, Bacteremia A. Tetanospasmin Clostridium, Peptostreptococci B. Tetanus Bacteroides, Prevotella, Brain Abscesses VI. Clostridium botulinum (Canned Good Bacillus) Porphyromonas, Fusobacterium, A. Botulism Toxin Clostridium spp. 1. Foodborne Botulism Peptostreptococcus, Bacteroides, Female Urogenitary Tract 2. Infant Botulism Clostridium, Prevotella bivia, Infection B. Clinical Infections of C. botulinum Actinomyces israelii VII. Clostridioides difficile Bacteroides, Fusobacterium, C. Intraabdominal infections, A. Biochemical Test perfringens, Peptostreptococci Liver Abscess, Peritonitis, Perineal and Perirectal VIII. Collection, Transport , Storage of Anaerobic Infections Bacteria С. perfringens, C. novyi, C. septicum Myonecrosis IX. Laboratory Diagnosis Peptostreptococcus, Oral, Sinus, Dental Infections A. Culture Porphyromonas, Fusobacterium B. Other Laboratory Tests Porphyromonas, F. nucleatum, Aspiration Pneumonia and X. Actinobacteria Peptostreptococcus, B. fragilis, Pleuropulmonary infections XI. Other Species of Anaerobic Bacteria Actinomyces A. Bacteroides fragilis B. Actinomyces israelli CLOSTRIDIUM SPP. C. Cutibacterium acnes Obligate Aerobes, G (+) Spore-forming rod; Catalase (-) D. Lactobacillus spp. Frequently encountered in exogenous anaerobic infections E. Prevotella melanogenica or intoxications F. Porphyromonas Virulence: Collagenase, Hyaluronidase (spreading G. Fusobacterium factor), Lecithinase (Cell Destruction), Phospholipase H. Veilonella Histotoxic Clostridia (Myonecrosis): C. perfringens, C. I. Bacterial Vaginosis novyi, C. septicum, C. histolyticum, C. bifermentans 1. Gardnerella vaginalis Clostridium septicum - Marker for malignancy in the Gl 2. Mobiluncus spp. Tract ("Smooth swarming" on plated media) XII. Other Related Infections of Non-sporeforming Clostridium sordelii - Only Urease Positive Clostridia Anaerobic Bacteria XIII. Identification of Nonsporeforming Anaerobic GENERAL CHARACTERISTICS OF CLOSTRIDIA Bacteria Form endospores anaerobically XIV. Treatment of Anaerobic Infections Peritrichous flagella except: C. perfringens, C. ranosum, C. innocum Swollen Sporangia except C. perfringens and C. INDICATORS OF ANAEROBIC BACTERIA bifermentans 1. Foul odor upon opening an Non-encapsulated except C. perfringens anaerobic jar of bag (Clostridium Single Hemolytic Reaction except C. perfringens difficile, Fusobacterium, CHO Fermenters except C. tetani and C. histolyticum Porphyromonas) Killed immediately (C. novyi) in Oxygen presence 2. Presence of sulfur granules Notes: (Actinomyces, Cutibacterium, o Clostridium perfringens – Most pathogenic Eubacterium nodatum) among clostridium spp. because it has 3. Brick-Red Fluorescence endospores, capsule and double hemolytic (Prevotella/Porphyromonas) reaction. 4. Absence of Superoxide Dismutase o Sporangia – microorganism containing terminal (SOD) to subterminal endospore 1 TRINITY UNIVERSITY OF ASIA – COLLEGE OF MEDICAL TECHNOLOGY | 3MT03 LESSON #15: ANAEROBIC BACTERIA 2. CLOSTRIDIUM TETANI (“TACK HEAD BACILLUS”) Soil and environment inhabitant Endospores found in hospital, soil dust, feces of farm animals Microscopy: Terminal spore and swollen sporangia ("Drumstick/Lollipop/Tennis Racket" Appearance) Culture: 1. CLOSTRIDIUM PERFRINGENS o BAP - Heavy "smooth swarming" anaerobic: (“GAS GANGRENE BACILLUS”) grow slowly Former: Clostridium welchii o BAP - Colonies with matte surface with Most commonly isolated Clostridium in blood cultures narrow zone of B-hemolysis Produce DNase Biochemical Tests: Lacks ability to produce essential amino acids o Motile; (+) Gelatinase and Indole; (-) Virulence Factors: Alpha Toxin and Enterotoxin A & C Lecithinase and Lipase Microscopy: "Boxcar-shaped" bacilli; Endospores Notes central to subterminal o Oxidation of iron in the rust with the dust of Can cause bacteremia or sepsis Clostridium tetani makes its pathogenesis, not Culture: the rust itself o BAP: Dome-shaped, gray to white colonies; o Only C. tetani does NOT cause food poisoning Double zone of hemolysis (Alpha and Beta o Has gelatinase Zones) o Different from perfringens = C. tetani is motile; o Litmus Milk - "Stormy Fermentation of Milk" C. perfringens is non motile (because of gas) Biochemical Tests: o Very fermentative; Non-motile o (+) Lecithinase (Egg Yolk Agar) o (+) Nagler Test (Lecithovitalin Reaction on EYA) - Detects the enzyme lecithinase which is an alpha toxin TETANOSPASMIN o (+) Reverse CAMP Test Virulence factor of C. tetani (Neurotoxin) Endopeptidase selectively cleaving synaptic vesicle membrane protein Synaptobrevin Cause tension or cramping and twisting in skeletal muscles surrounding wounds and tightness of jaw muscles CLINICAL INFECTIONS OF C. PERFRINGENS TETANUS TETANIC TRIAD = Trismus ("Lock Jaw"), Risus GAS GANGRENE/MYONECROSIS (“EATING SORE”) Sardonicus ("Distorted Grin"), Opisthotonos (Muscle Life threatening destruction of muscles and other spasms) tissues; necrotizing infection of skeletal muscles Symptoms: Muscular rigidity (Jaws, Neck, Lumbar Cause: Alpha Toxin Region), difficulty in swallowing, abdominal, chest, back, Entry via wounds, trauma, frostbite, surgery and limb rigidity Accompanied by bullae (fluid-filled blisters), pain, Incubation period: 3-21 days long swelling, serous discharge, discoloration, tissue necrosis Incubation related to the distance of injury to the CNS TREATMENT: Hyperbaric Oxygen Therapy Tetanus Neonatorum - Contaminated instruments used for newborns FOOD POISONING/ENTERITIS NECROTANS (“PIG-BEL”) Ingestion of Enterotoxin in contaminated food (Improper storage) Symptoms: Foul-smelling stool (Diarrhea), Crampy abdominal feeling Types of Food Poisoning: o Type A - Mild, Self-limiting: with diarrhea and cramping pain o Type C - Serious, rarely encountered; bloody 3. CLOSTRIDIUM BOTULINUM diarrhea and cramping pain (“CANNED GOOD BACILLUS”) First identified as enteritis; first isolated from pigs Found in soil and aquatic sediments Potential Bioterrorism agent Subterminal spore; B-Hemolytic (BAP) Proteolytic bacterium - ability to oxidize one Amino Acid with the 2nd amino acid as the electron acceptor ("Stickland fermentation/reaction") 2 TRINITY UNIVERSITY OF ASIA – COLLEGE OF MEDICAL TECHNOLOGY | 3MT03 LESSON #15: ANAEROBIC BACTERIA BOTULISM TOXIN COLLECTION, TRANSPORT, STORAGE Virulence factor: Most potent toxin known to man OF ANAEROBIC BACTERIA Selectively cleaves synaptic vesicle membrane protein Synaptobrevin (prevent exocytosis and release of GUIDELINES Acetylcholine) Specimens held at Room Temperature pending 7 Antigenically different types (A to G) - A,B,E in human processing (Refrigeration can oxygenate specimen) infections Food and Feces (C. difficile) transported at 4C Toxin Type A - Botox Treatment; used to treat Improper collection - growth of other anaerobes Strabismus (Wandering Eye) Specimen collected from actual site of Infection and not a swab of a mucosal surface Swabs used ONLY when aspiration and biopsy is not possible Swabs placed in 0.5 mL sterile Thioglycolate broth Crucial factor in final success of anaerobic culture is the transport of the specimen (lethal effect of Oxygen) - transported quickly under anaerobic conditions/minimum exposure CLINICAL INFECTIONS OF CLOSTRIDIUM BOTULINUM Refrigeration is a form of OXIDATION causes for Botulism main features: Double or Blurred vision, Impaired microorganisms to die; sensitive to oxidation speech, difficulty in swallowing, weakness, paralysis Unacceptable Specimens for Anaerobic Bacteria o Swabs - OPS/NPS, Vaginal, Cervical, Urethral, FOODBORNE BOTULISM Rectal, Decubitus Ulcers, Perirectal Abscess, Ingestion of preformed toxin on non-acidic vegetables, Foot Ulcers, Exposed wound, Eschars, Sinuses, preserved food, meat-based food, mushroom foodstuffs Gingival, Intraoral Surface collections Caused by: Botulism Toxin Type A o Sputum - Nasotracheal/Endotracheal Suction; Prevention: heat or cook the canned goods properly Expectorated o Bronchial Wash INFANT BOTULISM o Feces, Ileostomy, Colostomy Effluents Actual Infection caused by ingesting the organism from o Gastric and Small Bowel Contents honey or via breastfeeding 4. CLOSTRIDIOIDES DIFFICILE Most common cause: Antibiotic-associated diarrhea and Pseudomembranous enterocolitis "Infection Control Dilemma" among hospitalized patients Found as GIT Microbiota in 5% of individuals Produce Glutamate Dehydrogenase - important for the toxin determination of C. difficile Virulence: Enterotoxin A (Antibiotic associated diarrhea), Cytotoxin B (Pseudomembranous enterocolitis) Causes ANTIBIOTIC diarrhea BIOCHEMICAL TESTS Ferments Fructose forming Formic Acid (CCFA) - Pink- colored medium turns yellow Microscopy: o Chains up to 6 cells aligned end-to-end o LABORATORY DIAGNOSIS o Endospores: Oval, Subterminal Culture: CULTURE o Cycloserine-Cefoxitin-Fructose-Agar (CCFA) - Media used: Anaerobic Blood Agar, Thioglycolate, Egg Yellow, Ground Glass Colonies Yolk Agar (EYA), Cycloserine Cefoxitin Fructose Agar o BAP - "Horse stable" or "Barnyard" odor; (CCFA), Peptone-Yeast Glucose (PYG) Broth, Nonhemolytic Brucella/Blood Agar (BRU/BAP), PEA, CNA o BAP - Fluoresce chartreuse under longwave UV Transport Media: Pre-reduced Anaerobically Sterilized light (PRAS), Amies o PRAS - For aspirates and tissues o Amies 1mL Pre-reduced: Anaerobic and Aerobic Bacteria Primary Plates - freshly prepared or used within 2 weeks of preparation Air removed from sealed jar using Mercury Growth Inhibitors - Peroxides and Dehydration ‘ (prolonged storage) BRU/BAP - Observe swarming and double zone of hemolysis; support nearly all obligate and facultative anaerobes 3 TRINITY UNIVERSITY OF ASIA – COLLEGE OF MEDICAL TECHNOLOGY | 3MT03 LESSON #15: ANAEROBIC BACTERIA EYA - Detect activity of Lipase and Lecithinase (+ insoluble opaque whitish precipitate within the agar) PYG - Detect volatile fatty acids CCFA - Selective and Differential Medium o Cycloserine, Cefoxitin - Inhibit Gram Negative Coliforms o pH Indicator: Neutral Red (Yellow in presence of LABORATORY DIAGNOSIS Acid pH) Reducing Agents - Thioglycollate, Cysteine, Dithiothreitol OTHER LABORATORY TESTS (Added to culture media to obtain low RedOx potential) Direct Nagler Test C. septicum - "Smooth swarming" growth on plated media o Using EYA Plate + C. perfringens Type A C. tertium - Grow minimally on the aerotolerance plate, Antitoxin mistaken for a facultative anaerobe o (+): Yellowish-Egg color (Inhibition of Lecithinase reaction produced by C perfringens) Table No. 2 Non Selective Culture Medium Mouse Neutralization Test NON-SELECTIVE USE o Definitive Identification test for C. botulinum CULTURE MEDIUM o Detects presence of Neurotoxins in serum or Cooked Meat Broth Anaerobic organisms; with Glucose can be used for Gas-Liquid Chromatography feces Anaerobic Blood Agar Anaerobes and Facultative Anaerobes Reverse CAMP Test EYA Lecithinase and Lipase o Confirm presence of C. perfringens Peptone-Yeast Extract Gas-Liquid Chromatography o (+): Reverse Arrowhead at Intersection of 2 Lecithinase and Lipase streaks (towards Clostridium isolates) Anaerobic PEA Cell Culture Cytotoxicity Test Glucose Broth o Gold Standard Test for detection of C. difficile Thioglycolate Anaerobes, Facultative Anaerobes, toxin Type B Aerobes o 2-3 days to achieve (+) result Spot Indole Test Table No. 3 Selective Culture Medium o Differentiate C. bifermentans and C. sordelii SELECTIVE USE (Indole +) from other Clostridium spp. (Indole -) CULTURE MEDIUM Bacteroides Bile Esculin Selective and Differential for Agar (BBE) B. fragilis Laked Kanamycin- Prevotella, Bacteroides spp. Vancomycin Blood Agar (LKVB) Anaerobic PEA Selective for inhibition of G- rods and swarming by some Clostridium spp. Cycloserine-Cefoxitin Selective for Clostridioides difficile ACTINOBACTERIA Fructose Agar (CCFA) 1. Actinomyces 2. Bifidobacterium LABORATORY DIAGNOSIS 3. Cutibacterium 4. Eggerthella Table No. 4 Laboratory Diagnosis 5. Mobiluncus TEST USE Gram Stain Spores - Not observed in GS smears of clinical specimens or in smears of colonies UNLESS plate OTHER SPECIES OF ANAEROBIC BACTERIA has been incubated for many days C. ramosum, C. clostridioforme - Routinely stained 1. BACTEROIDES FRAGILIS as G- Bacilli Most commonly isolated anaerobe from blood cultures - Vancomycin special - potency antimicrobial Significant cause of intraabdominal abscesses and aerotolerance tests - used to determine B. fragilis + B. thetaiotaocimron - PID & Ovarian Abscess true GS reaction of pink-staining anaerobic Microscopy: Pleomorphic with vacuoles; bacillus Encapsulated Catalase Differentiate Clostridium (Catalase negative) from Biochemical Tests: (+) Catalase, (+) Growth in 20% Bile; Test Bacillus (Catalase positive) Reagent: 15% H2O2 B-Lactamase Producer; Saccharolytic; Non-motile (+): Formation of Bubbles Lipase and Medium: Egg Yolk Agar 2. ACTINOMYCES ISRAELII Lecithinase Lipase - Hydrolyze Diglycerides and Triglycerides Most common cause of Actinomycosis Test to Fatty Acids and Glycerol Microscopy: Thin, Non-sporeforming, Branching G+ o (+): Colony covered in an iridescent Bacilli multicolored sheen ("Mother-of-Pearl" Anatomic Site: Microbiota of the Oral Cavity or Gasoline on water appearance) Diagnosis: Sulfur Granules o Organisms: C. Botulinum, C. novyi type A, C. sporogenes Lecithinase - Cleave lecithin in EYA releasing 3. CUTIBACTERIUM ACNES insoluble fat (Diglyceride) Frequently isolated from blood cultures o (+): Opaque Zone around colonies Presence: contamination from patient's skin from o Organisms: C. perfringens, C. poor site preparation during phlebotomy bifermentans, C. sordelii, C. noyvi type A 4 TRINITY UNIVERSITY OF ASIA – COLLEGE OF MEDICAL TECHNOLOGY | 3MT03 LESSON #15: ANAEROBIC BACTERIA Rarely cause infection because they cannot grow in the 8. VEILLONELLA urine Gram-negative cocci Produce lipases → split free fatty acids from skin lipids → Normal flora of the mouth, nasopharynx & intestines Inflammation → acne Isolated in polymicrobic anaerobic infections Frequent cause of post-surgical wound infections, Rarely a sole cause of infections especially those that involve the insertion of devices (IV lines or phlebotomy sites) 9. BACTERIAL VAGINOSIS 4. LACTOBACILLUS SPP. 9A. GARDNELLA VAGINALIS Gram (+) Pleomorphic rod (Coccoid or Spiral-shaped; Gram-variable-staining rod, facultative anaerobic bacteria Aerotolerant Anaerobe; Non-motile o Cell wall very thin - can appear either Species: L. acidophilus, L. fermentum, L. vaginalis, L. Gram-positive or Gram. negative under the salivarius, L. plantarum microscope Lactobacillus acidophilus Small (1-1.5 pm diameter) non-spore forming, non-motile o Normal flora of the mouth, GIT, Vaginal Canal coccobacilli o Maintain the pH of vaginal area Wet smears → "clue cells" → vaginal epithelial cells with o Important in Female Genitalia - Protect from numerous bacilli but no yeasts or trichomonas Urogenital "Fishy odor" vaginal discharge o Infections (Produce Lactic Acid from Glycogen = Low pH ( erupt to surface and drain pus that may contain Associated with dental, pulmonary, Ob-Gyne & soft tissue "sulfur granules" infections Endocarditis - Lactobacillus P. bivia & P. disiens - occurs in the female genital tract Diabetic Foot Ulcers and Decubitus Pressure sores - Usually caused by G- bacilli 6. PORPHYROMONAS Gingivitis, Meningitis, Lung, Brain Abscesses – by Gram (-) Bacilli Anaerobic cocci Normal oral flora Enophthalmos after cataract surgery; antiseptic Frequently isolated from gingival & peri-apical tooth phlebotomy contaminant – Cutibacterium acnes infections, breasts axillary, perianal & male genital Lemierre Disease - Fusobacterium necrophorum infections Finegoldia magna - most pathogenic anaerobic cocci and the one most often Isolated in pure cultures of 7. FUSOBACTERIUM endocarditis, meningitis, and pneumonia Human infections are caused by F. necrophorum & F. Peptostreptococcus - Reclassified into Anaerococcus, nucleatum Finegoldia, Peptoniphilus F. nucleatum - thin rods with tapered ends → needle-shaped morphology IDENTIFICATION OF Part of the gingival, gastrointestinal & genital microbiota NON-SPORE FORMING ANAEROBIC BACTERIA → F. necrophorum Anaerobic G- bacteria major normal flora of the colon, More virulent; very pleomorphic outnumbering aerobes 1000:1 Virulence factors: Inoculation of organisms start with CAP followed by BAP o Leukotoxin PRAS provides semisolid medium and anaerobic o Hemagglutinin environment o Endotoxin (LPS) Bile Disk Test - Identify B. fragilis (Bile-tolerant) Capable of inducing platelet aggregation SPS Disk Test - Rapid Identification of Cause severe infections of head & neck Peptostreptococcus anaerobius (Sensitive) Lemierre's disease against Peptostreptococcus asaccharolyticus (Resistant) NOTE B. urealyticus require Formate and Fumarate for growth in ALL three species (Prevotella melanogenica, Broth Cultures Porphyromonas, Fusobacterium) are ORAL anaerobic Selective Medium (Anaerobes): Laked bacteria Kanamycin-Vancomycin Agar Kanamycin, Colistin, Vancomycin - Preliminary Antibiotics for testing 5 TRINITY UNIVERSITY OF ASIA – COLLEGE OF MEDICAL TECHNOLOGY | 3MT03 LESSON #15: ANAEROBIC BACTERIA Table No. 5 Preliminary Antibiotics Results KANAMYCIN VANCOMYCIN COLISTIN Bacteriodes R R R REFERENCES fragilis Bacteroides S R S 1. Notes from the discussion by SIR NATHANIEL ureolyticus VERON M. CASAMINA, MSMT, RMT, ASCPI Fusobacterium S R S Porphyromonas R S S Veilonella S R S SUSCEPTIBLE: ≥10mm Zone of Inhibition RESISTANT: 1 month o B. bacilliformis - Inoculation on Columbia Blood Agar with 5% defibrinated blood or hemin-supplemented media after 18 days SERODIAGNOSIS Only test performed to diagnose Rickettsial Confirm diagnosis in convalescent Antibodies to rickettsia (except R. rickettsii) o Not reliably detected until at least 2 weeks after patient becomes ill Cat Scratch disease - confirmed by PCR testing of Table No. 7 Bartonella Lymph Node Aspirates and Biopsies and analysis of fatty SPECIES AGENT OF acid composition B. quntana Trench Fever/Louse-borne disease 1. Indirect Immunofluorescent Antibody (IFA) Assay B. henselae Cat Scratch Disease (Main) o Gold Standard; Scrub Typhus - 1:400 B. elizabethae Infective Endocarditis 2. Weil-Felix Reaction - presumptive test; agglutinate B. bacilliformis Oraya Fever (Chronic Verruga Fever) certain strains of P. vulgaris by serum from patients with B. clarridgeiae Cat Scratch Disease (2nd) Rickettsial diseases o Q Fever, Ehrlichiosis, Rickettsial Pox - do not CAT SCRATCH DISEASE (CSD) / CAT SCRATCH FEVER induce Weil Felix Antibody in infected patients Most common cause: Bartonella henseleae 3. Microimmunofluorescence Dot Test - Excellent 2nd common cause: Bartonella clarridgeiae sensitivity for detecting Rickettsial antibodies; early Other cause: Bartonella quinatana diagnosis of RMSF (7-10 days after symptom onset) Infection via: o Human Transmission - Cat Scratches, Licks, WEIL-FELIX TEST Bites Heterophile agglutination test using Proteus strains to o Cat and Kitten Transmission - Ctenocephalides detect rickettsial antigen felis (Cat Flea) P. vulgaris - source of OX-19 and OX-2 Other species of Bartonella vectors and animal hosts: P. mirabilis - source of OX-2 o Bartonella henselae - Flea, Cat o Bartonella clarridgeiae - Flea, Cat Table No. 8 Weil-Felix Test o Bartonella vinsonii subsp. vinsonii - Car Mits, DISEASE WEIL-FELIX TEST Voles OX-19 OX-2 OX-K o Bartonella vinsonii subsp. berkhoffii - Ticks, Rocky mountain spotted + + - Dogs, Coyotes fever o Bartonella vinsonii subsp. arupensis - Ticks Rickettsialpox - - - Epidemic typhus + - - Endemic typhus + - - Brill-Zinsser disease +1- - + Scrub typhus - - - Trench Fever - - - 3 TRINITY UNIVERSITY OF ASIA – COLLEGE OF MEDICAL TECHNOLOGY | 3MT03 LESSON #17: RICKETTSIACEAE, ANAPLASMATACEAE, COXIELLACEAE REFERENCES 1. Notes from the discussion by SIR NATHANIEL VERON M. CASAMINA, MSMT, RMT, ASCPI Table No. 9 Summary of Arthropod-Borne Bacterial Infection ORGANISM DISEASE VECTOR/MODE OF TRANSMISSION INCUBATION PERIOD 1. Spotted Fever Group Rickettssia conorii Buotenneus Fever or Ticks (Rhipcephalus sanguineus) Mediterranean Spotted Fever Rickettsia rickettsii Rocky Mountain Spotted Fever Wood Ticks (Deracentor andersoni) Approximately 7 (RMSF) Dog Ticks (Dermacentor variabilis) days Brown Dog Ticks (Rhipicephalus sanguineus, Ambyloma cajennense) Rickettsia akari Rickettsial Pox Mouse Mite (Liponyssoides sanguineus) Approximately 10 days 2. Typhus Group Rickettsia prowazekii Epidemic Typhus/Brill-Zinsser Louse (Pediculus humanus corporis) Approximately 7 Disease/ Recrudescent Squirrel Flea (Orchopeas howardii) days Typhus/Flying Squirrel Typhus Squirrel Louse (Neohematopinus sciuriopteri) Ricketssia typhi Endemic Murine Typhus Rat Fleas (Xenopsylla cheopis) 3. Scrub Typhus Group Orientia tsutsugamuchi Scrub Typhus Chigger Bite (Letotrombidium deliense) Approximately 2 weeks 4. Ehrlichia Ehrlichia chaffeensis Human Monocytic Ehrlichiosis Lone Star Tick (Amblyomma americanum) 5 - 10 days (Resembles RMSF) Ehrlichia ewingii Canine Ehrlichiosis Tick Bite Anaplasma phagocytophilum Human Granulocytotropic Deer Tick (Ixodes scapularis, Ixodes pacificus) 5 - 11 days Anaplasmosis Neorickettsia sennetsu Sennetsu Fever Not Known 5. Other Rickettsial Fever Rickettsia akari Rickettsial Pox Gramasid Mite (Liponyssoides sanguineus) Rickettsia felis Flea-brone spotted Fever Flea Bite or Feces 6. Coxiella burnetii Q Fever Inhalation of Aerosol from infected animals 7. Bartonella quintana Trench Fever Feces of Pediculus humanus corporis 8. Bartonella henselae Cat Scratch Disease: Bacillary Kitten Scratch or Bite Angiomatosis 9. Bartonella bacilliformis Oraya, Fever, Verruga Peruana Sandfly (Lutzomyia) bite 4 TRINITY UNIVERSITY OF ASIA – COLLEGE OF MEDICAL TECHNOLOGY | 3MT03  MICR_111 NATHANIEL VERON M. CASAMINA, MSMT, RMT, ASCPI | 3MT03 3RD YEAR, 1ST SEMESTER BY: MANALO, A. & MILAR, K. [TRANS] LESSON #18: CHLAMYDIACEAE OUTLINE Table No. 2 Biovars and Serovars 3 BIOVARS 3 SEROVARS (BASED ON MOMP I. Chlamydiaceae ANTIGENIC DIFFERENCES) Trachoma A, B, Ba, C - Endemic Trachoma A. Chlamydia trachomatis Lymphogranuloma L1, L2, L2a, L2b, L3 - Lymphogranuloma 1. Clinical Infections Venereum (LGV) Venereum 2. Laboratory Diagnosis Mouse Pneumonitis D-K, Da, la, Ja - PIS, Urethritis, Cervicitis, B. Chlamydophila psittaci (Former Epididymitis, Inclusion Conjunctivitis Chlamydia psittaci) C. Chlamydophila pneumoniae D. Differential Characteristics CHLAMYDIACEAE Non-motile, small (0.2-1.5um), resemble Gram-negative cell wall, obligate intracellular parasites requiring living CLINICAL INFECTIONS cells for growth. o Also known as an “Energy Parasites” Table No. 3 Clinical Infections o Does not possess Cytochromes DISEASE DESCRIPTIONS ▪ Cytochromes - Important for energy Trachoma Chronic inflammation of the production conjunctiva leading to ▪ It is an obligate intracellular parasite blindness because it needs a eukaryotic host cell Cause distortion of the eyelids; eyelashes become misdirected to generate its own ATP. and turn in Have tropism for columnar epithelial cells Transmission: fomites, Species: Chlamydia trachomatis, Chlamydophila hand-to-hand contact with an psittaci, Chalymophila pneumoniae infected patient, by flies Repeated exposure: Chronic Table No. 1 2 Morphological Forms Follicular 2 MORPHOLOGICAL FORMS Keratoconjunctivitis, Reticulate Replicative, Non-infectious Form Conjunctival scarring, Pannus Body (RF) Intracellular, metabolically active form Lymphogranuloma Sexually transmitted diase Diverts host cell’s functions to their own metabolic Venereum Has a multisystem involvement needs Initially a small, painless, ulcer Elementary Infectious Form or papule (pimple-like) and Body (EB) Extracellular form; spherical; dense; resembles G- develops as nodules (buboes) bacilli; has rigid cell wall after several weeks. Infects host cells by requiring active phagocytosis Intradermal Skin Test: Frei’s 2 Components of outer membrane: MOMP Test (Transmembrane protein), LPS Antigen Inclusion Conjunctivitis Usually infects infants Copious, yellow discharge from the eye, an inflamed and swollen conjuctiva, edematous eyelids presence of large inclusion bodies in host cell 1. CHLAMYDIA TRACHOMATIS cytoplasm with an incubation period of 4-5 days Major cause of Pelvic Inflammatory Disease (PID) and Ocular Trachoma One of the major Sexually Transmitted Pathogens Can travel through birth canal (Can Infect Infants) Associated with infertility and ectopic pregnancy Natural Hosts: Humans Unique Feature: 10 stable plasmids 1 TRINITY UNIVERSITY OF ASIA – COLLEGE OF MEDICAL TECHNOLOGY | 3MT03 LESSON #18: CHLAMYDIACEAE LABORATORY DIAGNOSIS 2. CHLAMYDOPHILA PSITTACI (FORMER CHLAMYDIA PSITTACI) Table No. 4 Clinical Infections Causative agent of Psittacosis or Ornithosis LAB DESCRIPTIONS Endemic pathogen of all bird species (Parrots, DIAGNOSIS Parakeets, Chicken, Ducks) Specimens Swabs - Dacron or Rayon tipped swabs Had outbreaks among turkey-processing workers and Urethra and Cervical secretions, pigeon aficionados - can only be cultivated under BSL-3 Conjunctiva discharges, NPS and Rectal conditions. swabs; Fallopian Tube and Epididymis Chlamydiophila felis, Chlamydophila caviae, aspirates Chlamydophila abortus - C. psittaci strains that cause Culture Reference Method: Cell Culture conjunctivitis, rhinitis on cats guinea pig conjunctivitis, and McCoy cells, HeLa 229, Buffalo Green abortion among ruminants. Monkey, Kidney Cells, Cycloheximide-treated Acquisition: Inhalation of infected aerosols from dried McCoy cells Prior to culture: Shell vial technique bird excreta or handling infected birds. (Centrifuge specimen onto cell monolayer, Diagnosis: organisms are growing on a coverslip in the o Complement Fixation: Tite > 1:32 (Presumptive bottom of the vial) Psittacosis) After 48-72 hours: monolayers stained with o Direct Microimmunofluorescence: Sensitive iodine or IF stains to examine the presence of Method inclusions o PCR-RFLP: Identify and distinguish all 9 Others Cytologic Examination - cell scrapings from chlamydial spp. the conjunctiva of newborns or persons with ocular trachoma Enzyme Immunoassay - most rapid antigen assay o Detects either outer membrane LPS chlamydial antigen or the MOMP Antigen More reliable in patients with symptomatic and 3. CHLAMYDIA PNEUMONIAE Detection and shedding large numbers of organism Formerly Chlamydia penumoniae - TWAR (Taiwan Nucleic Acid DFA staining method using Acute Respiratory Agent) strain Amplification Fluorescein-Isothiocyanate-conjugated o TWAR-183: Taiwanese child with Trachoma Monoclonal Antibodies: Identify in infected o AR-139: Patient with Pharyngitis cells Human Pathogen; Transmission via aerosol droplets via Chemiluminescent DNA probe, ELIS, DFA: the respiratory route Detect chlamydial antigen 3rd most common cause of infectious respiratory NAATs: Most sensitive method for detection disease LPS Antigen: Major antigen detected Associated with pneumonia, bronchitis, pharyngitis, Specimen: Endocervical or Urethral Swabs sinusitis Serodiagnosis Primary antigen detected: LPS with Isolated from patients with Otitis media with effusion Ketodeoxyoctonated (EB) Culture Media: HL or Hep-2 cell lines If negative in sero tests: Reliably exclude Method of Choice: Micro-IF assay (Sensitive and chlamydial infection COMPLEMENT FIXATION: Specific) o Detect family-reactive antibody; used to diagnose LGV o (+): Single point titer > 1:64 (presumptive for LGV) MICROIMMUNOFLUORESCENCE ASSAY (MICRO-IF) o Used for type 0 specific antibodies for C. trachomatis o Used to diagnose LGV, Trachoma, Inclusion conjunctivitis REFERENCES o (+): IGM titer of 1:32 1. Notes from the discussion by SIR NATHANIEL VERON M. CASAMINA, MSMT, RMT, ASCPI 2 TRINITY UNIVERSITY OF ASIA – COLLEGE OF MEDICAL TECHNOLOGY | 3MT03 LESSON #18: CHLAMYDIACEAE CHLAMYDIACEAE: DIFFERENTIAL CHARACTERISTICS Table No. 5 Differential Characteristics PROPERTIES C. TRACHOMATIS (GROUP A) C. PSITTACI (GROUP B) C. PNEUMONIAE Natural Hosts Humans Birds, Lower Animals Humans Elementary Body Morphology Round Round Pear-shaped Inclusion Morphology and Bodies Round, Vacuolar, Variable, Dense, Round, Dense **Halbertstadler-Prowazek **Levinthal-Cole-Lillie** bodies*** Stains used Lugol’s Iodine Machiavelo and Giemsa Giemsa Glycogen-containing inclusions Present Absent Absent Sulfonamide susceptibility Susceptible Resistant Resistant Diseases Trachoma, LGV, Inclusion Psittacosis Pneumonia, Pharyngitis, Bronchitis conjunctivitis Number of serovars 20 10 1 3 TRINITY UNIVERSITY OF ASIA – COLLEGE OF MEDICAL TECHNOLOGY | 3MT03  MICR_111 NATHANIEL VERON M. CASAMINA, RMT, MSMT, ASCP | 3MT03 3RD YEAR, 1ST SEMESTER BY: MILAR, K. [TRANS] LESSON #19: CELL WALL DEFICIENT BACTERIA OUTLINE INFECTION IN HUMANS I. Cell Wall Deficient Bacteria Table No. 1 Infection in Humans II. Infection in Humans ORGANISM INFECTION IN HUMANS III. Mycoplasma pneumoniae Mycoplasma pneumonia Pharyngitis, Tracheobronchitis, Primary Atypical Pneumonia IV. Mycoplasma hominis, Ureaplasma urealyticum Mycoplasma hominis Postpartum Fever, Pyelonephritis V. Other Mycoplasma Species Mycoplasma genitalium Non-Gonococcal Urethritis, Pelvic A. Mycoplasma genitalium Inflammatory Disease B. Mycoplasma fermentans Ureaplasma urealyticum Non-Gonococcal Urethritis, VI. Laboratory Diagnosis Pyelonephritis, Premature Birth, VII. Culture Abortion VIII. Pathogens in Class Mollicutes IX. Treatment and Prevention of Mycoplasmas MYCOPLASMA PNEUMONIAE (EATON AGENT) X. Comparison between Characteristics of First isolated from a cow with pleuropneumonia in the Common and Atypical Bacteria 1800s Named Pleuropneumonia-like organism (PPLO) when first isolated from humans CELL WALL DEFICIENT BACTERIA "Eaton Agent" - From Monroe Eaton (Embryonated Class Mollicutes ("Soft") - Smallest self-replicating Chicken Eggs for cultivation) organisms, formerly thought as viruses (Lack a Cell Wall) Agent of Primary Atypical Pneumonia (PAP)/Walking Pleomorphic - Coccold to Rods Pneumonia May grow on artificial cell-free media; requires complex o Waste Products: H O., Ammonia - Ciliostasis and media sloughing of superficial cells o Unlike Rickettsia and Chlamydia which require May be associated with neurologic and systemic live cell culture media symptoms Major Antigenic Determinants: Membrane Glycolipids & P1 Protein - For attachment Proteins cross-reacting with human tissues and other o Interacts with neuraminic acid-containing bacteria glycoproteins (Ciliostasis) → Destroy Epithelial Important families Cells → Inhibit bacterial clearance → Irritation of o Mycoplasmataceae - Mycoplasma, LRT → Persistent cough Ureaplasma Initiation of disease: Attach to Respiratory Mucosal o Acholeplasmataceae - Acholeplasma (A. Cells; Escape Phagocytosis, modulate immune system laidiawii) o Spread via respiratory droplets Species: M. pneumoniae, M. hominis, M. fermentans. M. o Cause outbreaks in closed populations pirum, M. penetrans, M. genitalium, U. urealyticum, A. (families, military recruit camps) laidiawii) Contains Sterol (Cholesterol) for membrane function and growth Cell-wall deficiency: o Resistant to cell-wall antibiotics (Penicillin, Cephalosporins, Vancomycin) o Prevents staining by Gram Staining; Can pass MYCOPLASMA HOMINIS, UREAPLASMA UREALYTICUM thru 0.45 um filters (GENITAL MYCOPLASMA) o Slow-growing (Generation time: 1-16 hours), Cause infections of the Urogenital tract and might play a highly fastidious, facultative anaerobes role in bacterial vaginosis (Asymptomatic Sexually active ▪ M. preumonice - Strict Aerobe individuals) ▪ M. hominis - More rapidly growing Recovered also from nose and throats of infants Mycoplasma o Occur through an infected birth canal Requires cholesterol and fatty acids for growth Can cause invasive disease in immunocompromised patients (Agammaglobulinemia), Prostatitis, Bacterial Vaginitis, Non-Gonococcal Urethritis Mycoplasma hominis colonial characteristic - "Fried Egg" appearance; Seen in 50% of cases of NGU o Cervicitis occur more in patients with HIV than those without o More frequently causing infection in urethra and rectum of homosexual men with HIV infection 1 TRINITY UNIVERSITY OF ASIA – COLLEGE OF MEDICAL TECHNOLOGY | 3MT03 LESSON #19: CELL WALL DEFICIENT BACTERIA Mycoplasma hominis - cause postabortal and postpartum fever Ureaplasma urealyticum, Ureaplasma parvum - Do not cause disease in the female lower genital tract o U. urealyticum - Has the ability to metabolize Urea (T-Strain of Mycoplasma) o U. urealyticum - NGU in men (U. parvum has not) o U. urealyticum - Isolated from tracheal aspirates CULTURE of low-birth-weight infants with respiratory Culture Media: Beef/Soybean Protein with serum, Fresh diseases, causes Arthritis, Cystitis Yeast Extract, Biphasic SP-4, PPLO Broth/Agar with Yeast Extract and Horse Serum, Modified NYC Medium pH Requirement: 5.5-6.5 (Ureaplasma, Seen in Shepherd 10B Arginine Broth), and 6-8 (Mycoplasma) Mycoplasma - "Fried Egg colonies" on plate Ureaplasma - Dark Brownish clumps on A7/A8 Agar (Solid) M. hominis - Stained with Dienes or Acridine Orange; (-) OTHER MYCOPLASMA SPECIES Urease M. pneumoniae - Requires biphasic medium (sealed MYCOPLASMA GENITALIUM container up to 3 weeks in 5-10% CO) Associated with NGU, Cervicitis, Endometriosis, PID Glucose (Dextrose) incorporated in media selective for M. Linked to tubal sterility pneumonia and M. genitalium Found to be more frequently in urethral samples taken Urea and/or Arginine - incorporated in media to detect U. from men with acute NGU than in those from men without urealyticum and M. hominis Urethritis SPS: Inhibitory to mycoplasma (Overcome inhibition: 1%) Lesser linked to HIV than M. hominis MYCOPLASMA FERMENTANS Respiratory opportunistic pathogen (Throats with LRT infections) Also in AIDS patients with respiratory illness Isolated from synovial fluid of Rheumatoid Arthritis patients PATHOGENS IN CLASS MOLLICUTES Also in urine of homosexual males with HIV Table No. 2 Pathogens in Class Mollicutes FEATURE MYCO- UREA- ACHOLE- PLASMA PLASMA PLASMA Cell Wall + + + Deficient Gram Stain, - - - Susceptibility to Penicillin LABORATORY DIAGNOSIS Urease - + - SPECIMEN Activity o M. pneumoniae - Throat swab, Serum, BAL, Exists in nature - - + Sputum, Lung Tissue as free-living o Genital Mycoplasma - Urethral, Vaginal or organism Endocervical swab, Blood, Urine, Prostatic Pleomorphic + + - shape secretions, Semen Other shared 1. Smaller than other bacteria (close size NO DIRECT METHOD FOR IDENTIFICATION! characteristics to Myxoviruses) DEFINITIVE IDENTIFICATION OF M. pneumoniae 2. Smaller genome than other bacteria o Overlaying suspicious colonies with 0.5% Guinea 3. Lower GC ratio than most bacteria pig RBC in PO, buffered saline 4. Limited metabolic activity SERODIAGNOSIS 5. Many mollicutes contain DNase o ELISA - Most widely used method (Detect IgM in serum sample) TREATMENT AND PREVENTION OF MYCOPLASMAS ▪ IgM is for present infection Drugs of choice: Erythromycin, Doxycycline, Newer RAPID IDENTIFICATION: Manganous Chloride Urea Fluoroquinolones Test (U. urealyticum) Immunity is not lifelong o Reaction is observed under a dissecting Vaccines are ineffective microscope o Unlike in other causative agents of pneumoniae o (+): Dark Brown precipitate of Manganous that have vaccines Chloride around colonies o U. urealyticum utilize Manganous Chloride in the presence of Urea 2 TRINITY UNIVERSITY OF ASIA – COLLEGE OF MEDICAL TECHNOLOGY | 3MT03 LESSON #19: CELL WALL DEFICIENT BACTERIA REFERENCES 1. Notes from the discussion by SIR NATHANIEL VERON CASAMINA, MSMT, RMT, ASCP COMPARISON BETWEEN CHARACTERISTICS OF COMMON AND ATYPICAL BACTERIA Table No. 3 Comparison between Characteristics of Common and Atypical Bacteria CHARACTERISTICS BACTERIA RICKETTSIACEAE CHLAMYDIACEAE MYCOPLASMAS Obligate Intracellular - + + - DNA and RNA + + + + Contains Ribosomes + + + + Peptidoglycan in Cell Wall + - + - Replication by Binary Fission + + + + Growth on Non-living Medium + - - + Sensitivity to Antibiotics + + + + Sensitivity to Interferon - - + - 3 TRINITY UNIVERSITY OF ASIA – COLLEGE OF MEDICAL TECHNOLOGY | 3MT03  MICR_111 NATHANIEL VERON M. CASAMINA, MSMT, RMT, ASCP | 3MT03 3RD YEAR, 1ST SEMESTER BY: MANALO, A. & MILAR, K. [TRANS] LESSON #20: SPIROCHETES OUTLINE TREPONEMA SPP. Greek word: “Turning Thread” I. Spirochetes Dark Field Microscopy II. Treponema spp. Only infects humans A. Treponema pallidum subsp. pallidum Killed rapidly at 4C (Blood Bags): Bases for Syphillis B. Laboratory Diagnosis therapy III. Syphilis Remain visible in whole blood or plasma for at least A. Stages of Syphilis 24 hours (Blood Transfusion): Cold Sterilization B. Congenital Syphilis Technique C. Serodiagnosis for Syphilis Stain poorly with Gram’s stain or Giemsa stain IV. Non-Treponemal Serodiagnostic Tests Species: T. pallidum, T. carateum V. Other Species of Treponema spp. A. Treponema pallidum subsp. pertenue B. Treponema pallidum subsp. endemicum C. Treponema carateum D. Treponema denticola, Treponema TREPONEMA PALLIDUM SUBSP. PALLIDUM socranskii Agent of Syphillis VI. Borrelia spp. (Blood Spirochetes) Microaerophllic (3-5% Oxygen) A. Borrelia recurrentis Composed of Phospholipids Bilayer and Protein Antigens B. B. hermsii, B. turicatae, B. cutoni, B. (Outer Membrane) parkeri Has the ability to cross intact mucous membranes and C. Borrelia burgorderi sensu stricto, placenta Borrelia garinii, Borrelia afzelii Inhibited rapidly by heat and dryness; susceptible to D. Pathogenesis/Diseases disinfectants E. Laboratory Diagnosis Has tropism to arterioles VII. Leptospira spp. Microscopy: Long, slightly larger than RBCs, fine spirals A. Pathogenesis with 10-13 coils and 3 periplasmic flagella B. Laboratory Diagnosis Generation Time: 30 hours C. Other Tests Antibodies produced to T. pallidum subsp. Pallidum: o Treponemal o Nontreponemal (Reagin) SPIROCHETES Order Spirochaetales Unusual Morphologic Features - Long, Slender, Helically-curved unicellular bacteria Various types of motility patterns because of fibrils/axial filaments/periplasmic flagella (corkscrew winding) Multilayered outer sheath (similar to G- outer membrane) LABORATORY DIAGNOSIS OF TREPONEMA SPP surrounds protoplasmic cylinder. Table No. 2 Laboratory Diagnosis of Treponema spp. Generally GRAM-NEGATIVE LABORATORY DESCRIPTION DIFFERENTIATION OF SPIROCHETES GENERA: DIAGNOSIS o Number of axial fibrils Specimen Skin Lesions (Clean with saline) by o Number of insertion disks (Plate-like structure dark-field examination or fluorescent where fibrils are attached, located near the antibody staining terminal of the cell) present Oral Lesions - Not examined o Biochemical and Metabolic features (Nonpathogenic Spirochetes lead to False positive results) Table No. 1 Microscopic Direct examination of exudates by CHARACTERISTICS TREPONEMA BORRELIA LEPTOSPIRA Examination dark-field microscopy to observe Number of spirals 4-14 3-10, loose Tightly coiled motility for definitive identification Axial Filaments 3 15-20 2, long (Corkscrew) Insertion Disk 1 2 o Diagnostic for 1’ syphilis if Motility Corkscrew No specific Rapid motile treponemes seen from pattern translational, the chancre specimen rotational Stains used: Levaditi’s Impregnation (spinning) Stain or Fontana Tribondeau Other Cell ends Stain well with 2 Periplasmic Characteristics pointed, covered Giemsa, flagella, Direct Detection in Lesions: with sheath Visualize by “Question FITC-labeled antibodies Bright0Field mark-like” shape 1 TRINITY UNIVERSITY OF ASIA – COLLEGE OF MEDICAL TECHNOLOGY | 3MT03 LESSON #20: SPIROCHETES Histologic Hallmark: Endarteritis showing CONGENITAL SYPHILIS concentric endothelial and fibroblastic Transmission: Beginning in the proliferation with mononuclear cell infiltrate 10th - 15th weeks AOG rich in plasma cells. Early Congenital: Rhinitis + Molecular Test PCR (Neurosyphilis on AIDS patients) Desquamating maculopapular Western Blot: Detection of Congenital Syphilis rash Late Congenital: Interstitial Keratitis, Hutchinson Teeth, Saddle Nose, Mulberry molars, Periostitis, CNS Anomalies, Saber Shins, Symmetrical swelling SYPHILIS (FRENCH/ITALIAN DISEASE/THE GREAT POX) (Clutton’s joints) Also known as the “Great Imitator” Infect blood vessels and perivascular area; can cross SERODIAGNOSIS FOR SYPHILIS placenta Basis of diagnosis especially in tertiary syphilis Untreated: T. pallidum subsp. Pallidum disseminates to Antibodies against STORCH (specifically IgM) - diagnostic other parts of the body (ie. Bones) - ⅓ may exhibit for neonatal infection “biological cure” Can cause co-infection with HIV NON-TREPONEMAL (NON-SPECIFIC/SCREENING) TEST Transmission: Sexual contact/Congenital; Skin Detect presence of non-specific antibodies or contact with active infection (primary or secondary); antibody-like protein (Reagin or Wassermann Transfusion of fresh blood; Needle stick; Specimen Antibodies) to Lipoprotein material from damaged cells handling and cardiolipin from treponemes Symptoms: Hard chancre, fever, sore throat, headache, Used to monitor treatment - Demonstrate rise and fall of rash (palms and soles), gummas on skin titers o Chancre - Single, Erythematous, Painless lesion Tests: Rapid Plasma Reagin (RPR) Test, VDRL Test, that is non-tender, snd firm with a clean surface Unheated Serum Reagin (US), Toluidine Red Unheated and raised border Serum Test (TRUST), ELISA Drug of choice: Penicillin Sample: CSF or Serum o Early stages: Long-acing Benzathine Penicillin Reagent: Cardiolipin (Phospholipid) + Lecithin + o Congenital and Late Syphillis: Penicillin G Cholesterol + Choline Chloride + Charcoal (Neuro: Penicillin) (+): Flocculation o Alternatives: Tetracycline, Doxycycline TREPONEMAL (SPECIFIC/CONFIRMATORY) TEST Detects presence of antibodies to treponemal antigens FTA-ABS (Indirect Fluorescent Antibody Test) o Overlaying whole treponemes (T. pallidum Nichols strain) fixed in a slide with a heated STAGES OF SYPHILIS serum from patients suspected of having syphilis due to previous (+) VDRL or RPR test Table No. 3 Stage of Syphilis o (+): Fluorescence of the Treponemes STAGE DESCRIPTION Treponema pallidum Particle Agglutination Primary Symptoms: Chancre (Hunterian chancre), o Uses Gelatin particles sensitized with T. pallidum Syphilis Infectious lesion (usually at the site), Most antigens commonly at the genitalia Develops 10-90 days after infection No systemic s/s at this stage Secondary Develops 2-12 weeks after the appearance Syphilis of primary lesion Symptoms: Fever, sore throat, weight loss, headache, rash (palms and soles) All lesions are highly infectious. Latent Stage Disease becomes subclinical but not (Silent necessarily dormant Stage) Diagnosis ONLY by Serological Tests Tertiary Tissue Destructive Phase Stage/Late Appears 10-15 years after initial infection Syphilis Patients usually NOT infectious in this stage Complications: CNS Disease (Neurosyphilis, Eye Disease, Cardiovascular Abnormalities , Granuloma-like lesions (Gummas) 2 TRINITY UNIVERSITY OF ASIA – COLLEGE OF MEDICAL TECHNOLOGY | 3MT03 LESSON #20: SPIROCHETES NON-TREPONEMAL SERODIAGNOSTIC TESTS BORRELIA SPP. (BLOOD SPIROCHETES) RPR test - Detect antigen coated with Stain well with Giemsa stain, visualized by Bright-field Carbon (Most common) Microscopy; slow-growing Spirochetes o Does not require heating, not Multiply by Binary Fission recommended for CSF In vitro Borrelia are microaerophilic VDRL - Recommended for diagnosis of Species: B. recurrentis, B. dutoni, B. hermsi, B. turicata, B. Neurosyphilis using CSF parkeri, B. afzeli, B. burgdorferi, B. garinii o Reagents must be freshly prepared o Patient's serum heated at 56C for 30 minutes (Complement inactivation) IF REACTIVE OR WEAKLY REACTIVE - Undergo Titration and tested with Treponemal tests False Positive RPR and VDRL: Old age, Pregnancy, BORRELIA RECURRENTIS Hepatitis, Rheumatic Fever, SLE, Infectious Agent of Louseborne or Epidemic Relapsing Mononucleosis Fever/European Relapsing Fever Vector: Pediculus humanus (Louse) OTHER SPECIES OF TREPONEMA SPP. Reservoir: Humans TREPONEMA PALLIDUM SUBSP. PERTENUE B. HERMSII, B. TURICATAE, B. CUTONI, B. PARKERI Agent of YAWS/Frambesia Tropica/Pian/Parangi/Paru/ Agent of Tickborne Relapsing Fever/Endemic or Buba/Bouba American Relapsing Fever o Yaws - Non-venereal infection (chronic Vector: Ornithodoros (Soft Ticks) ulcerative sores anywhere on the body with eventual tissue and bone destruction); may lead to crippling if untreated Acquired by direct contact through breaks of skin Lesions: 1. Primary Lesion - Mother Yaws/Frambesia 2. Secondary Lesion - Daughter Yaws BORRELIA BURGDORFERI SENSU STRICTO, 3. Tertiary Lesion - Gangosa BORRELIA GARINII, BORRELIA AFZELII Agents of Lyme Disease TREPONEMA PALLIDUM SUBSP. ENDEMICUM Vector: Ixodes (Hard Ticks) - Ixodes pacificus, ixodes Causative agent of endemic non-venereal scapularis, Ixodes persulcatus, ixodes dammini Syphilis/Bejel Transmission: Bite of the Ixodes Ticks o Bejel - Non-venereal form of Syphilis prevalent in Ticks' Natural Hosts: Deer and Rodents (Peromyscus Middle Eastern and North African children leucopus - White-footed mouse) (Primary lesion on or near the mouth, pimple-like All stages of ticks (Larva, Nymph, Adult) - Harbor sores on trunk, arms, legs) spirochete and transmit disease Transmission: Direct contact with active lesions, Maintained in the environment by horizontal transmission contaminated fingers and utensils from infected nymphal ticks TREPONEMA CARATEUM Causative agent of Pinta/Carate/Mal De Pinto/Azul PATHOGENESIS/DISEASES FOR BORRELIA SPP. o Pinta - Skin infection; Primary lesion is a slowly enlarging papule with regional lymph node RELAPSING FEVER enlargement, followed in 1-12 months by a Acute infectious disease marked by recurrent febrile generalized red to slate-blue macular rash episodes (2-10 eps) Acquired through contact with infected skin Only spirochetal disease where the organisms are visible in blood with bright-field microscopy TREPONEMA DENTICOLA, TREPONEMA SOCRANSKII Symptoms: Fever, Headache, Myalgia (2-15 days after Causes Ulcerative Gingivitis and Chronic Periodontitis infection) Related to T. pallidum Fever persists for 2-6 days, patient appears to recover, only to relapse days or weeks later Relapse - due to Borrelia to alter its antigenicity, host will develop "new" immunity to each altered strain 3 TRINITY UNIVERSITY OF ASIA – COLLEGE OF MEDICAL TECHNOLOGY | 3MT03 LESSON #20: SPIROCHETES LYME DISEASE LEPTOSPIRA SPP. Acute, Recurrent Inflammatory infection involving the Obligate Aerobic Spirochetes, can be grown on Artificial large joints (knees), with local inflammation, Erythema media migrans (Bull's Eye appearance), and swelling Cannot be readily stained (Impregnate with Silver stain) First observed: 1975 at Old Lyme, Connecticut, USA Live in the Lumen of the Renal Tubules (shed into the urine) 3 STAGES OF LYME DISEASE Can survive in the neutral or slightly alkaline waters for 1. First Stage (Erythema Migrans) months o Characteristic red, ring-shaped lesion with a Animal of choice for cultivation: Hamsters, Guinea Pigs central clearing (appear at site of tick bite or other Species: L. interrogans (Pathogenic), L. biflexa sites) Microscopy: Thin, Flexible organisms, "Question o Presence of Erythema Migrans is mark-like" shape - both ends have hooks rather than DIAGNOSTIC! tapering off; Often seen as chain of cocci (Dark-Field o S/S: Headache, Fever, Muscle and Joint pain, Microscopy) Malaise Virulence Factor: Hemolysin 2. Second Stage Generation Time: 6-16 hours o May start weeks to months after infection; Animal Reservoirs: Rats and Dogs dissemination of the organisms o Symptoms: Meningitis, Nerve Palsy ("Bannwarth Syndrome") 3. Third Stage o Appearance of chronic arthritis, may continue for years o Patients may develop demyelination of neurons with symptoms of Alzheimer's Disease and Multiple Sclerosis PATHOGENESIS FOR LEPTOSPIRA SPP. LABORATORY DIAGNOSIS FOR BORRELIA SPP. LEPTOSPIROSIS (INFECTIOUS JAUNDICE) MICROSCOPIC EXAMINATION Zoonotic disease in humans by L. interrogans Darkfield Microscopy, used for detection in blood Enter host through breaks in the skin, mucous cultures after 2-3 weeks of incubation at 34-37C membranes, or conjunctiva Relapsing Fever Organism invades bloodstream, spread throughout CNS o Specimen: Peripheral blood and Kidney o Stained with Wright's or Giemsa stains, Pretibial rash - Associated with Serovar autumnalis (Fort organism is blue-colored Bragg Fever) o In wet preparations (1:1 blood:NSS) IgM detected within a week after disease onset Lyme Disease Mode of Acquisition: Contact with urine of carriers, o Specimen: Blood, Biopsy, CSF Indirectly by contact with bodies of water contaminated o Tissue section: Warthin-Starry stain with urine of carriers o Blood, CS: Acridine Orange or Giemsa Stain Symptoms: Fever, Headache, Myalgia, Anorexia, Vomiting CULTURE TYPES OF LEPTOSPIROSIS Kelley's Medium (Barbour-Stoenner-Kelly Medium), or Anicteric Leptospirosis Chick Embryo o Symptoms: Septicemic stage, High fever, Agents of Relapsing Fever cultured in nutritionally-rich severe headache (3-7 days), followed by the media under microaerophilic conditions immune stage Organism: Slow-grower, 7-14 days at 35C o Hallmark of Immune Stage: Aseptic Meningitis SERODIAGNOSIS, MOLECULAR TESTS Icteric Leptospirosis/Weil Disease Relapsing Fever - Increased titers in Proteus OX K o Severe form of the illness (Liver, Kidney, or Antigens (Up to 1:80) Vascular dysfunction Lyme Disease - Serology is the standard method for o Death occur in 10% of cases diagnostic testing o IgM and IgG are detected o Tests: ELISA, IFA (First-line test for Lyme Disease antibody) Molecular Tests - PCR (B. burgdorferi DNA in Urine) 4 TRINITY UNIVERSITY OF ASIA – COLLEGE OF MEDICAL TECHNOLOGY | 3MT03 LESSON #20: SPIROCHETES LABORATORY DIAGNOSIS FOR LEPTOSPIRA SPP. SPECIMEN, MICROSCOPY SPECIMEN o Blood, CSF, Tissues (Bacteremic Phase), Urine (Immune Phase) o Urine - obtained after 2 weeks of illness or up to 30 days after onset of symptoms MICROSCOPIC EXAMINATION o Detection of motile Leptospires in the specimens by dark-field microscopy CULTURE Fletcher's or Ellinghausen-McCullough-Johnson- Harris (EMJH) Medium, Bovine Serum Albumin, Stuart Broth, Noguchi's Medium Urine inoculated immediately (toxic to spirochetes) Few drops of patient's blood (Oxalate, Heparin) directly inoculated onto Fletcher or EMJH, incubate in the dark for 4-6 weeks at 25-30C Organisms grow below the surface - Examined weekly for growth Differentiate from saprophytes (saprophytes grow at 10C and lower) OTHER TESTS FOR LEPTOSPIRA SPP. SERODIAGNOSIS o Four-fold or greater rise in titer of agglutinating antibodies o Diagnosis of Leptospirosis made by demonstrating seroconversion o ELISA, RIA, Immunomagnetic Capture - Detect Antigens in most specimens o IF, IHC - Detect antigens in tissues o Microscopic Agglutination - Reference method using living cells MOLECULAR TESTS (Leptospiral DNA) o PCR, Hybridization Tests REFERENCES 1. Notes from the discussion by SIR NATHANI