TB Diagnosis Lecture 2021 Notes PDF

Diagnosis of TB Dr Marianne Black Mycobacteriology Referral Laboratory, Braamfontein, Johannesburg. Department of Clinical Microbiology and Infectious Diseases University of the Witwatersrand. July 2019  Lecture objectives List specimen types that are suitable f...

Diagnosis of TB Dr Marianne Black Mycobacteriology Referral Laboratory, Braamfontein, Johannesburg. Department of Clinical Microbiology and Infectious Diseases University of the Witwatersrand. July 2019  Lecture objectives List specimen types that are suitable for TB diagnostic investigations. List the advantages and limitations of direct microscopy as diagnostic method for TB. List the advantages and limitations of culture as diagnostic method for TB. List the advantages and limitations of phenotypic drug resistance determination in TB. List the advantages and limitations of genotypic drug resistance determination in TB. Compare the Xpert MTB/RIF assay and the Hain Line Probe assay. Grade TB diagnostic platforms in order of increasing sensitivity. Explain the principle of the diagnosis of latent TB, and list the available platforms. List the limitations of existing tests for the diagnosis of latent TB. Describe the principle of the urine lipoarabinomannan assay and the WHO recommendation for use. 2  Specimen Collection and Transport Specimen collection: – Safety for health care worker (HCW). – Clear labelling. – Good quality, representative specimen: Early morning is best (Not always feasible). Specimen transport: – Safe packaging of materials to minimise risk of leakage/contamination. – Should arrive at the laboratory within 4 hours. – Cold chain, out of direct sunlight: Want to avoid overgrowth of other bacteria that grow quicker than mycobacteria. High temperatures kill bacilli (For culture of M. tuberculosis). Transport delays: Refrigerate and transport on ice. Specimen Type (CULTURE): – Pulmonary TB: Sputum, Induced sputum, Gastric aspiration, Bronchial washings, Brochoalveolar lavage (BAL). – Disseminated TB/ Isolated organ TB: Blood, CSF, urine, lymph node aspirate, tissue and other body fluids, bone marrow, skin samples, other. 3  Specimen Collection and Transport Sputum: – Sterile, wide mouthed, tightly fitting screw top, disposable container: Provided by lab. – Induced sputum (nebulised hypertonic saline) – strict airborne precautions: Paediatric setting. Adult patients, difficulty with producing sputum. Gastric aspirates: – Early morning aspirate after fasting 4-6hr. Urine: – First morning, clean catch, midstream urine. – Minimum of 40ml (as much as possible). – 24-hr pooled specimen NOT ACCEPTABLE. Blood culture: – Aseptic collection. – Inoculate 5ml at bed site into broth media. – BACTEC MycoF Lytic. Cerebrospinal Fluid: – Minimum of 5 ml. Tissue and fluids: E.g. Lymph node/pleural fluid aspirate/biopsy. – Aseptic collection. – Sterile container/NO fixative (eg formalin) /NO preservative. 4  Active TB disease diagnosis Direct Microscopy – Auramine smear Culture – Specialised liquid culture media (mycobacteria growth indicator tube –MGIT) Drug susceptibility testing – Phenotypic methods – Genotypic methods 5  Direct Microscopy Advantages Limitations Inexpensive. Low sensitivity (25% – 65%): – Visual detection limit is 10’000 Rapid results. bacilli/ml – Influenced by specimen quality Quantitative measure of (sputum vs saliva), site (pulm vs bacterial load: extra-pulm), HIV status (pauci- bacillary disease) – Smear grade correlated with Cannot differentiate infectiousness (negative, mycobacterial species (MTB scanty, 1+, 2+ 3+). complex vs NTM). – Treatment monitoring. Cannot differentiate between viable / nonviable bacilli. Cannot determine drug susceptibility. 6  Culture Advantages Limitations Remains the gold standard – Mycobacteria divide ~ high sensitivity (requires only once/day: Lengthy time to 10-100 bacilli/ml), but does depend on specimen result. quality/type. Prone to contamination - Allows for precise Bacteria/fungi outgrow identification of species. mycobacteria. Allows for phenotypic (culture) based drug susceptibility Requires Biosafety Level III testing. laboratory infrastructure. Used to monitor treatment response. 7  Drug susceptibility testing (DST) Phenotypic (culture based) Genotypic methods methods Comparison between growth in the Detects mutation in the presence and absence of the drug being tested. DNA sequence, which Advantages confers resistance. – Gold standard for DST: Detects additional resistance that may have been missed by genotypic methods. Gene Xpert® MTB/RIF assay – Tests a larger repertoire of drugs (1st (GXP) / Xpert MTB/RIF Ultra line and 2nd line drugs). – MICs can also be determined. Hain Lifescience® Line Limitations probe assays (LPA) – Lengthy time to result. – Prone to contamination: DST cannot be reported if culture is not pure (bacteria/fungi) or if mixed infection with a NTM/MOTT. 8  Drug susceptibility testing – Genotypic methods (GXP) Gene Xpert MTB/RIF assay – Identify MTB complex and provides rifampicin susceptibility result. – Performed on pulmonary and selected extra-pulmonary specimens (eg purulent fluid, tissue, CSF) – Turn-around time of 24 hours 9 Xpert MTB/RIF /Ultra assay Minimal expertise needed Turn-around-time (TAT) of 24 hours (laboratory based) 10  Drug susceptibility testing – Genotypic methods (LPA) Hain Line probe assay (LPA) MTBDRplus and MTBDRsl Detects MTB complex 1st line LPA: rifampicin and isoniazid. 2nd line LPA: fluoroquinolones and second-line injectable agents. Performed on clinical specimens and culture. More complex process, expertise needed, laboratory infrastructure. TAT: 2-3 days 11  Drug susceptibility testing – Genotypic methods Limitations – Limited range of drugs tested. – Cannot be used to monitor response to treatment: PCR-based platforms will amplify residual DNA even if organism in non-viable. – Genotypic assays amplify limited gene regions: Some resistance-inducing mutations may be missed. 12  Limit of detection GXP Ultra ~10-100 bacilli/ml 13 National Tuberculosis Management Guidelines 2014 14 Laboratory request form 15  Latent TB infection - diagnosis Principle: Detect immune response: Cell mediated immunity to previous infection with M. tuberculosis (memory). Available platforms: – Skin: induration in response to inflammation. Tuberculin skin test (purified protein derivative [PPD], Mantoux test). – Blood: production of γ-interferon by white blood cells (Measure γ-IFN levels). Interferon Gamma Release Assays (IGRA) 16  Latent TB infection - diagnosis Limitations of existing tests: – They cannot differentiate between latent TB infection and active TB disease: May be positive in both disease and infection. Still have to rule out active disease with microbiological tests. – False negatives may occur in immunocompromised or in advanced TB disease. – Limited use in high endemic countries. – TST: Time consuming, subjective, less specific than IGRA (Injected antigens are found in most mycobacteria). – IGRA: Expensive, but more specific than TST (Injected antigens are specific to M. tuberculosis). 17  Lateral flow urine lipoarabinomannan assay (Urine LAM test) Based on the detection of mycobacterial lipoarabinomannan (LAM) antigen in urine. Released from metabolically active or degenerating mycobacterial cells. Advantages – Point-of-care test for TB. – LAM only present in people with active TB disease. – Urine is easy to collect and store, and lacks the infection control risks associated with sputum collection. – Improved sensitivity in HIV-TB co-infection, which further increases with lower CD4 counts (CD4