Respiratory Module Lab 1: Bacterial, Viral, & Fungal Upper Respiratory Tract Infections PDF

Respiratory module laboratory diagnosis of selected cases of bacterial, viral and fungal upper respiratory tract infection Lab 1 in microbiology for respiratory module, medical students Dr. Haneya Anani Prof. of Med...

Respiratory module laboratory diagnosis of selected cases of bacterial, viral and fungal upper respiratory tract infection Lab 1 in microbiology for respiratory module, medical students Dr. Haneya Anani Prof. of Medical Microbiology and Immunology Faculty of Medicine Prof. Haneya Anani Learning Outcomes On completion of this lab, the student will be able to: 1. To know the morphology and culture characters of Strept. pyogenes 2. To know the laboratory diagnosis of acute follicular tonsillitis 3. To know the laboratory diagnosis of Diphtheria, Vincent’s angina and oral candidiasis 4. To know the laboratory diagnosis of viruses causing respiratory tract infection Bacteria causing upper respiratory Viruses causing upper respiratory tract tract infections infections 1. Acute follicular tonsilitis 1. Adenovirus 2. Scarlet fever 2. Rhinoviruses 3. Diphtheria 3. Corona viruses 4. Vincent’s angina 4. EBV 5. Otitis media 5. Parainfluenza type 1 and 2 6. Respiratory syncetial virus in adult & children 7. Herpes simplex Upper respiratory tract infections Candidiasis  Hints about streptococci β-haemolytic streptococci Aerobic and facultative anaerobes are classified according to hemolysis on blood agar : Beta-haemolytic streptococci that produce complete lysis of red blood cells with release of haemoglobin due to production of streptolysin S Alpha-haemolytic streptococci that cause incomplete lysis of red blood cell with formation of green pigments Non-haemolytic streptococci The ß-haemolytic streptococci are further classified into serologic groups on the basis of carbohydrate cell wall antigens (Lancefield groups A- U). Group A, B, C, D, and G cause human disease. prof. Haneya Anani 4 Diagnosis of tonsilitis caused by Streptococcus pyogenes Specimens: throat swab Direct gram stained film showing gram positive cocci arranged in chains among pus cells Culture on A: B-haemolysis Best growth is achieved on blood agar B: α-haemolysis & chocolate agar. C:No haemolysis Blood agar: β- (complete) hemolysis = clear zone. Biochemical identification Catalase negative Bacitracin sensitivity : sensitive  Identification of Streptococcus pyogenes β- haemolysis on blood agar Bacitracin sensitivity : sensitive to bacitracin Streptococcus in culture lancefield grouping test will allow you to determine the type of beta hemolytic streptococci by clumping of the blue latex particles with one of the antibodies Ag are extracted from throat swab + Antibodies = (Agglutination)  Laboratory diagnosis of scarlet fever It caused by erythrogenic toxin of Strep. Pyogenes Spot diagnosis: tonsilitis, rash and strawberry tongue Laboratory diagnosis Specimen: throat swab Strawberry tongue of scarlet fever Rapid strep test Throat culture Direct smear stained with Gram shows Gram positive cocci arranged in chains Culture on blood agar for identification of organism specific test by in vivo toxin neutralization test ( Shultz-Charlton test ) The test is done by intradermal injection of antitoxin that cause disappearance of erythematous area injected by antitoxin  Specific test by in vivo toxin neutralization test ( Shultz-Charlton test ) The test is done by intradermal injection of antitoxin that cause disappearance of erythematous area injected by antitoxin Strep. in pus (Charlton test shows : A faint yellowish-brown discoloration over the blanched area, following intradermal injection of scarlet fever antitoxin.  Diagnosis of diphtheria is clinically Laboratory diagnosis for confirmation Laboratory diagnosis of diphtheria case  Diagnosis of carrier It is done by the throat or nasal swabs are diagnosed as in a case. Corynebacterium diphtheriae in culture ( Gram stain) Corynebacterium diphtheriae in culture ( Methylene blue stain)  Elek’s test: it is a double immunodiffusion test It is used for detection of the toxigenic strain of C. diphtheriae The test strain is streaked on a solid agar plate at right angle of filter paper containing the antitoxic serum A line of precipitation appears if the strain is toxigenic. Precipitation band Positive test Negative test  Vincent’s angina The normal spirochaetes with fusiform bacilli multiply acute necrotizing ulcerative gingivitis, associated with ulcerative lesions in oral Acute necrotizing ulcerative cavity and tonsillar area ( membrane) gingivitis) (Trench fever) Causative organisms: Spreads to tonsils It is caused by two anaerobic organisms Treponema or Borrelia vincentii Vincent’s angina Fusobacterium ulcerance which is anaerobic gram negative fusiform bacterium or cigar shaped bacterium. 13 dr. haneya 11/25/2024  Laboratory diagnosis Smear preparation from gingival lesion shows pus cells, Spirochaetes, and Gram -ve fusiform bacilli Gram stain is sufficient for diagnosis of Vincent angina Gram stained film of Treponema vincentii Treponema vincentii by Fontana stain 14 dr. haneya 11/25/2024 Laboratory diagnosis of Oral thrush ( oral candidiasis) It grows on nutrient agar, blood agar & Sabouraud’s dextrose agar Samples: throat swab 1. Direct microscopic examination of specimen shows Gram positive budding yeast cells with pseudohyphae 2. Culture on Sabouraud’s dextrose agar or blood agar or nutrient agar 3. The growing colonies identified by Morphology Germ tube test when colonies incubated in serum at 37C for 1 hours.( specific test for candida albicans) Biochemical test Gram stained film showing Gram positive yeast cells in exudate Germ tube for Candida Culture of Candida on albicans when colonies Gram stained film showing Gram Sabouraud’s dextrose agar incubated in serum at 37C positive yeast cells in culture 2019-2020 Viruses causing upper respiratory tract infections 1. Adenovirus 2. Rhinoviruses 3. Corona viruses 4. EBV 5. Parainfluenza type 1 and 2 6. Respiratory syncetial virus in adult & children 7. Herpes simplex  Laboratory diagnosis of viral infections ❑Virus isolation and identification. ❑ Detection of viral antigen in pathological specimens. ❑ Detection of viral specific antibodies. ❑ Other methods: - Electron microscopy - Molecular diagnosis Virus isolation require the use of living cells There are 3 main systems: I.Tissue culture II.Chick embryo III.Laboratory animals Laboratory diagnosis of respiratory diseases caused by adenovirus Specimen: secretion Detection of Ag by ELISA PCR Cell culture : Cells obtained from man or animal are grown in artificial culture media in glass bottle in a monolayer. These cells are living & metabolizing & support viral replication. Tissue culture bottle  Detection of viral growth in tissue culture Cytopathic effect (CPE) ❑Many viruses infect susceptible cell cultures and produce degenerative cellular changes (CPE) which can be observed by an ordinary light microscope and the responsible virus is said to be cytopathogenic. ❑ inclusion bodies includes (aggregation of viral material or cell debris may collect in area of the cell)  CPE of adenovirus in tissue culture → clumping or grape like cluster of large round cells. The characteristic CPE produced in tissue culture by Herpes virus Herpes virus → enlarged, ballooned cells & multi- nucleated giant cells  Other methods for detection of viral growth in tissue culture Some viruses may produce CPE slowly or not at all, so alternative methods for detecting the presence of these viruses are: a.Heamagglutination and heamadsorption for detection of heamagglutanating viruses b. haemoagglutinating viruses are mixed well with erythrocytes, viruses attach directly to the erythrocytes to produce haemagglutination.  Viral Heamagglutination test for detection of Influenza and Parainfluenza viruses Titer of virus haemagglutinin is 64 in patient No 1 Titer of virus haemoagglutinin is 8 in patient No 2  Result Unagglutinated cells settle at buttom of the well Haemagglutinated cells produce a layer of small clumps that cover the bottom of the wells Titer of the test : The titer is the inversion of the dilution in the last well in which there is a layer of small clumps Titer is highest dilution of virus causing complete haemagglutination Epstein- Barr Virus case A patient has presented to clinic with fever, sore-throat & enlarged lymph node. His peripheral blood smear shows atypical lymphocytes and diagnosed as glandular fever that caused by Epstein Barr virus. They infect B lymphocyte through CD21 - CD8 cell proliferate and make atypical lymphocyte to kill infected cells Laboratory diagnosis Atypical T lymphocyte in blood picture Detection of EBV nucleic acid by PCR Detection of heterophile antibodies by Paul Bunnell test or monospot test  Laboratory finding of infectious mononucleosis EBV viral infection Test Control Monospot test for Atypical T lymphocytes EBV infection EBV antibodies agglutinate sheep red blood cells in tube ( Paul- Bunnell test or monospot test ( slide agglutination )  Laboratory demonstrations S 3 Monospot test ( photo) Photo showing atypical lymphocytes Catalase test ( photo) Strept in culture (slide ) Viral hemagglutination test ( photo) Strept in pus (slide) Bacitracin test ( photo) Cell culture bottle Complete hemolysis on blood agar CPE of adenovirus Lancefield group test Shultz-Charlton test CPE of herpes virus Diphtheria gram stain (slide) Loffler’s serum (photo) Gram positive yeast cell ( candida) Diphtheria on blood tellurite Throat swab Germ tube test Elek’s test ( photo) Culture of candida on sabouraud’s agar Film showing spirochetes Gram stain ( slide) Film showing spirochetes Fontana stain ( photo) Lab 2 Laboratory diagnosis of selected pathogen causing lower respiratory tract infection Learning Outcomes On completion of this lab, the student will be able to: 1. To know the morphology and culture characters of Strept. Pneumoniae 2. To know the laboratory diagnosis of Pseudomonas infection ,Klebsiella infection, pulmonary anthrax and Mycobacterial tuberculosis 3. To know laboratory diagnosis of fungal pulmonary infection  Mycobacterium tuberculosis Morphology: Mycobacteria are thin straight or curved rods. They cannot be classified as either Gram-positive or Gram-negative. They are classified as acid-fast and alcohol- fast as demonstrated by Ziehl-Neelsen stain due to their impermeability by certain dyes and stains. They appear as thin pink rods single or in small groups. Culture Characteristics: M. tuberculosis grows only on selective medium (egg base Lowenstein-Jensen medium). It is an obligate aerobe. Slow growth rate 2-8 weeks.  Laboratory identification of M.tuberculosis Accepted specimen: sputum Rejected specimen : saliva ( high epithelial cells). Suitable container : sterile wide mouth cup Number of specimens: 3 consecuative morning sputum samples to optimize isolation of the organism It should be examined as follows: 1. Direct smears are made from the specimen, and stained with Ziehl-Neelsen stain. 2. Decontamination and concentration of sputum  Two sputum specimens were submitted to the Microbiology laboratory for investigation culture. A Gram stained smear was evaluated for each specimen prior to culture. A. Which one of the two results represents a sputum specimen that is acceptable for culturing Pus cell Epithelial cell Direct detection of actively growing bacilli 1.Microscopy 2.Culture Rapid diagnosis can be done by A. Molecular diagnosis by detection of DNA in the patient ’s specimens by PCR QuantiFERON TB test: it depend on measuring of interferon gamma released when the blood of tuberculosis patient or latent infection is mixed with specific protein derived from tubercle bacilli such as ESAT-6 or CFP or specific tuberculosis antigen B. BACTEC system C. Indicator tube D. Antigen detection methods Indirect methods for the detection of MTB Tuberculin skin test or PPD test Interferon-Gamma Release Assays (IGRA) Ziehl-Neelsen stain experiment Type of stain: differential stain it is used to differentiate between acid fast bacilli and non- acid fast bacilli Microscopy (M. tuberculosis stained by Ziehl-Neelsen from sputum specimen) that appears as red-pink long or slightly curved bacilli Non- acid fast appear blue. Ziehl-Neelsen stain Acid fast bacilli appear thin pink rod in blue background  Traditional culture culture should be done even if smear is positive, for confirmation L -J medium is the traditional method (6- 8 weeks) M.tuberculosis shows buff-colored culiflower colonies on Löwenstein–Jensen medium.  Rapid diagnosis by culture on fluid media Mycobacteria grow more rapidly and reliably in a liquid culture medium compared with the solid medium. (M. tuberculosis stained by Ziehl-Neelsen from mycobacterium culture fluid medium) that appears as cord like due to cord factor stick bacilli together  Rapid diagnosis Culture on fluid media by C.BACTEC culture system D. Mycobacteria indicator tube Culture on media containing C culture on fluid media containing Labeled palmitic acid. fluorescence sensor, when Mycobacteria consume O2 The growing bacteria utilize the palmitic acid produce fluorescence that detected and released radioactive CO2 detected by machine by UV. Time of results is 5-15 days M. tuberculosis in macrophages after staining with auramine-rhodamine dye The bacilli are bright yellow, and the macrophages are green under ultraviolet light when stained with rhodamine auramine stain  The mycobacterial growth indicator tube Mycobacteria is No Mycobacteria fluorescent The MGIT tube on the right contains growing mycobacteria and is fluorescent when exposed to UV light. In contrast, the tube on the left contains no mycobacteria.  Biochemical reaction The colonies on L J medium are identified by biochemical reaction Nitrate reduction into nitrite Principle of test: Inoculate the nitrate broths with bacterial suspension. Incubate the tubes at 37°C for 24 hours. After incubation Add 6-8 drops of nitrite reagent A and add the 6-8 drops of nitrite reagent B. Observe for at least 3 minutes for a red color to develop  Quanti-feron-TB Gold test What is the name of test reaction ? The method of measurement : ELISA method ELISA plate ESAT 6= Early Secretory Antigen Target 6 CFP= culture filtrate protein Quanti-feron-TB Gold test Tuberculin Skin Test  The test is tuberculin test  Type of hypersensitivity : delayed-type hypersensitivity  Injected antigen : Purified Protein derivative (PPD)  Time of reading: 48-72 hours  The best time of reading : 72 hours  Induration area: not less than 9mm  Laboratory diagnosis of lobar pneumonia caused by Strept. pneumoniae Direct gram stained film showing gram positive diplococci capsulated among pus cells. Capsules appear as unstained halos around the organism. Culture on Blood agar: alpha hemolysis The colonies identified by Gram stained film shows Gram positive diplococci capsulated Biochemical identification 1. Catalase negative 2. Quelluing test is positive 3. Tests for differentiation from Strept. viridans prof. Haneya Anani 44 Pneumococci in sputum Positive Quelluing test The test is done by mixing of sputum alpha haemolysis on or suspension of pneumoncoccal blood agar culture with specific antiserum on a microscopic slide and examined by oil Incubate in a 5-10% CO2 or candle – immersion lens shows swollen jar at 35˚ C capsule. Differentiation between pneumococci and Strept. viridans Optochin sensitivity test Bile solubility test Laboratory identifications of Klebsiella pneumoniae K.pneumoniae causes bronchopneumonia Morphology: Gram negative, non-motile, capsulated bacilli. Cultural characters: They give mucoid rose pink colonies on MacConkey’s agar ( lactose fermenter). Specimens : sputum 1. Direct smear stained with Gram stain 2. Culture on MacConkey’s medium 3. Culture on blood agar 4. The colonies identified by A.Gram stained film shows Gram negative bacilli B.Biochemical tests C.Slide agglutination test Laboratory identifications of klebsiella Mucoid Rose pink colonies on MacConkey’s medium (lactose fermenter ) Biochemical reactions Gram negative bacilli ( Klebsiella) Prof. Haneya Anani 48 Biochemical reactions Sugar fermentation with production of acid and gas Indole negative Methyl red negative TSI positive H2S –ve - Ve ornithine decarboxylase Citrate positive Urease positive Lysine decarboxylase positive Laboratory diagnosis of Pseudomonas infections Specimens: sputum Direct microscopic examination: Gram negative non- spore forming bacilli with Gram stained film Culture: MacConkey’s agar produce pale yellow colonies ( non-lactose fermenter). Blood agar produce β- hemolysis Pseudomonas produce exopigment on nutrient agar with grape like adour Pyocyanin pigment that give blue non-fluorescent color on agar Pyoverdin that give a greenish color on agar The colonies identified by A. Microscopy: Gram stained film: Gram negative bacilli non-spore forming B. Biochemical reactions Prof. Haneya Anani 50 Identification of pseudomonas Pseudomonas culture on Nut. Non lactose fermenter pale yellow agar showing exopigment +Ve -Ve gram negative bacilli) Oxidase test , positive reaction is purple color) Haemophilus influenza Haemophilus influenza (capsulated) causing acute epiglotitis Haemophilus influenza (non-capsulated) causing pneumonia Specimen: sputum Direct microscopic examination H. influenzae are Gram negative coccobacilli Direct detection of H. influenza Type b capsule in specimens either by the Capsular swelling (quellung) reaction Culture; on chocolate agar at 37 C in 5% CO2 ( candle jar). Colonies are identified by their morphology Haemophilus require certain growth factor ( X and V factors) for growth Laboratory diagnosis of acute epiglottitis caused by Haemophilus influenzae Specimen : sputum Direct stained smear of sputum show Gram negative coccobacilli capsulated Culture on chocolate agar Incubation condition : 5-10% CO2 at 37C Quellung reaction: swelling of Gram negative coccobacilli capsule after addition of Haemophilus antiserum 5-10% Candle jar Quellung test H. influenzae on Chocolate agar Satellite phenomenon Satellitism is growth of Haemophilus in the vicinity of staphylococcal growth on blood agar Haemophilus does not grow on blood agar  Laboratory diagnosis of pulmonary anthrax Specimens: sputum Direct microscopic examination 1.Gram stained smear: Gram positive large bacilli with square ends arranged in chains (bamboo stick appearance) and surrounded by unstained halo capsule, 2.Direct smear stained with polychrome methylene blue; for demonstration of polypeptide capsule: the organism appears blue while the capsule purple or pink. The specimen are culture on : Blood agar: non-haemolytic reaction with Medusa head colonies Gelatin medium produce inverted fire tree appearance (Due to slow gelatin liquefiers) maximum liquefication on the surface than at the bottom Gram stained film of Bacillus anthracis in Capsular stain of Bacillus anthracis in tissue culture. Bamboo-stalk" arrangement with showing square-ended, blue bacilli in short central spores chains surrounded by a pink capsule (polychrome methylene blue stain) Culture of Bacillus anthracis on blood agar shows non-haemolytic reaction with Medusa head colonies Inverted fire tree on gelatin medium Lab. Diagnosis of Atypical pneumonia caused by Mycoplasma pneumoniae Specimens – throat swabs, respiratory secretions and sputum. 1. Direct detection by PCR 2. Culture on enriched media contain serum shows Fried egg appearance colonies after 3-10 days Grow better at 10% CO2 3. Serological test Lab. Diagnosis of Chlamydia infection 1. Direct detection by PCR 2. Direct microscopic examination of smear stained by Giemsa stain to demonstrate Intracytoplasmic inclusion bodies 3. Direct immunofluorescence to demonstrate Intracytoplasmic inclusion bodies 4. Detection of Chlamydial Ag 5. Isolation in tissue culture Laboratory diagnosis of pulmonary Aspergillosis Aspergillus fumigatus Chest X ray : Fungal ball Laboratory diagnosis Specimen : Bronchial lavage, sputum , biopsy from fungus ball Direct smear stained by Lactophenol cotton blue) shows (Septate hyphae with characteristic aspergillus head) Culture on: SDA Identification from culture : Surface/Reverse color Surface color : green velvety/powdery colonies Reverse color : cream/ tan Molecular diagnosis by PCR  SDA 1. Conidia 2. Conidophore 3. Vesicle Reverse color: cream/tan surface color : green , velvety colonies Laboratory diagnosis of Coccidioidomycosis Diseases: community-acquired pneumonia Causative fungus is Coccidioides immitis Type of fungus is a dimorphic Spore shape :Arthroconidia A. Specimens : sputum or tissue biopsy B. Microscopic Examination microscopic examination shows spherules containing endospores ( Spherules containing spores in tissue diagnostic) C. Culture: culture on SDA medium in the test tube and incubated at 25-30°C. Fragmentation of hyphae into cells called Arthroconidia D. Serological test such as latex agglutination test ( the best diagnostic test)  Laboratory demonstrations Lab 2 1. Gram negative bacilli slide 2. Exopigment on nutrient agar 1. M. tuberculosis ZN stain (Cord factor) (photo) 3. Oxidase test 2. M. tuberculosis ZN stain slide+ ZN stain bottles 4. Non –lactose fermenter on Mac. Agar 3. Lowenstein –Jensen medium 5. Biochemical reaction of klebsiella 6. Mucoid rose pink colonies on Mac agar 4. Indicator tube of M. tuberculosis 7. Aspergillus on SDA 5. Tuberculin test ( photo) 8. SDA 6. Blood sample + ELISA plate ( QuantiFERON test) 9. Conidia stained by LPC blue 7. Catalase test ( photo) 10. Fried egg colony (Mycoplasma) 8. Pneumococci (slide )+ bile solubility test 11. Chlamydia ( Geimsa stain) 9. Strept in culture (slide) 12. Chlamydia ( immunofluorescence) 10. Optochin test (photo)+ quelling test 13. Aspergillus culture 11. Bacillus anthracis in culture (slide ) 14. Sputum samples 12. Bacillus anthracis in tissue ( capsular stain ( photo) 13. Inverted fire tree ( photo) 14. Medusa head colonies on blood agar ( photo) 15. Chocolate agar 16. Gram negative coccobacilli slide