THE ROLE OF THE MICROBIOLOGY LABORATORY IN THE DIAGNOSIS OF RESPIRATORY TRACT INFECTIONS

1. THE ROLE OF THE MICROBIOLOGY LABORATORY IN THE DIAGNOSIS OF RESPIRATORY TRACT INFECTIONS Dr Catherine Samuel Division of Medical Microbiology University of Stellenbosch National Health Laboratory Service

2. COMPLETING THE REQUEST FORM • A request form must accompany each sample submitted to the laboratory (or 1 form if multiple tests are requested on a pt) • Pt's surname + first name • Pt's hospital no. clinic number or ID no. • Pt's DOB & sex • Requesting physician's name • Contact no. • Person who took the specimen • Date + time of collection • Source of specimen • Diagnosis • Indicate the test(s) requested

3. LABELLING THE SAMPLE • NB: Lab will not process unlabelled specimens • Pt's surname + first name • Pt's hospital number, clinic number or ID number • If available, make use of the addressograph sticker provided • Use only sterile, screw cap, leak proof, disposable plastic containers for specimen collection

4. SAFETY AND INFECTION CONTROL •  Safety considerations with regard to the handling of specimens: • Treat all specimens as potentially hazardous • Do not contaminate external surface of the collection container or its accompanying paperwork • Minimize direct handling of specimens in transit from pt to lab. Ideally, specimens should be placed in plastic sealable bags with a separate pouch for specimen request form Protect yourself! • Practice universal precautions: • Wear gloves and a lab coat or gown when handling blood/body fluids • Change gloves after each patient or when contaminated • Wash hands frequently

5. Specimen quality Consequences of a poorly collected and/or poorly transported specimen include: i) failure to isolate causative organism ii) recovery of contaminants or normal flora which may be misleading & result in improper Rx of pt • Diagnosis of LRTI is frequently complicated by contamination of specimens with URT secretions during collection • Specimen quality is judged microscopically • A properly collected specimen should contain minimal numbers of squamous epithelial cells + significant numbers of neutrophils with bacterial infection

6. Throat (Pharyngeal specimens): • Routinely used for the isolation of Group A Streptococci • If diphtheria is suspected, a sample of the pseudomembrane should be collected • Do not obtain throat samples if epiglottis is inflamed, as sampling may cause serious respiratory obstruction • Depress tongue gently with tongue depressor • Extend sterile swab between the tonsillar pillars & behind the uvula. (Avoid touching the cheeks, tongue, uvula, or lips) • Sweep the swab back + forth across the post. pharynx, tonsillar areas, any inflamed or ulcerated areas to obtain sample

7. EPIGLOTTITIS • Culture of the throat not indicated • Touching inflamed epiglottis may precipitate complete obstruction of the airway SINUSITIS • Needle aspirate of sinuses • Do not submit a swab

8. LOWER RESPIRATORY TRACT SPECIMEN COLLECTION • Sputum, • Endotracheal aspirates (TA) • Bronchial washings, bronchial brushes, bronchial biopsy specimens • Bronchoalveolarlavage (BAL)fluid • Lung biopsy

9. It is best to obtain a sputum specimen early in the morning, before the patient has eaten or taken medication • Give pt clear instructions & explain the difference between sputum & saliva/spit • The patient should rinse his/her mouth with water prior to collection • NB: aerosols containing TB bacteria • It is best for the pt to produce a specimen either outside in the open air or away from other people • All specimens should be transported to the lab promptly. Failure to do this may result in the death of fastidious organisms & in overgrowth by more hardy bacteria. If prompt delivery is not possible specimens should be refrigerated at 4-8OC

10. General guidelines for specimen collection for TB analysis • Collect initial specimens before anti- TB meds are started • Collect sufficient quantity for the tests requested • Do not use any fixatives or preservatives • Transported to the lab asapafter collection. If not possible, the specimens should be refrigerated to inhibit the growth of unwanted micro-organisms • Do not freeze specimens. • Mycobacteria are killed by UV light, therefore specimens should not be placed anywhere where they may be exposed to direct sunlight or become too hot • Sputum microscopy & culture: Acid-fast microscopy is done on all specimens where TB microscopy is requested & where sample volume is sufficient If 3 consecutive sputum specimens are –vefor AFB & TB still clinically suspected, send 2 more sputum specimens on 2 consecutive days for TB culture

11. 3 Ways in which micro lab can id bacterial agents of RTI • Direct detection of the pathogen in a patient specimen • Culture of bacteria for identification and sensitivity testing • Taking measurements of patient responses to the presence of a pathogen by means of antibody detection (serology)

12. 3 Ways To Identify Bacterial agents of RTI • Direct detection of the pathogen in a patient specimen Microscopy Antigen detection • Molecular methods

13. 3 Ways To Identify Bacterial agents of RTI 2. Culture of bacteria for identification + sensitivity testing

14. 3 Ways To Identify Bacterial agents of RTI • Taking measurements of patient responses to the presence of a pathogen by means of antibody detection (serology) Serology is mainly used for pathogens that cannot be cultured in cell-free media

15. Diagnostic studies for specific agents of RTI • Bacteria • Streptococcus pneumoniae • Haemophilusinfluenzae • Moraxellacatarrhalis • Staphylococcus aureus • Gram-negative bacilli • Other • Available assays • Gram stain + Culture • Respiratory secretions • Pleural fluid • Blood cultures

16. Diagnostic studies for specific agents of RTI • Legionella species • Culture for respiratory secretion/tissues • Buffered charcoal yeast extract (BCYE) – “gold standard” (not performed at TBH, only at reference labs) • Urine Ag detection • Several kits available • Reliably detects only Legionellapneumophilaserogroup 1 • Serology • Acute-and convalescent-phase specimens • 1-3 months may be required for seroconversion • PCR with respiratory secretions – most promising

17. Legionellapneumophila. (A) Gram stain of a bronchial biopsy specimen in a patient with fulminant Legionnaires’ disease. (B) Culture plate showing white colonies on buffered charcoal yeast extract medium.

18. Diagnostic studies for specific agents of RTI • Mycoplasmapneumoniae • Serology – method of choice • Culture – rarely performed; requires specialized media, prolonged incubation • PCR – available through reference labs

19. Diagnostic studies for specific agents of RTI • Chlamydia pneumoniae • Serology – microimmunofluorescence (MIF) is best • Culture – not widely available • PCR • Chlamydia psittaci • Serology – culture not recommended in routine diagnostic labs due to safety risk

20. B. pertussis Charcoal cephalexin plates showing growth of “mercury drop” colonies of Bordetellapertussis • Contact laboratory • Special agar medium (charcoal-cephalexin plates) • Calcium-alginate swabs for the collection of pernasalspecimens • Insert swab into nasal passage, aiming towards the midline & down to posterior wall of nasopharynx • Take > 1 swab on consecutive days for optimal results • Plates are incubated for 7 days • Alternative: Nasopharyngeal aspirate (for PCR detection) • PCR: rapid, sensitive; calcium alginate swabs are inhibitory to PCR Whooping cough

21. Diagnostic studies for specific agents of RTI • Coxiellaburnetii • Serology – titer of antibody to phase I + II by immunofluorescent antibody (IFA)

22. Diagnostic studies for specific agents of RTI • Nocardia species • Gram stain & modified acid fast stain • Culture of respiratory specimens + tissues • PCR “chalky” white colonies on blood agar Gram positive filamentous branching bacilli

23. Diagnostic studies for specific agents of RTI • Mycobacterium species • Acid-fast stain • Culture : broth/solid media • Direct amplification techniques available Ziehl-Neelsen stain Auramine stain

24. Mycobacteriumtuberculosis Solid culture media for TB: Lewenstein-Jensen (LJ) media “Bread crumb” colonies of TB growing

25. Mycobacteriumtuberculosis BACTEC Midget® : automated liquid TB culture system

26. HAIN test®: Molecular based technique for the identification + sensitivity testing of TB

27. Diagnostic studies for specific agents of RTI • Fungi • Blastomyces • Histoplasma • Coccidioidesimmitis • Sporothrixschenckii • Fungal stains • Gomori’smethenanine silver (GMS) • Calcoflour white • Periodic acid-Schiff (PAS) • Culture • PCR • Serology – may be unreliable in immunocompromised host

28. Cryptococcus species • Gram stain, India Ink • Culture • Serum cryptoccocal latex antigen agglutination test (CLAT) • Opportunistic Fungi Candida species • Gram stain or fungal stain • Culture • 1-3-beta-D-glucan antigen detection in blood (Fungitell assay ®)

29. Opportunistic Fungi… Aspergillus species • GMS or calcofluor stain (histology) • Recovery in culture – recommended, as other molds may have similar histolopathologic appearance • Galactomannan Ag detection in blood • 1-3-beta-D-glucan antigen detection in blood (Fungitell assay ®) (A) Lactophenol cotton blue stained preparation showing characteristic conidiophores (B) Aspergilloma. Tomogram showing fungus ball contained within the lung cavity (C) Invasive aspergillosis. Histologic section showing fungal hyphae invading the lung parenchyma + blood vessels

30. Opportunistic Fungi… Zygomycetes species • GMS or calcofluor stain: non-septate broad hyphae suggestive of zygomycetes • Recovery in culture • Histopathology of tissue section

31. Opportunistic fungi Pneumocystisjiroveci (carinii) PCP • Giemsa stain, GMS, other • Direct fluorescent antibody stain • Induced sputum, BAL gives higher yield • PCR Methenamine silver stain of BAL: cysts of P.jiroveci Indirect immunofluorescence using monoclonal ab’s