Microbiological investigations in ent.pptx

Useful for 3rd mbbs course

1. Microbiological investigations in ent - Sonal

2. Microbiological investigations in ENT involve laboratory tests to identify microorganisms causing ear, nose and throat infections Accurate diagnosis and treatment of ENT infections.

3. Types of organisms Bacteria – streptococcus , pseudomonas aeuroginosa , corynebacterium diphtheriae Viruses – rhinovirus , adenovirus , coronavirus , influenza Fungi – candida albicans , aspergillus , mucor

4. Types of samples Nasal swabs Throat swabs Ear swabs Sinus aspirates Tonsillar swabs Laryngeal swabs Sputum samples Blood samples

5. Bacterial infections in ENT Staphylococcus aureus – furuncle , diffuse otitis externa , CSOM , acute suppurative parotitis , acute and chronic mastoiditis , septal abscess , pharyngitis , tonsillitis , parotitis , adenoiditis , peritonsillar abscess - haemolytic streptococci – otitis externa , otitis media , mastoiditis, sinusitis , septal abscess, pharyngitis , tonsillitis , parotitis, acute necrotizing otitis media Streptococcus pneumonia – AOM , CSOM , otitis externa , tonsillitis , Ludwig’s angina , laryngitis , rhinosinusitis , nasopharyngitis , rhinitis Haemophilus influenzae – ASOM , tonsillitis , Ludwig’s angina , epiglottitis ,laryngitis , rhinosinusitis , pharyngitis Pneumococcus – rhinitis , tonsillitis , nasopharyngitis Corynebacterium diphtheriae – faucial diphtheria , laryngeal diphtheria Pseudomonas aeruginosa – necrotizing otitis externa , CSOM , Mycobacterium tuberculosis – otitis media , nose ,oral granulomatous infection , TB of larynx Actinomyces – oral ulcers , granulomatous infection of oral cavity

6. Viral infections Adenovirus – rhinitis , sinusitis , pharyngitis Rhinovirus - rhinitis , sinusitis , pharyngitis , common cold Influenza virus – pharyngitis , rhinitis Parainfluenza – pharyngitis , laryngotracheobronchitis , HSV – otitis externa , labyrinthitis ,rhinitis , gingivostomatitis CMV – pharyngitis , cochlear implant infection EBV – infectious mononucleosis , tonsilliltis , pharyngitis

7. Fungal infections Candida – otomycosis , nose , fungal ball , fulminant fungal sinusitis , oral thrush ,pharyngitis , laryngeal mycosis Aspergillus – otomycosis , fulminant fungal sinusitis ,chronic invasive sinusitis , allergic fungal sinusitis , Aspergillosis of nose and sinuses Mucor – rhinocerebral mucormycosis , sinusitis Histoplasma - Laryngeal mycosis , tracheobronchitis

8. Staphylococcus aureus Specimen – pus , wound swab , sputum , blood , nasal swabs Direct smear microscopy – gram positive cocci in clusters Culture : Nutrient – circular , smooth , convex , produce yellow pigments Blood agar – similar to nutrient agar surrounded by zone of -haemolysis Peptone water – uniform turbidity Selective media – mannitol salt agar Culture smear microscopy – gram positive cocci in clusters ;hanging drop – non motile Biochemical identification : Catalase – positive ; coagulase- positive ; protein A detection Automated – MALDI-TOF Antimicrobial susceptibility testing

9. - haemolytic streptococci Specimen – pus swab , exudates , blood Direct smear microscopy – gram positive cocci in chains Culture : Blood agar – small , pinpoint , wide zone of - haemolysis Selective media – crystal violet blood agar ( S. pyogenes ) Culture smear microscopy – gram positive spherical cocci in chains ( hanging drop – non motile ) Biochemical tests : Catalase – negative Bacitracin sensitivity test – resistant ( group B ) Automated – MALDI-TOF Antimicrobial susceptibility test

10. Haemophilus influenzae Specimens: Sputum, blood, CSF Direct examination : Pleomorphic gram-negative coccobacilli Capsule detection: By Quellung reaction Antigen detection: By latex agglutination test, direct-IF or ELISA Culture : Blood agar with S. aureus streak line shows satellitism Others: Chocolate agar, Fildes agar and Levinthal’s agar Identification : Biochemical tests such as disk test for X and V factor Automated systems such as MALDI-TOF or VITEK Typing methods such as biotyping and serotyping (using specific antisera) Molecular method: Multiplex PCR ( BioFire FilmArray ), detecting common agents of pyogenic meningitis in CSF Antimicrobial susceptibility testing

11. pneumococcus Specimen collection : Sputum, CSF, pleural fluid Direct smear microscopy : Reveals pus cells and lanceolate shaped gram-positive diplococci, surrounded by a clear halo (due to capsule) Capsular antigen detection in CSF: By latex agglutination C-antigen detection in urine and CSF by ICT Culture : Blood agar: It forms draughtsman or carrom coin shaped colonies Chocolate agar: It produces greenish discoloration (bleaching effect) Culture smear : Reveals lanceolate-shaped gram-positive diplococci Identification : Biochemical identification: It is bile soluble, optochin sensitive, inulin fermenter Automation methods such as MALDI-TOF and VITEK Serotyping : By Quellung reaction or latex agglutination test Antimicrobial susceptibility testing.

12. Corynebacterium diphtheriae Isolation of the Corynebacterium diphtheriae Specimen: Throat swab and a portion of pseudomembrane Direct smear : Gram stain: Club shaped gram-positive bacilli with Chinese letter arrangement Gram stain: Club shaped gram-positive bacilli with Chinese letter arrangement Culture media : Enriched medium: Blood agar, chocolate agar and Loeffler’s serum slope Selective medium: Potassium tellurite agar and Tinsdale medium, produces black colonies Diphtheria toxin demonstration : Elek’s gel precipitation test Detection of toxin-by ELISA or ICT

13. Pseudomonas aeruginosa Sample collection: Pus, wound swab, urine, etc Direct smear: Gram-negative bacilli, and pus cells Culture : Nutrient agar: Opaque, irregular colonies with metallic sheen (iridescence) and blue green diffusible pigments Blood agar: β-hemolytic grey moist colonies Selective media: e.g. cetrimide agar Culture smear and motility: Motile, gram-negative bacilli Identification : Catalase positive and oxidase positive ICUT tests: Indole (–), Citrate (+), Urease (–), TSI:K/K, gas (–), H2 S(–) Antimicrobial susceptibility testing

14. Mycobacterium tuberculosis Specimen collection : In pulmonary TB: Sputum (2 specimens—spot and early morning), gastric aspirate (in children) ‰ EPTB: Specimens vary depending on the site involved Digestion, decontamination and concentration of specimen Direct microscopy by acid-fast staining : Ziehl- Neelsen (ZN) technique—long slender, beaded, less uniformly stained red colour acid-fast bacilli Kinyoun’s cold acid-fast staining Fluorescent (auramine) staining—it is more sensitive and smears can be screened more rapidly than ZN stain Conventional culture media —take 6–8 weeks Culture identification : MPT 64 antigen detection—by ICT

15. Molecular methods : PCR detecting IS6110 gene CBNAAT (GeneXpert) and Truenat —for identification and detection of resistance to rifampicin; has a turnaround time 2 hours Line probe assay (e.g. Genotype TB)—for identification and detection of resistance to 1st and 2nd line ATDs; has a turnaround time of 2–3 days Diagnosis of latent tuberculosis By tuberculin skin test (e.g. Mantoux test) and interferon gamma release assay (IGRA).

16. mucor Histopathological staining such as H&E or methenamine silver stain of tissue biopsies shows broad aseptate hyaline hyphae with wide angle branching Culture on SDA at 25°C: Reveals characteristic white cottony woolly colonies with tube filling growth (hence called lid lifters) Microscopic appearance : broad aseptate hyaline hyphae, from which sporangiophore arises and then ending at sporangium which contains numerous sporangiospores Rhizoid : Some species bear a unique root like growth arising from hyphae Mucor: rhizoids absent.

17. candida Direct microscopy : Gram-positive oval budding yeast cells with pseudohyphae Culture on SDA : Produces creamy white and pasty colonies Tests for species identification : Germ tube test (positive for C. albicans) Growth on CHROMagar Growth at 45°C (positive for C. albicans) Molecular methods such as PCR Immunodiagnosis : Antibody detection against cell wall mannan antigen Antigen detection such as cell wall mannan and cytoplasmic antigens Enzyme detection, e.g. enolase

18. aspergillus Direct examination : KOH (10%) mount or histopathological staining (Fig. 69.6) of specimens reveals characteristic narrow septate hyaline hyphae with acute angle branching Culture : inoculated into SDA Antigen Detection β- d-Glucan antigen assay: Galactomannan antigen ( specific ) Antibody Detection

19. virus Direct Demonstration of Virus Electron microscopy Immunoelectron microscopy Fluorescent microscopy Light microscopy: Histopathological staining: To demonstrate inclusion bodies ¾ Immunoperoxidase staining. Detection of Viral Antigens By various formats such as ELISA, direct IF, ICT, flow through assays. Detection of Specific Antibodies : Newer diagnostic formats such as ELISA, ICT, flow through assays Molecular Methods to Detect Viral Genes : Nucleic acid probe—for detection of DNA or RNA by hybridization PCR—for DNA detection by amplification Reverse transcriptase-PCR—for RNA detection Real time PCR—for DNA quantification Real time RT-PCR—for RNA quantification. Isolation of Virus by : Animal inoculation Embryonated egg inoculation Tissue cultures: Organ culture, explant culture, cell line culture (primary, secondary and continuous cell lines).